Cerebral neurothropic factor

1. introduction

The present invention relates to nucleic sequences encoding brain neurotrophic factor (BDNF), substantially pure protein, peptide fragments or derivatives derived therefrom, as well as antibodies directed against BDNF protein, peptide fragments or derivatives. The invention further relates to genes that are new members of the newly defined BDNF / NGF gene family and products of these genes. The invention also relates to pharmaceutical compositions containing active doses of BDNF gene products or, conversely, antibodies directed against BDNF gene products, and, individually, the BDNF gene products of the invention are valuable for the treatment and diagnosis of dopaminergic neuronal disorders such as like Parkinson's disease, as well as sensory neuronal damage and retinal degenerative diseases.

2. Preconditions of the invention

2.1. Nerve cell death and the role of neurotrophic factors in the development of the nervous system

In the early stages of development, many parts of the vertebrate nervous system contain significantly more neurons than those found in adult animals. Early developmental periods are characterized by periodic natural killing of nerve cells (Carr and Simpson 1978, J. Comp. Neurol. 182: 727-740; Cohan et al. 1984 Science 225: 1258-1265). Survival, differentiation, and maturation of developing neurons can be regulated more rapidly by external environmental factors or epigenetic factors than by a strict hereditary genetic program. For example, experimental work on chicken embryos has shown that transplantation or dissolution of peripheral field-targets, such as limb plexus or eye, in the early stages of chick development may result in a corresponding increase or decrease in sensory sympathetic and motor neurons adjacent to the increased or decreased field target. decrease (Hamburger 1934, J.Exp.Zool

68, 449; Holiyday and Hamburger. 1976, J. Comp. Neurol. 170: 311-321; Zandmesser and Pilar 1976, J.Cell. Biol., 68: 357-374). The field-target can only support a limited number of neurons, and excess neurons can be selected during normal development to adapt to the tissue-target neurotrophic power. The discovery and release of a protein now called nerve growth factor (NGF) has led to the molecular hypothesis that the target can regulate the amount of neurons that survive and innervate this tissue (Levi-Montalcini et al. 1968, Physiol. Rev. 48: 524- 569; Thoenen and Barde 1980, Physiol. Rev. 60: 1284-1335).

It is now well established, at least in the peripheral nervous system, that nerve tissue targets synthesize and release a limited number of various neurotrophic molecules that are critical for the survival of certain types of neurons (Korsching and Thoenen, 1983, Proc. Natl. Acad. Sci. USA, 80: 3513-3516; Neumann et al. 1984, EMBO, 3: 3183-3189; Shelton and Reichardt 1984, Proc. Natl. Acad. Sci. U.S.A. 81: 70517955; Korsching and Thoenen 1985, Neurosci. Lett. 54: 201- 205).

2.2 Nerve growth factor

Nerve growth factor is currently the most fully characterized neurotrophic molecule of this type and has been shown to be essential for the early survival of sympathetic and descendent sensory neurons in both chickens and rats, both in vitro and in vivo (Levi- Montalcini and Angeletti, 1963, Develop Biol 7: 653-659; Levi-Montalcini et al., 1968, Physiol. Rev 48: 524-569). Injections of purified NGF into developing chicken embryos have shown that NGF causes severe hyperplasia and hypertrophy of spinal cord sensory neurons and sympathetic neurons (Levi-Montalcini and Booker, 1960 Proc. Natl. Acad. Sci. USA 46: 373-384; Hamburger et al. , 1981 J. Neurosci 1: 6071). In contrast, removal or destruction of endogenous NGF by daily administration of anti-NGF antibodies neonatal rats was ultimately associated with disruption of the sympathetic nervous system (Levi-Montalcini and Booker, 1960, Proc. Natl. Acad. Sci. USA, 46: 384-391; Levi-Montalcini and Angeletti, 1966, Pharmacol Rev. 18: 619-628). The use of NGF antibodies, even at the earliest stage of development, during delivery to the uterus or during passive transplacental maternal antibody delivery, resulted in a significant reduction in sensory neurons originating from the nerve compartment, such as the dorsomedial and dorsomedial trigeminal nerve sensory neurons (Goldert et al., 1984, Proc. Acad. Sci. USA, 81: 1580-1584; Gorin and Johnson 1979, Proc. Natl. Acad. Sci. USA, 76: 5382-5386). Until recently, almost all NGF studies have been concerned with its role in the peripheral nervous system, but it is now clear that NGF also affects the development and function of some populations of neurons in the central nervous system (Thoenen et al., 1987, Rev. Physiol. Biochem. . Pharmacol. 109: 145-178; Whittmore and Senger 1987, Brain Res. Rev. 12: 439-464).

Successful discovery of high levels of NGF protein in the adult mouse mouse forearm gland (Cohen, 1960, Proc. Natl. Acad. Sci. USA 46: 302-311) and even earlier discovery of high levels of NGF in snake venom (Cohen and LeviMontalcini 1956, Proc. Natl. Acad. Sci. USA, 42: 571-574) gave sufficient amounts of NGF to be studied in physiology, protein chemistry and later molecular biology (i.e., molecular cloning of NGF and its receptor). The function of high levels of NGF in the adult rat male gonadal remains unexplained, but it is clear that such a rich source cannot play any role in the development and maintenance of the peripheral and central nervous system. In tissue-target innervated neurons sensitive to NGF (neurons that have been found to depend on NGF for their survival, high affinity for NGF receptors, high specificity NGF internalization, and retrograde transport), constant measurable levels of NGF are very low, at picograms and nanograms per grams of tissue compared to a thousand times the level of adult rats in the peritoneal gland. NGF was not detected in any significant amount in the serum and is therefore apparently not a circulating growth factor or hormone (Suda et al., 1978, Proc. Natl. Acad. Sci. USA 75: 4042-4046).

In addition to the discovery of a major source of NGF protein in the murine forearm, the development of sensitive, reliable, and efficient biological experiments played an important role in elucidating the biology and biochemistry of NGF. Neurons of the developing chicken germ dorsal root node (DRG) were among the first to detect the ability to respond to NGF in vitro. Chicken E8-E12 DRG explant cultures in plasma clot and subsequently neuronal enriched chicken DRG cultures have been shown to be very useful in bioassays investigating NGF activity (for example, during purification) and in vitro bioassays of NGF (Levi-Montalcini et al., 1954, Cancer Res 4: 49-57; LeviMontalcini and Angeletti 1963, Develop-Biol 1: 653-659; Greene 1977, Develop Biol 58:96, ibid. 58: 106). The availability of more than 40 DRGs from a single chicken embryo through dissection has led to widespread proliferation of bioassays with NGF in many laboratories.

In addition to the availability of large amounts of NGF protein and efficient testing of NGF systems, the third major factor that has made an important contribution to our understanding of NGF biology is the relative ease with which NGF antibodies are obtained from guinea pig, rabbit, sheep, etc .; murine NGF appears to be highly immunogenic. Cohen (1960, Proc. Natl. Acad. Sci. USA 46: 302311) received antibodies against NGF secreted from the mouse forearm and, together with Levi-Montalcini and Booker (1960, Proc. Natl. Acad. Sci USA, 46: 384-391) that these antibodies cause sympathetic node destruction or immunosympathectomy when administered daily to rat neonates (Levi-Montalcini and Angeletti 1966, Pharmacol. Rev. 18: 619-628).

The abundance of NGF protein allows its primary sequence to be determined using relatively conventional chemical assays for protein (Angeletti and Bradshaw 1971, Proc. Natl. Acad. Sci. USA 68: 2417-2420). Many animals, including mice (Scott et al., 1983, Nature, 302: 538-540), human (Ullrich et al., 1983, Nature 303-821-825), cows and chickens (Meier et al., 1986). , EMBO 5: 1489-1493) and rats (Whittmore et al. 1988, J. Neurosci. Res. 20-402-410) The NGF gene was cloned using conventional molecular biology techniques based on the generation of appropriate oligonucleotide probes using the mouse NGF protein sequence. NGF availability also significantly facilitated NGF receptor assay, ultimately leading to molecular cloning of the human and rat NGF receptor (Johnson et al., 1986, Cell 47, 545-554; Radeke et al., 1987, Nature 325: 593-597). ).

It is now well known that NGF is not a ubiquitous neurotrophic factor. As found in in vitro and in vivo studies, NGF is not likely to be a survival factor of the parasympathetic neurons derived from the sensory neurons of the nerve patch or the intestinal neurons. Furthermore, NGF is probably not a survival factor for developing motoneurons (Appengeim, 1982, J. Comp. Neurol. 210: 174-189), although these neurons express at least a weakly affinity form of the NGF receptor during their development (Raivich et al., 1985, EMBO, 4: 637-644). Inactivity of NGF for this type of neuron has led to the search for other neurotrophic factors, particularly factors that support the survival of spinal motor neurons and / or parasympathetic neurons of the dorsal root ganglia.

Other neurotrophic factors

In recent decades, there have been numerous reports of neurotrophic activity in various tissue extracts and in many types of cell-conditioned growth media. However, in almost all cases, the purification and characterization of those active substances was hampered by their very small amounts, in the range of pico and nanograms per gram of tissue.

Furthermore, although adequate bioassays with peripheral neurons have been performed, the development of robust, repetitive, and specific experiments on central nervous system neurons has proven problematic. Whereas separate types of peripheral neurons are found in distinct, easily susceptible nodal nodes, the distribution of central nervous system neurons is always very heterogeneous. Yes, specific markers are required to identify and isolate individual classes of CNS neurons. Progress in obtaining markers such as antibodies directed against the cell surface or cytoskeletal components or specific histological staining has been very limited. Accordingly, it has proved very difficult to characterize neurotrophic factors that:

(I) not as abundant as NGF (II) difficult to experiment with, (III) not sufficient to produce antibodies.

2,3.1. Comparison of Brain Ncurotrophic Factor (BDNF) with Nerve Growth Factor

Neurotrophic activity capable of supporting neuronal survival of the dorsal root node of the chicken embryo was identified in vitro in conditioned media cultured in rat C-6 glioma cells (Barde et al., 1978, Nature 274: 818). This activity was not neutralized with antibodies to murine NGF, suggesting another neurotrophic factor in the conditioned medium. Similar activities not blocked by NGF antibodies were subsequently detected in adult rat cerebral astroglial normal cell cultures (Lindsay 1979, Nature 282: 80-82 Lindsay et al. 1982, Brain Res 243: 329-343) and in developing and adult rat cerebellum. extracts (Barde et al., 1980, Proc. Natl. Acad. Sci. USA, 77: 1199-1203) and in the spinal cord of developing and mature chickens (Lindsay and Peters 1984, Neurosci 12: 45-51). However, in neither case were active factors isolated and identified, and it remains unclear whether the observed activity belongs to the same or several factors.

Using a porcine brain as a starting material, Barde et al., (1982, EMBO, I: 549-553) reported a factor, now referred to as brain neurotrophic factor (BDNF), which improved neuronal survival of the dorsal root node from the E10 / E11 chicken embryo. Neurotrophic activity was found to be highly alkaline protein (with an isoelectric point pi greater than 10.1), which migrated on the electrophoresis gel with sodium dodecyl sulfate (SDS) to form a single band corresponding to a molecular weight of 12.3 kD. The purified factor was estimated at 1.4 x 10 3 but its yield was very low, only about 1 µg of purified BDNF per 1.5 kg. pig brain. In addition, since the final step of the purification process was gel electrophoresis, BDNF activity could not be repeatedly completely renatured, due to the presence of residual SDS (Barde and Thoenen, 1985, Hormones and Cell Regulation, T 3, Eds., Alsvir Science Publishers, 385-390). It has been pointed out that BDNF has a high alkalinity and molecular size very similar to NGF monomer. However, BDNF exhibits properties that differ from known NGF properties in that (a) in the bioassay with chicken dorsal root nodule, anti-NGF antibodies had no appreciable effect on BDNF biological activity; (b) the BDNF and NGF activities appeared to be complementary; and (c) unlike NGF, BDNF was found to have no effect on the survival of sympathetic neurons in the E12 chicken embryo. In addition, neurotrophic activity of these sources has been shown in earlier studies of brain extract to affect sensory neurons at a later stage than activity associated with NGF. Using dissociated chicken embryonic neuronal cultures grown in a polycationic substrate such as polylysine or polyiornitine, BDNF was found to support survival in more than 30% of E10-E11 (tenth or eleventh day of embryonic development) neurons from the dorsal root node but with little effect on the same. neuronal survival at E6 (Barde et al., 1980, Proc. Natl. Acad. Sci. JSV, 77: 1199-1203, supra). NGF has been reported to support the survival of 30-40 percent of E6 DRG neurons under similar conditions. Interestingly, both NGF and BDNF were found to grow at approximately 50% of DRG neurons from chicken embryo E6-E12 ages when cultured in a substrate coated with extracellular matrix glycobaltamin (Lindsay et al., 1985, Develop Biol. 112: 319-328). Subsequent studies have found that the activities of NGF and BDNF are complementary when both substances are at saturating concentrations.

Early studies on the neuronal specificity of NGF by Levi-Montalcini (1966, Harvey Lectures 60: 217-259) suggested that NGF was not a ubiquitous neurotrophic factor, even among sensory neurons, because NGF did not affect neurons from certain sensory nodes in the chicken head, tenth nodal ganglion of the maxillary nerve. Subsequently, in vitro studies (Johnson et al., 1980, Science 210: 916918; Pearson et al., 1983, Develop. Biol. 96: 32-36) showed that NGF deficiency during embryogenesis does not affect the survival of neurons from many rat head sensory nodes, whereas similar effects strongly reduce the number of neurons in the sensory nodes originating from the nerve compartment. More detailed in vitro studies (Lindsay and Rohrer 1985, Develop. Biol. 112: 30-48; Davies and Lindsay, 1985, Develop. Biol. 11: 62-72, Lindsay et al., 1985, J. Cell. Sci. Suppl. . 115-129) have clearly shown that NGF supports the survival of most sensory neurons in the neural crest, but does not appreciably affect the survival of cerebral sensory neurons derived from nerve plaques.

The first evidence for the neural specificity of BDNF as distinct from NGF activity was the in vitro demonstration that purified BDNF supports 40-50% of sensory neurons dissociated from the chick embryo ganglion of E6, E9 or E12 ages (Lindsay et al., 1985, J Cell Cell Sci Suppl 3: 115-123). NGF had no appreciable effect on these neurons, acting alone or in combination with BDNF. Later, explant cultures have shown that BDNF supports axon survival and regrowth in sensory nodes derived from the nerve patch, including the vagal nerve, cranial and ventrolateral nodes (Davies et al., 1986, J. Neurosci. 6: 18971904), none of which was sensitive to the effects of NGF. In all of the above assays, NGF antibodies had no appreciable effect on BDNF activity. In addition to these effects obtained with peripheral node neuronal cultures, BDNF has been found to promote survival and cellular neuronal differentiation in quail neural crest neuronal cultures (Kalcheim and Geandreau 1988, Develop. Brain Res. 41: 79-86).

Prior to the present invention, it was not possible to obtain a sufficient amount of BDNF to induce an immune response that inhibited the production and comparison of anti-BDNF antibodies to NGF antibodies against their effects on neuronal populations and prevented BDNF / NGF experiments with their cross-neutralization. However, two recent studies of BDNF (Kalcheim et al., 1987, EMBO 6: 2873; Hofer and Barde 1988, Nature 331: 261-262) have nonetheless demonstrated the physiological role of BDNF in the development of the peripheral nervous system in birds. When a mechanical barrier was placed in the egg between the E3 / E4 DRG and their CNS target in the neural tube, the death of many DRG neurons was observed (Kalcheim and Le Donarin 1986, Develop Biol. 116: 451-466). It has been postulated that this neuronal death may be due to the loss of the neurotrophic factor in the CNS (neural tube). It was later observed that BDNF attached to a laminin-coated sialastic membrane was able to prevent this death (Kalcheim et al., 1987, EMBO 6: 2871). Injections into quail egg were found to reduce naturally occurring cell death in nodal ganglia, which was not observed with NGF (Hofer and Barde 1986, Nature 331: 261-262). In addition to this effect obtained with peripheral sensory neurons originating from both the nerve scapula and nerve plaques, BDNF was found to support the survival of developing CNS neurons. Johnson et al {l 986, J. Neurosci. 6: 3031-3938) provided data showing that BDNF supports retinal ganglion cell survival in cultures prepared from rat E17 embryos. This is consistent with previous studies showing that conditioned media and brain extracts prepared from area-targeted, innervated retinal ganglion cells support the survival of these neurons (Mecaffery et al., 1982, Ex. Brain Res. 48: 37-386; Sartley et al. et al., 1983, J. Neurosci 3: 2532-2544; Turuer et al., 1983, Dev. Brain Res. 6: 77-83).

In addition to this effect on the survival of developing neurons in cultures, BDNF has been shown to affect adult peripheral and CNS neurons in their cultures. BDNF, like NGF, stimulates axonal regeneration in adult rat DRG neuronal culture (Lindsay 1988, J. Neurosci 8: 2394-2405), although adult sensory neurons do not require neurotrophic factors for their maintenance in vitro for 3 or 4 weeks. In addition, BDNF has been shown to promote retinal ganglion cell survival and axonal extension in adult rat retinal cultures (Thonos et al., Eur. J. Neurosci 1: 19-26). A comparison of the biological effects of NGF and BDNF is presented in Table 1.

table

Comparison of biological activities of BDNF and NGF *

Survival **

<td colspan="2">Peripheral nervous system</td><td>BDNF</td><td>NGF</td>

<td>(I)</td><td>E6 chicken DRG</td><td> -</td><td> + +</td>

<td></td><td>E10 chicken DRG</td><td> -</td><td> + +</td>

<td></td><td>E12 chick sympathetic neurons</td><td> -</td><td> + +</td>

<td>(II)</td><td>E6-E12 chicken DRG</td><td> + +</td><td> + +</td>

<td></td><td>E6-E12 chicken knot g.</td><td> + +</td><td> -</td>

E12 chicken sympathetic neurons E12 chicken pineal node (Lindsay et al., 1985, supra).

(III) E3-E14 chicken:

neck + / + +

DM-tee + / + + beige + / + + elbow + / + +

VL-Triangular (ventrolateral) + + Anterior mesencephalic ++ (Davies et al., 1986, see above) (Barde et al., 1987, Proc. Natl. Res. 71: 185189).

Central nervous system (I) E17 rat retinal ganglion cells ++ (Johnson et al., 1986, J. Neuroaci. 6: 30313038) * chronological publication, effects found in vitro ** no survival (-), median survival (+ ), good survival (++)

2.3.2. Neural targets of brain neurotrophic factor

The sensory neurons of the peripheral nerve nodes have been found to originate from two different temporal embryologic derivatives, namely, the nerve splint and nerve patches. The neural crest starts the neurons of the autonomic nodes and the sensory nodes of the spinal cord and satellite cells ie DRG. The contribution of nerve splinters and nerve patches to the formation of sensory nodules in the cranial nerve has been studied using a quail / chick chimeric transplant system according to Le Donarin (1973, Develop. Biol. 20: 217-222; Noden 1987, Develop. Biol. 67: 313-329; Narajanan and Narajanan 1980, Anat Rec 196: 71-82; Ayer-Le Liuze and Le Donarin 1982 Develop Biol 34: 291-310; D'Amico-Maratel and Noden 1983 Am Am ​​Anat 166 : 445-468). As stated

Lindsay et al. in the review (1985, J.Cell. Sci. Supp. 3: 11-129), it is now believed, at least for birds, that the neurons of the eighth, ninth, and tenth distal nodes of the cranial nerve (cranial, incisal, and nodular ganglia, respectively) and eighth neurons of the vestibular-acoustic complex of the cranial nerve are derived exclusively from nerve plaques. The trigeminal node of the fifth cranial nerve has neurons of both squamous and squamous origin (with neurons of squamous origin predominantly in the ventrolateral pole of the maxillary and mandibular lobes), whereas the satellite cells of all nodal nodes originate from the sciatic.

In vitro experiments using both explant and dissociated, neuronal enriched, spinal cord and cerebral nerve sensory neuron cultures have shown that sensory neurons originating from the nerve scaffold respond to NGF and neurons derived from platelets (including neurons from the ventrolateral nerve in the trigeminal node and the entire neuronal population in the vestibular, elbow, mandibular and nodal ganglia) were predominantly insensitive to NGF during embryonic development. Despite their differences in needs and sensitivity to NGF, both posterior and sciatic sensory neurons have been found to respond to BDNF, promoting survival and axonal growth (Lindsay et al., 1985, J.Cell. Sci. Supp. 3: 115-129; Lindsay et al. 1985, Develop Biol 112: 319-328; Kalcheim and Geandreau 1988, Develop Brain Res 41: 79-86). Tebar and Barde (Tebar and Barde 1988, J. Neurosci 8: 3337-3342) studied how neurons in the dorsal root node of the chicken embryo bind labeled radioactive BDNF; these results are consistent with the existence of two classes of BDNF receptors, one with high affinity for BDNF and the other with low affinity. No high affinity receptors were found in sympathetic neurons.

Known neural targets of BDNF have been further explored by Barde et al. review (1987, Prog. Brain Res. 71: 185-189). Prior to the present invention, identification of cells synthesizing BDNF was not possible due to the absence of nucleic acids or antibody probes that are specific to BDNF. Attempts to prepare poly- or monoclonal antibodies against BDNF failed. This failure to obtain antibodies prevented molecular cloning of BDNF, determination of physiological activity in neurons in vivo after removal of BDNF, quantification of BDNF in tissues by immunoassays, and localization of BDNF by immunocytochemistry.

Table II

List of BDNF neurons responsive and non-responsive *

A. Responsive neurons

I Chicken sensory neurons derived from the scythe:

(a) dorsal root nodule,

(b) at the neck node

(c) in the dorsomedial nerve of the trigeminal nerve

(d) in the mesencephalic triangular nucleus **

Π Chicken sensory neurons derived from the ectodermal patch:

(a) In the nodular ganglion

(b) in the vestibule

(c) in the sandbox

(d) at the crank assembly

(e) in the ventrolateral nerve of the trigeminal nerve

IH Rat retinal ganglion cells

B. Non-responsive neurons

I Rat and chick sympathetic neurons

II Chick Parasympathetic Tumor Neurons * from Barde et al., 1987, Prog. Brain Res. 71: 185-189 ** see Ref. Davies et al., 1986, Nature 319: 497-499

3. Brief Description of the Invention

The present invention relates to nucleic acid sequences encoding brain neurotrophic factor (BDNF), a purified protein, as well as peptide fragments or derivatives derived therefrom, and antibodies directed against the BDNF protein, peptide fragments and derivatives. The present invention provides for the first time sufficient amounts of BDNF to produce anti-BDNF antibodies and support diagnostic and therapeutic use of BDNF.

In various embodiments of the invention, the BDNF nucleic acids, proteins, peptides, derivatives, and antibodies provided by the present invention can be used in methods of diagnosing and treating a variety of nerve disorders and disorders, and, individually, in the diagnosis and treatment of disorders associated with sensory neurons. as well as retinal degeneration. In addition, in certain embodiments of the invention, BDNF nucleic acids and BDNF gene products may be used in the diagnosis and treatment of neuroblastoma tumors, Parkinson's, and Alzheimer's disease. BDNF gene products in further specific embodiments of the invention may also be used to facilitate implantation into the neural tissue of implants, or conversely, to promote nerve regeneration following injury caused by trauma, infarction, infection, and post-surgical intervention.

The invention also relates to pharmaceutical compositions comprising an active amount of BDNF gene products or, on the other hand, antibodies directed against BDNF gene products, which can be used for the diagnosis and treatment of various nerve diseases and disorders.

Additionally, by providing a complete BDNF nucleotide sequence, the present invention enables the alignment of BDNF and NGF genes, while identifying homologous fragments and defining the BDNF / NGF gene family. Accordingly, the present invention relates to a method for identifying additional members of the BDNF / NGF genetic family. In a particular embodiment, the method according to the invention is used to identify a novel non-BDNF and non-NGF member of the BDNF / NGF genetic family. The invention further supports novel members of the BDNF / NGF gene family as determined by the method described, as well as their gene products.

3.1. Abbreviations and their meanings

<td>BDNF</td><td>brain neurotrophic factor</td>

<td>hBDNF</td><td>human BDNF</td>

<td>CAT</td><td>cholinacetyltransferase</td>

<td>CNS</td><td>Central nerve system</td>

<td>DRG</td><td>dorsal root node (s)</td>

<td>EDTR</td><td>ethylenediaminetetraacetic acid</td>

<td>NGF</td><td>nerve growth factor</td>

<td>PFS</td><td>saline was phosphate buffered</td>

<td>PCR</td><td>chain polymerization reaction</td>

<td>RNG</td><td>peripheral nervous system</td>

<td>SDS</td><td>sodium dodecyl sulfate</td>

<td>Three</td><td>3- (hydroxymethyl) aminomethane</td>

4. Description of the drawings

FIG. nucleotide sequence and the deduced amino acid sequence from porcine prepro BDNF cDNA. In this FIG. full sequence of two overlapping cDNA clones is shown. Underlined peptide sequences obtained by micro-sequencing (Table III). The only consensus sequence for N-glycosylation is underlined in two lines. The start of a mature BDNF sequence is marked by two asterisks.

FIG. 2. Comparison of NGF and BDNF sequences

Areas highlighted, with more than two amino acids detected at identical sites. These sequences start with the first amino acids of the mature protein and end with the last amino acids before the stop codons. 51 amino acids are common to BDNF and various NGF (Schwarz et al., 1989, J. Neurochem. 52: 1203-1209), including six cysteine ​​residues.

FIG. 3. Eco-RI Southern Southern blotting autoradiography of human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast genomic DNA hybridized with 32P NGF and BDNF probes.

FIG. 4. Northern blot analysis. 20 µg total RNA from each tissue was used per lane and cross-linked with the mouse BDNF 32P-labeled cRNA probe. In the brain tissue, unlike the other seven tissues, a strong signal is seen at about 1.45 kD.

FIG. 5. Human BDNF cDNA sequence and deduced amino acid sequence and porcine, rat and chicken DNA sequences.

FIG. 6th BDNF-expressing plasmid pCM VI - pBDNF.

FIG. 7. (a) Results of antiserum binding to B5 peptide as determined by ELI SA using serial dilution of antisera.

b) Quantification of binding by dilution of 1: 500 in various antisera with 50 ng BDNF.

FIG. 8. Results of BDNF (dotted line) and control (solid line) cultures from ventral midbrain, immunohistochemical staining, tyrosine hydroxylase.

FIG. 9. Dopamine consumed by ventral midbrain cultures. Cultures with BDNF are marked with dotted lines, control cultures are marked with solid lines.

FIG. 10. (a) Effect of BDNF on multiple CAT-positive cells from terminal brain cholinergic neuron cultures.

(b) Cultures of terminal brain cholinergic neurons with a density of 260000 cells (black bar) or 150000 cells (dotted bar) per well were treated with 150 ng / ml NGF. Quantities of CAT immunostaining cells were compared between treated and untreated NGF cells.

FIG. 11. Changes in CAT enzyme activity in cultures of terminal brain cholinergic neurons expressed in picomoles of substrate catalyzed per minute as a function of BDNF concentration,

FIG. 12. Astroglial cell cultures with approximately 60% adhesion were exposed to (a) epidermal growth factor or (b) BDNF for 46 hours and then incubated with [³H] methylthymidine. Bound pH] was measured relative to EGF and BDNF concentrations (EGF epidermal growth factor).

FIG. Southern blotting of EcoRI sections of chicken, mouse, rat BDNF / NGF crossed with RIB / 20 probe. Positions of Eco RI fragments of NGF and BDNF genes labeled N and B, respectively.

FIG. 14. Comparison of BDNF and NGF Sequences with a New BDNF / NGF Gene Family Member Identified by PCR Using Mouse DNA with Primers Box 3 / Box 4: A New Gene (Here designated M3-4), Also Known as Neurotrophin 3 (NT- 3); showing only the coding strand sequence as well as the deduced amino acid sequence for comparison with murine mature NGF and porcine, rat, mouse or human BDNF. The dash represents positions using a deletion of a single codon from NGF to optimize comparison

For BDNF and M3 / 4. Written font overlaps and / or conservative amino acid substitutions.

FIG. Northern blotting of human tumor cell lines RNA hybridized with the human BDNF probe.

FIG. 16. Effect of depolarization of BDNF mRNA levels in ammonium horn neurons.

(a) Temporal expression of BDNF mRNA in primary cultures of ammonium horn neurons in the presence of 50 mM potassium chloride. Total cellular RNA was extracted from 0.5x10 4 cells of glycosylin and analyzed by 1.3% agar gel electrophoresis (Biziere, K. and Coyk, T. Neurosci., 1978, Lett. 8: 303; McGeer et al., 1978). , Brain Res. 139: 381). RNA transfected on Hybond N-filters with a 32-Labeled cRNA probe (specific activity 109 cpm / µg) specific for murine BDNF and obtained by in vitro transient transcription. The top two bands correspond to (4 and 1.5 kD) BDNF mRNA; the two BDNF bands (4 kb and 1.5 kb) are ribonuclease-A-resistant and correspond to two different transcripts which, however, are regulated in the same way, and the bottom band (700 b) corresponds to 10 picograms of short BDNF mRNA standard yield, was added to the samples prior to RNA extraction.

(b) Calcium dependence. Neurons were incubated for 3 hours in normal growth medium (CM), modified medium without calcium (-Ca) or medium with 10 mM nifedipine (NIF). Where indicated (+ K), 50 mM KCl was added.

FIG. 17. Elevation of NGF mRNA levels in ammonium horn neurons with potassium and cinic acid. Neurons were incubated for 3 hours in control medium (C) or in the presence of 50 mM calcium chloride (+ K) or 25 μΜ cinic acid (KA). RNA was extracted and determined by NGF mRNA level by quantitative PCR reaction (H). Ratings given: mean ±.

FIG. 18. Dose-response curve showing effect of cinic acid. Ammonium horn neurons were incubated for 3 h. at various concentrations of cinic acid. RNA was extracted and analyzed as described in Figs. 16 Estimates are the mean ± SEM of three experiments.

FIG. 19. Time-dependent increase in BDNF and NGF mRNA levels induced by treatment with cost acid. Total cellular RNA was extracted and analyzed as described in Fig.16 from ammonium horn (a) or bark (b) at the indicated time points (hours) after injection of cinic acid (12 mg / kg) into the abdominal cavity. A single dose of diacepam (10 mg / kg) was administered 90 minutes later by injection. after cainic acid (two BDNF bands (4 kb and 1.5 kb) are ribonuclease A resistant and correspond to two different transcripts which, however, are regulated in a similar manner). Values ​​shown correspond to 1.5 kb BDNF mRNA (o) and 1.3 kb NGF mRNA (o). A similar increase in level was obtained with 4 kb BDNF mRNA. Values ​​given in the figure correspond to the mean ± SEM of 3-4 experiments.

FIG. 20. Effects of anticonvulsants on cinic acid-induced BDNF mRNA expression. Rats were injected intraperitoneally with (10 mg / kg) diacepam (DZ), MK801 (1 mg / kg) (MK) or ketamine (20 mg / kg) (KET) for 15 min. before injection of cainic acid (12 mg / kg) (+ Ka) or saline (control). After 3 or. Ammonium horn tissue was used to isolate total RNA as shown in Fig. 16 The values ​​shown in the figure correspond to the mean ± SEM of 3 experiments.

FIG. 21. Partition cell cultures. A-Phase contrast micrograph of cell growth in time-dependent culture. Cells were plated at a density of 1.3 x 10 ląstelių cells per cm ^ and maintained at 5 HS / NS as described in the text. The indicated time points are: A, C - 1 day, C, D - 2 days and D, F - 4 days.

Cell type-specific markers were used to identify cell populations, ACL histochemically stained neurons (G, H) and NGF receptor-positive neurons (I). The scale is 25 pm.

FIG. 22. Comparison of dissociated septal cell responses to effects with BDNF and NGF based on cellular AChE levels.

A. Comparison of responses to BDNF (25 ng / ml), NGF (50 ng / ml), or a combination of both ligands at various densities on plates.

B. Comparison of responses of cholinergic neurons to various concentrations of BDNF and NGF (0 to 50 ng / ml). The cells with which these results were obtained were grown for 11-12 days in sulfur-containing medium. Values ​​given in the figure correspond to the mean ± SEM of 6-9 experiments.

FIG. 23. Bar graph depicting the effect of BDNF and NGF addition retention on dissociated septum cultures. BDNF (50 ng / ml) and NGF (50 ng / ml) were added to the dissociated cell cultures with 5-6 hours (+12), 5 days (5 / + - 7) or 7 days (-7 / + 5). . Cultures were maintained for 12 days. Cells were counted based on positive histochemical staining. Results are expressed as mean ± SEM of 4-5 replicates.

FIG. 24. Bar graph depicting the ability of BDNF and NGF to regulate the amount of NGF receptor-positive cells. Immunoassay of NGF receptors was performed with monoclonal antibody 192-IgG at a dilution of 121000. Dissociated septum cells were placed in a vessel with a density of 1.3 x 10 $ cells / sq. cm and operated without interruption for 12 days. Comparisons were made between the saturated NGF dose and the various BDNF doses (0 to 100 ng / ml). Mean ± SEM of 4 replicates are presented.

FIG. 25. Responses of septal cholinergic neurons to doses of BDNF (A) and NGF (B) that induce CAT activity. Cultures were placed in plates coated with polyiornithine and laminin with 6 mm wells and maintained for 12 days with 5 HS / NS. Cells were exposed to BDNF and NGF 5-6 hours after plating. The factor would change every 3 days when changing medium. Mean ± SEM of 5-6 replicates are shown.

FIG. 26. Dose dependence of enzyme activity of AChE on BDNF and NGF. Rat septal cholinergic neurons were cultured for 12 days in sulfur-containing medium with hormonal additives. Cells were exposed to BDNF (blank squares) and NGF (filled squares) as described in Figs. 25. presented mean ± SEM of 6-9 replicates. AChE activity in untreated cultures was 16.4 ± 2 nmole (HR) well.

FIG. 27. Time dependence of CAT enzymatic activity induced by BDNF compared to activity induced by NGF. Determination of the time required to induce CAT activity was performed with cells plated at a density of 2.3 x 10 $ cells / sq. cm., which were grown for different periods of time in media containing sulfur and hormonal additives and exogenous factors. Cells were initially exposed to BDNF (blank squares) and NGF (filled squares) 5-6 hours after plating. Results are the activity in the affected cultures expressed as a percentage of the activity in the untreated cultures (n = 6, BDNF 12 days n = 3).

FIG. 28. Comparison of the effect of glial cells on the ability of BDNF to induce CAT enzymatic activity.

The septal cells were plated at 2.3 x 10 4 cells / sq. cm in density. 5-6 hours after seeding, the medium was replaced with either sulfur-free medium or sulfur-containing medium, each containing 1% NS, as described in the specification. Addition of cytosine-arabinoside (1 μΜ) for 24 hours to further reduce astrocyte count. These cultures are then maintained for 12 days. Mean + SEM of 4 replicates are presented.

FIG. 29. Bar graph depicting the effect of BDNF and NGF on high affinity choline consumption levels. Choline intake assay was performed on cells maintained for 11 days with a density of 1.3 x 10 ^ cells per sq. cm. BDNF (50 ng / ml) or NGF (50 ng / ml) was exposed throughout the growing period. Mean ± SEM of 5 replicates with BDNF and 10 replicates with NGF are presented.

FIG. 30. SDS sheet containing 35S-labeled reaction products after digestion of human brain neurotrophic factor with trypsin and Arg-G endoproteinase.

FIG. 31. Graphical representation of bioassay with DRG comparing unaffected CHO-hBDNF and cleaved endoproteinase CHO-hBDNF. Axon regrowth was scored on a scale from 0 to 5, indicating maximum bioactivity.

FIG. 32. Photomicrographs, phasocontrast (A), and light field (B, C) of E14 rat ventral mesencephalic dissociated cell cultures stained after 9 days of culture with monoclonal antibody against tyrosine hydroxylase (TH).

A and B. Phase contrast and light field photographs of the same field showing low numbers of TH + neurons in these cultures. Only one ΤΉ + neuron (arrow) is visible in a field rich in phasorphic neurons with long axons. Several non-neural cells based on morphology or staining with GFAP (not shown) are obvious.

C. Bright field photograph showing 2 ΊΉ + neurons (small arrows) in a field containing about 98 phasic cells with neuronal morphology. Scale 100: 1M.

Fig. Strongly enhanced photomicrographs of 6-day cultures of E14 rat ventral mesencephalic cells showing different TH + neuron morphology, some with extensive axonal growth and highly developed cones of growth (A, B). Cultures were developed and stained as described in Figs. 32. in the explanation. Scale scale = 25 ohms.

FIG. 34. BDNF improves the survival of TH + dopaminergic mesencephalic neurons in cultures in a dose-dependent manner that is evident when cultured with three-day periods.

A. Comparison of the number of TH + neurons in cultures of E14 rat ventral mesencephalic cells exposed to BDNF (solid line) or without BDNF (dotted line). In the affected cultures, BDNF (50 ng / ml) was added once on the second day of cultivation. On the third day, no difference was detected between control and exposed cultures, but on day 8, TH - + - neurons in BDNF-exposed cultures were 1.8-fold higher than controls. Ratings are given as the average of two examples.

B. Effect of BDNF on TH + neuronal survival improvement is dose dependent. TH + neurons were quantified in double cultures after 8 days of growth in BDNF-free medium, or medium containing increasing concentrations of BDNF added on day two.

C. NGF had no effect on the survival of ΊΉ + neurons. To assess the specificity of BDNF, cultures were also cultured with or without NGF (50 ng / ml) for 8 days and analyzed for TH + cells. NGF added on day two did not increase survival of TH + neurons in cultures tested on days 6 and 8. Results presented are averages of two plates.

FIG. 35. Repeated doses of BDNF further increase the survival of TH + neurons compared to a single addition of BDNF on the second day. Cultures were prepared as described in the text. Recombinant human BDNF was purified from COS M5 cell supernatant. Cultures were exposed to either a single addition (SA, dashed) of BDNF (50 ng / mM) on day two or repeated doses of BDNF (50 ng / ml) on days 1, 2, 4, 6 and 8 (MA-multiple additions; solid line). or grown without BDNF (control; blank bars). At the indicated times, cultures were fixed and stained for TH immunoreactivity.

Multiple additions of BDNF resulted in a 2.7-fold increase in TH + neurons at Day 11 compared to controls, whereas the maximal increase obtained with a single addition of BDNF was slightly less than two-fold at 11 days. Representative averages for several experiments from two examples are presented.

FIG. 36. Delayed addition of BDNF did not increase the amount of ΤΉ + neurons to the same degree as the addition on the second day. Cultures were prepared as described in the text except for the time points of exogenous BDNF addition. On the second day, all cultures were transferred to sulfur-free medium and BDNF (50 ng / ml, porcine BDNF) was added only once on the second or fifth or seventh day (CD2, CD5, CD7). On the sixth, eighth and tenth day of cultivation, TH + neurons were quantified in double cultures. In this experiment, the addition of BDNF alone on day 2 increased TH + neuron levels by day 10 by a factor of 3. When BDNF was added with retention on day 5, the increase in TH + neurons on day 10 was less than 2-fold relative to control, and this difference in TH + cell count between the affected and control cultures did not occur with continued culturing. Controls are blank bars, BDNF addition on day two - dashed, BDNF addition on day 5, solid line, BDNF addition on day 7, diagonal dashed lines.

FIG. 37. Dose-dependent effect of BDNF on high affinity GABA uptake in ammonium horn cultures. The cultures were supplemented with partially purified hBDNF (rotofor-purified from COS M5 supernatants) of different dilutions. It was found that, at a dilution of 1 x 10'2, BDNF maximally promoted axonal growth in the dorsal root nodules of the chicken E8. The consumption of high affinity ^ H-GABA was measured 8 days after treatment with BDNF in vitro.

FIG. 38. BDNF protects black matter dopaminergic neurons grown in culture from neurotoxic effects of MPP +. Cultures were generated from E14 rat embryos as described in Fig.1 After 24 hours. after in vitro cultivation, the medium was replaced by a sulfur-free medium. After 3 days of cultivation, these cultures were divided into 4 groups of 6 plates (35 mm) each. One group was kept as a control and the other was exposed to: (I) 10 ng / ml major fibroblast growth factor (BFGF, bovine brain), Boerninger-Mannheim; (Π) 50 ng / ml mouse NGF and (III) 50 ng / ml BDNF. The cultures were maintained for another 24 hours and then 1 μΜ MPP + (Research Biochemicals, Ine., Natick. MA) was added to three plates of each group for 48 hours. At the end of this experiment, which lasted a total of 6 days, all plates were treated for TH immunoreactivity determination and the amount of nustatytas + cells in each group was determined. Data presented represent the number of TH + neurons after treatment with MPP +, which was calculated as a percentage of the amount of TH + neurons in similar cultures not receiving MPP +. Mean ± SEM from three cultures are presented.

5. Detailed Description of the Invention

The present invention relates to nucleic acid sequences encoding brain neurotrophic factor (BDNF) as well as to BDNF protein, peptide fragments and derivatives obtained in large amounts using these nucleic acid sequences. The invention further relates to pharmaceutical compositions and therapeutic applications of BDNF, for the first time providing means for producing sufficient quantities of essentially pure BDNF for clinical applications. The invention also relates to antibodies directed against BDNF or fragments thereof, and provides a method for obtaining a sufficient amount of an immunogen. Further, by making it possible to compare the BDNF and NGF nucleic acid sequences, the present invention enables the identification of homologous regions of the BDNF and NGF nucleic acid sequences while defining the BDNF / NGF gene family. The invention provides a further method of identifying and isolating members of this gene family.

For purposes of clarity of description, but without limiting the scope of the invention, it is further described in the following sections:

(I) Purification of BDNF (II) Bioassays of BDNF (III) Micro-sequencing of BDNF protein (IV) Cloning of DNA encoding BDNF (V) Expression of BDNF (VI) BDNF genes and proteins (VII) Obtaining of anti-BDNF antibody (VIII) BDNF / Identification of Additional Members of the NGF Gene Family (IX) Benefits of the Invention (Applications) (X) Pharmaceutical Compositions

5.1. Purification of brain neurotrophic factor

In order to identify the BDNF-encoding nucleic acid, micrograms of BDNF protein can be extracted from the tissues to determine the amino acid sequence, which can then be used to generate oligonucleotide probes. Because the BDNF protein is extremely rare, the portion of the amino acid sequence that can be detected is, in practice, very limited. BDNF can be extracted from pig brain by Barde et al. (1982, EMBO, 1: 549-553) or Hofer and Barde (1988, Nature 331: 261-262).

It is preferable to extract BDNF according to the following detailed description of the process, which is provided by way of example, rather than to limit the scope of the invention; the person skilled in the art can employ various modifications. Because of the rarity of BDNF, it is better to use about 6 kilograms of brain tissue in the purification process. Brain tissue is homogenized in sodium phosphate buffer at a concentration of 0.2 M sodium sulfate and approximately pH 6 containing 1 mM EDTR and 1 mM freshly added phenylmethanesulfonyl fluoride so that the ratio between brain tissue and fluid is 1 kg cerebrospinal fluid. Hydrochloric acid may then be used to adjust the pH of the mixture to 4, and then the mixture may be stirred for about 2 hours at 4 ° C. The mixture can be further centrifuged at 20000 for 25 minutes. After centrifugation, the supernatant is adjusted to a pH of about 6 with caustic soda and then mixed with 1 liter of pre-swollen carboxymethylcellulose (six kg of brain tissue) and again adjusted to pH 6 with 0.1 M sodium phosphate solution . After several washes, a total of approximately 20 liters of 0.1 M sodium phosphate, pH 6, is poured into the column and rinsed with the same buffer containing 0.13 M NaCl, preferably until morning. The active fractions were identified by a BDNF sensitive bioassay (see section 5.2), NaCl and subsequent dialysis, in batches of approximately 5 liters of 5 mM potassium phosphate at pH 6.8. The dialyzed fractions may then be placed on a hydroxyapatite column having a volume of about 20 ml for each initial kilogram of brain tissue; the pH of the hydroxyapatite column should be adjusted to 6,8 in the presence of 5 mM potassium phosphate prior to loading. This column is then eluted in a linear gradient from 500 mL of 5 mM potassium phosphate to 500 mL of 700 mM potassium phosphate, each at a pH of about 6.8. BDNF activity is optimally leached to approximately 500 mM potassium phosphate. The resulting active fractions can subsequently be brought to a final molarity of about 700 mM potassium phosphate, and transferred to a phenyl sepharose column (containing about 5 mL volume for each 6 kg of starter brain tissue), adjusted to pH 6.8 with 700 mM potassium phosphate solution. After washing with approximately 40 ml of the same buffer, BDNF activity can be washed with 0.1 M potassium phosphate, pH 6.8, and then BDNF activity can be dialyzed in distilled water and then lyophilized. For performing SDS gel electrophoresis on a sample, the lyophilized material is placed in a buffer containing 0.1% SDS, preferably mercaptoethanol free, and then placed on an SDS gel with an acrylamide gradient (linear) of 10 to 25%. After completion of electrophoretic separation, the gel can be stained for about 10 min. with COOMASSIE BLUE and then bleached for about 20 min. The band migrating at the level of the cytochrome-c marker can be cut out and gel electrophoresed. SDS can be largely removed according to Veber and Kuter (Weber and Kuter 1971, J. Biol. Chem. 246: 45044509). It should be noted that, in general, SDS is not completely removed. For sequencing, the BDNF purification process was modified according to Hofer and Barde (1988, Nature 331: 261-262) so that gel electrophoresis is not used in the final purification step.

5.2. Bioresearch of brain ncurotrophic factor

Any system which qualitatively or quantitatively detects BDNF activity can be used according to the present invention. Such bioassays can be used to identify and / or measure activity of natural or recombinant BDNF.

Any known bioassay that detects BDNF can be used here. For example, DRG neurons (dorsal root node of the chicken embryo) may be used in a BDNF assay as described by Barde et al. (1980, Proc. Natl. Acad. Sci. USA 77: 1199-1205).

Specifically, the dorsal root nodules of a chicken embryo can be harvested for 6-14 days, preferably 10-12 days, using a known dissection in the art, and immediately placed in a small volume of F-14 medium (made from GIBCO F-12 with Vogei and etc. - Vogei 1972, Proc. Natl. Acad. Sci. USA, 69: 31803184), which would need to be converted to a buffered phosphate-free Ca² + and Mg²⁺ ionic saline (PBS) containing about 0.1% trypsin. After about 20 minutes. After incubation at 37 ° C, these units can be centrifuged and washed twice with F14 medium containing about 10% (v / v) heat-inactivated horse serum. These assemblies can then be disassembled by gentle trituration (about 10 to 15 aspirations) using a small, about 1 mm diameter silicone paste pipette. The remaining tissue clumps are then preferably removed by passing the cell suspension through a nylon sieve having pores of about 40 µm diameter. Cell suspension is now available for 210 min. placed in a container of plastic material in which many non-neuronal cells must adhere to the plastic surface of the container during this time, leaving a population of cells enriched in neurons in suspension. Thereafter, the cells can be diluted to a concentration of approximately 5χ10²cells per milliliter of medium in the vessel (preferably F14 medium with added 10% v / v heat inactivated horse serum with antibiotics) and can be dispensed into plates coated with polyiornithine or polyiornithine / laminin (see below).

On the other hand, without limitation, bioassays of BDNF that are relatively insensitive to NGF may, under certain conditions, perform better than systems similar to the DRG system described above, which can, under conditions, also react to BDNF and NGF. Relatively BDNF-specific systems should include cultures of retinal ganglion cells as well as cultures of neurons derived from nerve plaques.

Perinatal retinal cells can be grown according to the methods described by Johnson et al. (Johnson 1986, J. Neurosci. 6: 3031-3038). For example, the retina may be removed from a perinatal animal (in rats, the term "perinatal" refers to the period from day 17 of the embryo and up to 48 hours postnatal pups), followed by washing the retina with PBS and incubating with 0.05- 0.1% trypsin for about 15 minutes at about 37 ° C. After proteolytic cleavage, the retina is rinsed with F14 medium containing about 10% v / v heat inactivated horse serum (F14-HS). The retinas are then disassembled by gentle pipetting in a small volume (about 1-10 mL) of F14-HS alone. Undissociated tissue is allowed to settle and pipette to remove remaining cells and culture media.

On the other hand, the BDNF bioassays of the invention may use cells derived from nerve plaques. Embryonic cerebral nerve explants, such as the ventrolateral portion of the trigeminal node or the anterior, cranial, caudal, or nodular nodes, may be grown on collagen gel coated with growth medium as described by Davies et al. (Davies, 1986, J. Neurosci 6 18971904), or disaggregated according to Barde (Barde, Proc. Natl. Acad. Sci. 1980, 77: 11991203).

Since it has been found that the reaction to BDNF can be enhanced tenfold by changing the polyiornithine growth substrate to a laminin-polyiornithine substrate (Barde et al., 1987, Prog. Brain Res. 71: 185-189), experiments with BDNF using laminin-containing substrates are substrates. For cultivation, surfaces may be prepared, for example, by coating them with sterile polyiornithine solution for 8 to 10 hours; (II) rinsing several times with sterile water; (III) Coating the surface with PBS solution containing approximately 25 µg / ml laminin for approximately two hours (Johnson et al., 1986, J. Neurosci 6: 3031-3038).

In any bioassay system according to the invention, the dose dependence curve can be determined by methods known in the art. Half-maximal survival to approximately 5 ng / ml of purified BDNF was detected using dissociated chicken sensory nodules and maximal survival was between 10 and 20 ng / ml of purified BDNF (Barde et al., 1987, Prog. Brain Res. 71: 185-189).

5.3. Micro-sequencing of brain neurotrophic factor protein

The sequence of BDNF protein derived from the brain can be determined by conventional methods, but it must be emphasized that due to its extreme rarity, a significant portion of the BDNF protein sequence cannot be reliably identified. This protein sequence can be sequenced directly or initially cleaved using proteases or other compounds known in the art, including but not limited to Staphylococcus aureus V8, trypsin and cyanogen bromide. Peptide sequences can be determined by automated Edman degradation using a gaseous micro-sequencer according to Hewick et al. (1981, J. Biol. Chem. 256: 7990-7997) and Hunkapillar et al. (1983, Methods Enzymol. 91: 227-236). Phenylthiogidanthionine-amino acid detection can then be performed according to Lottspeich (1985, Chromatography 326: 321-327). Overlapping sequence fragments can be detected and subsequently used to isolate longer fragments in the adjacent sequence.

5.4. Cloning of DNA encoding brain neurotrophin factor

The rarity of BDNF strongly hinders the use of standard methods for BDNF gene cloning. For example, if the available protein sequence was used to generate a complementary labeled oligonucleotide probe, and this probe was used to screen libraries of tissues presumed to synthesize BDNF, the number of positive clones would likely be incredibly low. This invention allows the BDNF gene to be cloned using a combination of techniques combining appropriate BDNF protein purification, BDNF protein micro-sequencing, oligonucleotide probe generation, cDNA library construction, amplification based on the resulting BDNF amino acid sequence, and finally, BDNF gene selection. In a preferred embodiment, this method involves amplification of tissue-derived nucleic acid sequences using a chain polymerase reaction (PCR) (Saiki et al., 1985, Science 230: 1350-1354) to increase the amount of BDNF sequences for cloning. Below is a detailed description of this method.

In particular, the amino acid sequence of the purified BDNF protein is used for deduction with oligonucleotide primers used in PCR reactions. Because of the degeneracy of the genetic code, multiple nucleotide triplets can represent the same amino acid, requiring the synthesis of several oligonucleotides for a given amino acid sequence to obtain the full set of potential nucleotide sequences; the resulting oligonucleotides are referred to as degenerate primers.

The polymerase chain reaction requires both a sense primer and an anti-sense primer. Accordingly, the amino acid sequence can be used as a primer for a single strand of DNA, and a second primer homologous to a commonly found DNA sequence, such as a reverse thymidine residue obtained by reverse transcription of polyadenosine mRNA tails, can be used as a primer for a second DNA strand. These primers are subsequently used in a polymerase chain reaction with a nucleic acid matrix that is believed to contain BDNF coding sequences, such as genomic DNA or, preferably, cDNA derived from mRNAs that are presumed to synthesize BDNF. The DNA reaction products are then analyzed by electrophoresis to determine if the DNA reaction product has a molecular size that matches the expected size of the BDNF gene and, preferably, the nucleotide sequence.

However, due to the use of two degenerate primers in a chain polymerase reaction that increases the probability of amplification for those nucleic acid sequences that do not encode BDNF, a more acceptable method variant provides for the use of only one degenerate primer and the other primer exactly matches the BDNF sequence. To identify the exact BDNF sequence, the amino acid sequence obtained using the purified BDNF protein is used to construct both degenerate primers, both sense and anti-sense. The DNA reaction product of these primers in a PCR reaction using a nucleic acid as a template encoding BDNF is a nucleic acid fragment encoding the amino acid fragment used to generate the primer and can be of expected length (i.e. as a minimum, the number of base pairs in length, which is calculated by multiplying the number of amino acid residues by three). Once the sequence of the DNA reaction product has been determined, it can be compared to a validated amino acid sequence to reassert that the amplified nucleic acid sequence can actually encode the BDNF peptide fragment. Although any nucleic acid sequencing method known in the art can be used, the most suitable method for terminating a deoxynucleotide strand is (Sanger et al., 1979, Proc. Natl. Acad. Sci. USA, 72: 3918-3921). Sequencing can be accomplished using a purified gel or better cloned DNA reaction product. The resulting DNA reaction product sequence can be used to generate an oligonucleotide primer corresponding to the exact sequence encoding BDNF. This primer can be used with a second primer, which may be degenerate, to increase the amount of BDNF sequences compared to the original number of exact sequence fragments. For example, but not limited to, the sense primer may correspond to the exact nucleotide sequence of BDNF, and the anti-sense primer may be a degenerate primer homologous to a region downstream of a fragment with a defined nucleotide sequence, e.g. . After that, it may be necessary to use a similar method to restore the sequence above the fragment, with the established nucleotide sequence; e.g., the anti-sense primer corresponds to the exact BDNF sequence and the sense primer is an abnormal primer homologous to the region of the BDNF sequence above the fragment with a defined nucleotide sequence, e.g., the 5 'end of the polyadenosine can be linked to the 5' end of the cDNA. deoxynucleotide transferase. Accordingly, the entire BDNF gene or mDNA sequence can be obtained by similar synthesis.

The DNA reaction products can be cloned using any of the methods known in the art. A large number of known vector host systems can be used. Possible vectors include, but are not limited to, cosmids, plasmids, or modified viruses, and the vector system must be compatible with the host cell. Such vectors include, but are not limited to, bacteriophages, such as those derived from the liam, or plasmids, such as pBR 322, pUC or Bluescript (Stratagene), plasmid derivatives.

Recombinant molecules can be introduced into the host cell by transformation, transfection, infection, electroporation, etc.

The BDNF gene is inserted into a cloning vector which is used for transformation, transfection or infection of the appropriate host cell to generate multiple copies of the gene sequences. This can be achieved by attaching a DNA fragment to a cloning vector which has complementary sticky ends. However, if the complementary restriction sites used for DNA fragmentation do not exist in the cloning vector, then the ends of the DNA molecule may be enzymatically modified. Insertion of restriction sites on endonuclease cleavage and sites on oligonucleotide primers used in the polymerase chain reaction may prove useful to facilitate insertion into the vector. On the other hand, any desired linkage may be obtained by attaching nucleotide sequences (linkers) to the ends of the DNA; these linked linkers may include specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In another embodiment of the method, the cleaved vector and the BDNF gene can be modified using homopolymeric enhancement.

In specific embodiments, transforming a host cell with recombinant DNA molecules that insert an isolated BDNF cDNA gene, or a synthesized RNA sequence, allows multiple copies of the gene to be generated. In this way, the gene can be obtained in large amounts by growing transformants, isolating recombinant DNA molecules from the transformants and, if necessary, recovering the inserted gene from the isolated recombinant DNA.

In a more preferred embodiment of the invention, once a clone derived from cDNA and encoding BDNF has been generated, it can be isolated using standard, known technology. For example, a labeled nucleic acid probe can be obtained from a BDNF clone and used to screen for nucleic acid hybridization genomic DNA libraries using, for example, the Benton and Davies method (1977, Science 196: 180), which they used for bacteriophage libraries, and Grunstein et al. Hognes (1975, Proc. Natl. Acad. Sci. USA, 72: 3861-3865) for plasmid libraries. The reconstituted clones can then be analyzed by mapping restriction fragments and nucleotide sequencing using well known techniques.

Further, additional cDNA clones can be identified from the cDNA libraries using the sequences obtained in the invention.

5.5. Expression of the brain neurotrophic factor gene

The nucleotide sequence encoding the BDNF protein or portion thereof may be inserted into a suitable expressive vector, i.e., a vector containing the necessary elements for transcription and translation of the inserted protein coding sequence. Necessary transcriptional and translational signals can be obtained from and / or from the native BDNF gene of the BDNF gene Many vector-host systems can be used to express a protein coding sequence. Examples include, but are not limited to, systems with mammalian cells infected with viruses (e.g., cowpox virus, adenovirus, etc.); insect cell systems infected with viruses (such as baculovirus); microorganisms such as yeast containing yeast vectors or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. The expressive elements of these vectors differ in their strength and specificity. Depending on the vector host system used, any of a number of suitable transcriptional and translational elements can be used.

Any of the above described methods for inserting DNA fragments into a vector can be used to construct expressive vectors having a chimeric gene consisting of the appropriate signals controlling the transcription and the protein coding sequence. These include in vitro DNA recombination and fusion technology, as well as in vivo recombination (genetic recombination).

The expression of the nucleic acid sequence encoding the BDNF protein or peptide fragment may be regulated by a second nucleic acid sequence such that the BDNF protein or peptide is expressed in a host transformed with a recombinant DNA molecule. For example, expression of BDNF can be controlled by any promoter / entachanserter element known in the art. Among the promoters capable of controlling BDNF expression include, but are not limited to, the early promoter region of SV40 (Bernast and Chambon,

1981, Nature 290: 304-310), a promoter with a long 5 'end repeat of the Raus sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), a herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl Acad Sci USA 78: 1440-1445), the regulatory sequence of the metallothionine gene (Brinster et al., 1982, Nature 296: 39-42), prokaryotic expression vectors such as the β-lactamase promoter (Willa-Kamaroff et al. , 1978, Proc Natl Acad Sci. USA 75: 37273731) or the tac promoter (DeBaer et al. 1983, Proc. Natl. Acad. Sci. USA 80: 21-25), see U.S. Pat. as well as Scientific American article Useful proteins from recombinant bacteria (SA 1980, 242: 74-94), plant expression vectors including the promoter region of nopaline synthetase (Herrera-Estrella et al., Nature 303: 209-213) or cauliflower mosaic. disease virus RNA 35S promoter (Gardner et al., 1981, Nucl. Acids. Res. 9: 2871) and the photosynthetic enzyme ribulosobiphosphate carboxylase promoter (Herrera - Estrella, 1984, Nature 310: 115-120), promoter elements from yeast or other fungi such as the Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerin kinase) promoter, the phosphatase promoter, and the following areas that control animal transcription, have tissue specificity, and have been used in transgenic animals: the spanning domain of the elastase-I gene, active in pancreatic acinar cells (Swift et al., 1984, Cell, 38: 639-646; Ornitz et al., 1986, Cold Spring Harfor Symp. Qant. Biol. 50: 399-409; MacDonald 1987, Hepatology, 7: 425-515), insulin gene control domain active in pancreatic beta cells (Hanahan, 1985: Nature 315: 115-122), immunoglobulin gene control domain active in lymphoid cells (Grosschedl et al., 1984). , Cell, 38: 647-658; Adams et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol Cell Biol. 7: 1436-1444), mouse mammary gland tumor virus control region, active in testis, breast, lymphoid derivatives and feces (Leder et al., 1986, Cell 45: 485495), albumin gene control region, active in liver (Pinkert et al. ., 1987, Genes and Devel., 1: 268-276), a control region of the alpha-fetobaltic gene active in the liver (Krumbauf et al., 1985, Mol. Cell. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58), alpha-I-antitrypsin gene control region, active in liver (Kelsey et al., 1987, Genes and Develop 1: 161-171), betaglobin gene control region, active in myelin cells (Morgan et al., 1985, Nature 315: 338-340; Kalbas et al., 1986, Cell. 46: 89-94), a control region of a major myelin protein active in cerebral oligodendrocytes (Readhead et al., 1987, Cell, 48: 703-712), a control domain of myosin light 2-chain gene active in skeletal muscle (Sani 1985, Nature 314: 283-286), and a gonadotropic hormone-releasing gene-controlling region active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

Expression vectors containing BDNF gene inserts can be identified in three main ways: (a) DNA-to-DNA hybridization, (b) detecting the presence or absence of marker gene function, and (c) expression of inserted sequences. In the former case, a foreign gene inserted into an expression vector can be detected by hybridizing DNA to DNA using probes containing sequences homologous to the inserted BDNF gene. In the second case, the recombinant vector / host system is identified and isolated based on the presence or absence of a particular marker gene function (e.g., thymidine kinase activity, antibiotic resistance, transformational phenotype, occlusion body baculophages, etc.) determined by foreign gene insertion into the vector. For example, if the BDNF gene is inserted into a vector marker gene sequence, recombinants containing the BDNF insert can be identified by detecting the absence of marker gene function. In the third case, recombinant expression vectors can be identified by experimentally investigating a foreign gene product expressed by the recombinant. Such assays may be based on the physical and functional properties of the BDNF gene product in a bioassay system as described in Section 5.2 above. section.

Once a particular recombinant DNA molecule has been identified and isolated, several known methods can be used to propagate it. When a suitable host system and growth conditions are in place, the expression vectors can be propagated and obtained in large numbers. As explained above, expression vectors may be used, including, but not limited to, human and animal viruses such as cowpox virus or adenovirus, insect viruses such as baculoviruses, yeast vectors, bacteriophage vectors (e.g., liamda), and plasmid plasmids. and cosmic DNA vectors, and that's far from it.

In addition, a host cell strain can be selected to modulate expression of the inserted sequence in a desired manner or to modify and produce a gene product. Expression induced by certain promoters may be enhanced in the presence of some inducers; this can control the expression of BDNF protein produced by genetic engineering. In addition, various cellular hosts have their own specific and specific mechanisms for translational and post-translational processing and modification of proteins (e.g., glycosylation, cleavage). Appropriate cell lines or host systems can be selected that perform the desired modification and processing of the foreign protein being expressed. For example, expression in a bacterial system may be used to make the product in the form of a non-glycolized protein. Expression in yeast yields a glycolized product. Expression in mammalian cells is used to obtain native glycolysis of the BDNF heterologous protein. In addition, various expression vector / host systems influence processing reactions such as proteolytic cleavage to varying degrees.

In a particular embodiment of the invention, the DNA encoding prepro-BDNF can be cloned in the plasmid pCMY, amplified and further used for transfection of COS cells using the calcium phosphate method (Chen and Okayama 1987, Mol. Cell. Biol. 7: 2745-2752); BDNF activity can then be harvested from the cell culture medium (see example in section 10, below).

5.3.1. Identification and purification of the expressed product

After identification of the recombinant that expresses the BDNF gene, the product of this gene should be analyzed. This is done experimentally using the physical and functional properties of the product.

After identification, the BDNF protein may be isolated and purified by standard techniques including chromatography (ionic, affine, and filter column chromatography), centrifugation, differential solubility, or any other standard protein purification method. Functional properties may be assessed using any of the known, sensitive BDNF experiments, including but not limited to, chicken embryo dorsal root nodules, perinatal rat retinal cells, or neurons derived from nerve plaques.

Importantly, the methods used for the preparation of BDNF from brain tissue produce partially inactive BDNF due to the presence of residual SDS, since these methods utilize gel electrophoresis with SDS in the final stage (Barde and Thoenen et al., 1985, Hormones and Cells Regulation, vol. 9 , Diumon et al., Elsvire Science publications, 383-390). In contrast to the known methods, the present invention allows the isolation of BDNF, which is produced by recombinant nucleic acid molecules and which does not contain SDS and therefore has full activity. For example, but not limited to, the anti-BDNF antibodies of the invention (such as antibodies directed to the porcine BDNF B5-33 amino acid fragment described in Section 11 below) can be used to harvest recombinant BDNF by immunoprecipitation or affinity chromatography, and thus produce detergent and full-activity BDNF molecules.

In another embodiment of the invention, prepro BDNF is enzymatically converted to mature BDNF using, for example, Arg-C endoproteinase (see, e.g., Chapter 17 below).

5.6. Brain neurotrophic factor genes and proteins

Using the methods described above (and see also the examples in Chapters 6 and 9 below), the corresponding nucleic acid sequences were identified and deductively derived amino acid sequences. The porcine BDNF cDNA sequence of Fig. 1 was determined. The human genomic DNA BDNF sequence of Fig. 5, which also contains porcine, rat and chicken DNA sequences, was determined. Each of these sequences, or functional equivalents thereof, may be used according to the invention.

The invention further relates to BDNF genes and their proteins isolated from porcine, bovine, equine, birds, cats or dogs, as well as primates or other animal species in which BDNF activity is detected. The invention further relates to BDNF nucleic acid subsets containing at least ten nucleotides, and further comprising the hybridizing portions of the BDNF sequence used, for example, in nucleic acid hybridization experiments, Southern and northern blotting, etc. The invention also provides the BDNF protein, and fragments and derivatives thereof corresponding to the amino acid sequences set forth in Figures 1 and 5, or functional equivalents thereof. The invention also provides fragments and derivatives of BDNF proteins which contain an antigenic determinant (s) or are functionally active. As used herein, the term "functional activity" refers to the detection of positive activity in experiments sensitive to known BDNF functions, such as chicken embryo DRG assays.

For example, the nucleic acid sequences described in Figures 1 and 5 can be substituted, alternately, by addition or by deletion, into functionally equivalent molecules. Because of the degeneracy of the nucleotide coding sequences, in practice, the present invention utilizes other DNA sequences that encode substantially the same amino acid sequences depicted in Figures 1 and 5, including, but not limited to, nucleic acid sequences containing all or part of the BDNF genes depicted. 1 and Fig. 5, which replace some codons encoding functionally equivalent amino acid residues within this sequence, i.e., sequences with silent mutations. Similarly, the BDNF proteins, or fragments and derivatives thereof, of the present invention, including, for example, but not limited to, contain substantially all or part of the amino acid sequence depicted in Figures 1 and 5 in their primary amino acid sequence. also altered sequences in which one amino acid residue is replaced by a functionally equivalent amino acid residue corresponding to a silent mutation. For example, one or more amino acids may be replaced by other amino acids of similar polarity, which function as functional equivalents, causing silent mutations. Within the sequence, the amino acid substitution may be selected from other members of the same class to which the amino acid substitution belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, histidine. Negatively charged (acidic) amino acids include aspartic and glutamic acids. The invention also relates to BDNF proteins, or fragments and derivatives thereof, which have been differentially modified during or after translation, e.g., by glycosylation or proteolytic cleavage by incorporation into an antibody molecule or other cellular Ugandan, etc.

In addition, mutations in this BDNF can be induced in vitro or in vivo, for the generation and / or disruption of translation sequences, for initiation and / or termination, or for generation of coding region variations and / or formation of new restriction enzyme endonucleases, modification in vitro. Any mutagenesis technology known in the art can be used, including but not limited to in vitro mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253: 6551), use of TAB® linkers (Pharmaceutics). etc

5.7. Generation of an antibody directed against brain ncurotrophin factor

According to the invention, the BDNF protein or fragments and derivatives thereof can be used as mutagens to generate anti-BDNF antibodies. Previous attempts to induce an anti-BDNF immune response have failed, possibly due to the presence of small amounts of pure BDNF. The problem of obtaining sufficient amount of BDNF has now been solved by the use of recombinant protein synthesis technology (based on the BDNF nucleic acid sequences of the invention) to produce a relatively large amount of BDNF in the production of BDNF proteins.

To further increase the likelihood of an immune reaction against BDNF, the amino acid sequence of BDNF is analyzed to identify those portions of the molecule that have an enhanced ability to immunize. For example, computer-assisted amino acid sequences identify surface epitopes using the Hopp and Woods method (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA, 78: 3824-3828), which has been successfully used for antigenic peptide identification in surface hepatitis antigen. On the other hand, it is possible to compare derived animal BDNF amino acid sequences and identify relatively inhomogeneous regions; these areas are also likely to be mutagenic in different species of animals.

Any technology that can generate antibodies using cell lines stable in cultures can be used to produce monoclonal antibodies directed against BDNF. For example, the present invention includes: hybridoma technology developed by Kohler and Milstein (1975). Nature 256: 495-497), trioma technology-human B-cell hybridoma (Kozbar et al., 1983, Immunology Today 4:72) and EBN-hybridoma technology for the production of human monoclonal antibodies (Cole et al., 1985, Monoclonal). antibodies and Cancer Therapy, Alan P. Liss, Ink., 77-96) and the like.

Human monoclonal antibodies or chimeric human-mouse (or other animal species) monoclonal antibodies are suitable for therapeutic applications. Human monoclonal antibodies can be obtained by any of a number of technologies known to those skilled in the art (e.g., Teng et al., 1985, Proc. Natl. Acad. Sci. USA, 80: 73087312; Kazbor et al., 1983, Immunology Today 4: 72- 79; Olsson et al., 1982, Meth. Enzymol 92: 3-16). Chimeric antibody molecules may contain an antigen-binding domain derived from a mouse and constant regions derived from a human (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 6851; Takeda). et al., 1985, Nature 314: 452).

Polyclonal antibodies against BDNF epitopes can be made using a variety of techniques known in the art. For the production of antibodies, various host animals, including but not limited to, species such as rabbits, mice, rats, and 1.1, are immunized by the administration of BDNF proteins or fragments or derivatives thereof. Various adjuvants may be used to enhance the immunological response dependent on the host type, including but not limited to Freund's complete or incomplete adjuvant, mineral gels such as aluminum hydroxide; surfactants such as isolecithin, pluronium polyols, polyanions, peptides, oily emulsions, Keyhale Limpet isocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

A molecular clone of the antibody against the BDNF epitope can be made using known techniques. Recombinant DNA methodology can be used to construct a nucleic acid sequence encoding a monoclonal antibody molecule or antigen-binding region thereof.

Antibody molecules may be purified using known techniques, such as immunoabsorbent or immunoaffinity chromatography, as well as chromatographic techniques such as HPLC (high performance liquid chromatography) or combinations thereof. Antibody fragments having the idiotype of a molecule are generated by known techniques. For example, but not limited to such fragments include the F (ab ') 2 fragment obtained by digestion of an antibody molecule with pepsin, the Fab' fragments obtainable by reduction of disulfide bridges of the F (ab ') 2 fragment, and the 2Fab and Fab fragments. obtained by treating an antibody molecule with papain and a reducing reagent.

An eleventh section example describes the production of a polyclonal antiserum directed against the peptide fragment of BDNF protein B5-33.

5.8. Identification of additional members of the gene family

Sequence analysis of the BDNF coding gene and deduction of its BDNF amino acid sequence revealed that this protein has many structural overlaps with NGF (Fig.2). The primary mature BDNF sequence, as well as the overall structure and possible processing mode of the protein precursor, suggests that NGF and BDNF genes can be derived from a common parent gene from the mature polypeptide region if three defects are introduced into the NGF sequence to optimize overlap. A total of 51 identical amino acids are common to previously known NGFs from many animal species and porcine and human BDNFs. These overlaps include all six cysteine ​​residues, suggesting that NGF proteins and BDNF have very similar secondary structures. In addition, four segments of six or more amino acids can be detected, in which the overlapping or non-conservative amino acid changes between NGF proteins and porcine BDNF of all animal species mentioned above. Thus, one would think that NGF and BDNF are probably highly related members of the same gene family.

The rational search for additional members of this BDNF / NGF gene family can be accomplished by the existence of unexpected conservative fragments with strong homology between BDNF and NGF, for example additional members of the BDNF gene family are identified by selecting sequences homologous to both BDNF and nucleic acid sequences. NGF. The following are used to identify those which, in addition, have nucleic acid sequences inhomogeneous for NGF and BDNF. The term inhomologous is used herein to designate a region that has at least about six contiguous nucleotides and in which at least two nucleotides differ from NGF and BDNF.

A more acceptable embodiment of the invention contemplates such a way. Consistent with each of the four conservative segments described above, a group of degenerate oligonucleotides can be synthesized that includes all possible coding sequences for the amino acids found in both BDNF and NGF through six adjacent codons. By numbering them at the amino terminus of the mature polypeptides (so that His 134 prepro BDNF corresponds to His I in the mature protein), these four conservative segments (boxes) can be characterized as follows (numbering relative to the human mature protein, DNA sequence depicted in Figure-1).

Table ΠΙ

<td>1 boxing</td><td>NGF</td><td>Gly10-Ser19</td><td>DNA sequences</td>

<td></td><td>BDNF</td><td>Gly8-Ser17</td><td> 587-616</td>

<td>Boxing 2</td><td>NGF</td><td>Lys50-Cys58</td><td></td>

<td></td><td>BDNF</td><td>Lys50-Cys58</td><td> 715-739</td>

<td>Boxing 3</td><td>NGF</td><td>Gly67-Asp72</td><td></td>

<td></td><td>BDNF</td><td>Gly67-Asp72</td><td> 764-781</td>

<td>Boxing 4</td><td>NGF</td><td>Trp99-Cysll0</td><td></td>

<td></td><td>BDNF</td><td>TrplOO-Cyslll</td><td> 863-898</td>

Synthetic oligonucleotides derived from the pairs of boxing sequences shown in Table 3 can be used as primers to amplify sequences from a potential source (RNA or DRN) by PCR. It can be mRNA or cDNA or genomic DNA from any eukaryote that can express a protein close to BDNF or NGF. Only six PCR reactions (namely: Box 1 primer with Box 2 primer; Box 1 with Box 3; Box 1 with Box 2; Box 3 with Box 2; Box 4 with Box 3) can detect a gene or gene a product having any two of the four specified segments with conservative sequences in combination with NGF and BDNF. If you want to synthesize several different abnormal primers for each box, you can still do a full re-selection with a relatively small number of PCR reactions. It is also possible to vary the stringency of the hybridization conditions used to prime PCR reactions to achieve a greater or lesser degree of similarity between the unknown gene and NGF or BDNF. If a segment of a previously unknown member of the BDNF / NGF gene family has been successfully amplified, this segment can be cloned and its primary sequence can be used and used as a probe to isolate a complete cDNA or genomic clone. This, in turn, will allow the complete nucleotide sequence of an unknown gene to be determined, its expression analyzed, and its protein product produced for functional analysis.

The method described above was used to identify a novel gene related to BDNF and NGF, described in the example in Chapter 13, below. Additionally, the present invention permits the use of BDNF / NGF sequence homology to generate novel recombinant molecules that are members of the BDNF / NGF gene family but are not found in nature. For example, but without being limited, the recombinant molecule of the invention can be constructed by combining parts of both NGF and BDNF genes. Such a molecule could have both BDNF and NGF properties and exhibit new biological activity, including both agonists and antagonists. The primary NGF and BDNF sequences can also be used to predict the tertiary structure of molecules by computer simulations (Hoor and Woods 1981, Proc. Matl. Acad. Sci, JSV, 78: 3824-3828); BDNF / NGF chimeric recombinant genes can be generated by assessing the correlation between tertiary structure and biological function. Similarly, chimeric genes having portions of any one or more members of the BDNF / NGF gene family, including the novel member described in Section 13, may be constructed in this manner.

5.9. Application of the Invention

The present invention relates to a BDNF nucleic acid sequence and substantially pure protein, peptide fragments or derivatives thereof. Now, for the first time, BDNF can be obtained in amounts sufficient for diagnostic and therapeutic applications. Similarly, antibodies and BDNFs obtained by the present invention, as well as nucleic acid probes, can be used for the first time in diagnostics and therapy. In most cases, the use of BDNF genes or gene products of the same species is more acceptable for diagnostic and therapeutic purposes, although it may be useful in certain embodiments of the present invention to use intergenic BDNF.

5.9.1. Diagnostic applications

The present invention relates to nucleic acids encoding BDNF and to proteins and peptide fragments or derivatives thereof, as well as to antibodies directed against the BDNF protein, peptides or derivatives which can be used in diseases and disorders of the nervous system associated with expression of BDNF. paternal lesions, diagnostics.

In various embodiments of the invention, the BDNF genes and related nucleic acid sequences and subsets, including complementary sequences, can be used in diagnostic hybridization assays. Nucleic acid sequences of BDNF and their subsets containing about fifteen nucleotides can be used as hybridization probes. Hybridization experiments can be used to detect, predict, diagnose or monitor disorders and diseases associated with alterations in BDNF expression, including sensory neurons and retinal neuronal degeneration. Such diseases and cases include, but are not limited to, injuries to the central nervous system, infarctions, infections, neurodegenerative disorders, malignancies, and postoperative changes, including but not limited to Alzheimer's, Parkinson's disease, Huntington's disease and degenerative retinal disease. For example, total RNA from a sample of a patient's tissue can be analyzed for BDNF mRNA, and a decrease in BDNF mRNA indicates neuronal degeneration. Relatively high BDNF mRNA levels have been detected in neuroblastoma tumor cells (see section 14 below); accordingly, in a particular embodiment of the invention, detection of increased mRNA levels using BDNF nucleic acid probes can be used in the diagnosis of neuroblastoma tumors. Because most tissues synthesize very small amounts of BDNF, BDNF mRNAs are particularly useful tumor markers.

In alternative embodiments of the invention, antibodies directed against the BDNF protein, peptide fragments or derivatives are used in the diagnosis of nervous system disorders and disorders, including sensory disorders and retinal degenerative diseases, as well as the disorders and diseases listed above. The antibodies of the invention may be used, for example, in situ hybridization technology, using tissue samples obtained from patients, if such evaluation is required. In another example, the antibodies of the invention may be used in ELISA procedures for detecting and / or measuring the amount of BDNF present in tissue and fluid samples; analogously, the antibodies of the invention may be used in Western blotting for the detection and / or measurement of BDNF in tissue and fluid samples. The antibodies of the invention that bind to BDNF in the ELISA and Western blotting procedures are described in Section 11 below.

In other embodiments of the invention, the BDNF protein, peptide fragments, or derivatives may be used to diagnose disorders of the nervous system. In a particular embodiment, such as, but not limited to, labeled BDNF protein or labeled peptide fragments may be used to detect tissues and cells that express BDNF receptors to identify BDNF receptor expression aberrations and, consequently, potential tissue and cell responses to BDNF. norms.

5.9.2. Therapeutic applications

This invention, which is directed to nucleic acids encoding BDNF and its proteins and peptide fragments or derivatives, as well as antibodies directed against BDNF protein, peptides or derivatives, can be used to treat diseases of the nervous system and disorders associated with paternal expression of BDNF expression. or resulting from the activity of BDNF or antibodies against it.

In various embodiments of the invention, the BDNF protein and peptide fragments or derivatives thereof are administered to patients with compromised nervous system trauma, surgery, ischemia, infection, metabolic disorders, food deprivation, malignancy, and toxic agents. Specifically, the invention can be used to eliminate conditions where sensory neurons and retinal ganglion cell lesions occur by administering a therapeutically active amount of BDNF protein or peptide fragments or derivatives. In various specific embodiments of the invention, BDNF is administered locally in the vicinity of damaged sensory neurons, including, but not limited to, the dorsal root node neurons or any of the tissues: cranial, mandibular, and nodular; ventrolateral pole of upper and lower jaw of ventrolateral pole of vestibular acoustic complex of cephalic nerve; of the middle cerebral trigeminal nerve. It may be desirable to introduce cognate BDNF peptides and BDNF proteins using membrane absorption, such as a soul membrane, which can be implanted near a damaged nerve. The present invention can also be used to accelerate patient recovery following diabetic neuropathy, such as multiple neuropathy. In other embodiments of the invention, BDNF proteins, or peptide fragments or derivatives thereof, can be used to treat congenital or neurodegenerative disorders, including but not limited to, Alzheimer's disease, Parkinson's disease, Parkinson's Plus syndrome (in which Parkinson's symptoms are caused by dopaminergic neurons). , such as progressive supranuclear palsy (Stil-Richardson-Olshevsky syndrome), oil-bridge vertebral atrophy, Shy-Drager syndrome-multiple systemic atrophy and Guaman's Parkinsonism dementia complex, as well as Huntington's chorea; alone, the invention can be used in the treatment of congenital or neurodegenerative disorders associated with sensory nerve dysfunction and retinal degenerative diseases. For example, the BDNF proteins, or peptide fragments or derivatives thereof, of the invention can be used in the treatment of hereditary elastic paraplegia with retinal degeneration (Kjelin and Bernard-Scholz syndromes), retinal pigmentosis, Stargardt's disease, Asher's syndrome (retinal pigmentosa with congenital deafness) pigmentosa with congenital deafness and polyneuropathy) and this is only part of the disease.

A defect in or synthesis of BDNF synthesis may be the cause of syndromes characterized by retinal degeneration and other sensory dysfunctions.

In particular embodiments of the invention, the administration of the BDNF protein or peptide fragments and derivatives thereof is combined with surgical tissue implantation in the treatment of Alzheimer's and / or Parkinson's disease. As discussed in Chapters 12 and 18, BDNF is dose-dependent (Figs. 34.) can be used to improve the survival of dopaminergic black matter neurons while utilizing BDNF in the CNS for dopaminergic neuronal diseases, including but not limited to Parkinson's disease (especially following the data in section 21 below showing that BDNF can be used n for the treatment of euro toxicity caused by the elimination of MPP toxin which causes Parkinsonism syndrome). Additionally, BDNF has been shown to support CNS cholinergic neuronal survival (see Figs. below to Chapters 12 and 16), including neurons from the basal cerebellum, suggesting that BDNF may be useful in the treatment of disorders associated with cholinergic neurons, including, but not limited to, Alzheimer's disease. About 35% of patients with Parkinson's disease have been shown to have Alzheimer's dementia; BDNF obtained according to the invention may be useful as the only therapeutic agent for this type of disease complex. Similarly, the BDNF produced by the present invention can be used to treat Alzheimer's disease associated with Down syndrome. The BDNF produced by the present invention can be used to treat a variety of dementias, as well as congenital inability to learn. It has also been found that BDNF is most likely to support astroglial cell proliferation, and that BDNF can be used to reduce CNS scar formation (for example, after surgery, trauma, or infarction), and BDNF can be used to treat CNS tumors of astroglial origin. In another embodiment of the invention, BDNF can be used to regulate NGF receptor expression and, therefore, may be useful for administering to a patient prior to or in association with NGF if required for treatment.

As shown in the example below in Chapter 15, BDNF expression (and slightly less NGF expression) in neurons, including ammonium horn and cortex neurons, can be enhanced by the introduction of cost acid. It has been observed (see section 15 below) that inhibition of non-NMDA receptors can block this cinic acid-induced BDNF expression. Accordingly, expression of cognate BDNF peptides and BDNF itself according to the invention can be induced by in vitro and in vivo administration of a cinic acid or cognate compounds, or carbachol, histamine or bradykinin, or cognate compounds that have been found to have similar effects in vitro or in vivo. in vivo, or by using non-NMDA glutamate receptor agonists or other drugs that potentiate or weaken the action of acetylcholine, histamine or bradykinin. NGF expression can also be induced by the introduction of cost acid or related molecules or non-NMDA receptor agonists.

In other embodiments of the invention, the BDNF protein, fragments or derivatives thereof are used in combination with other cytokines to obtain the desired neurotrophic effect. For example, but not limited to, BDNF according to the invention is used in combination with NGF or skeletal muscle extract to produce synergistic growth stimulation of sensory neurons, the term synergistic being understood to mean that the combination of BDNF protein, its peptide moieties and derivatives with a second reagent is stronger than acting on the same amount of these substances separately. It is anticipated that BDNF may function synergistically with other peptide-derived CNS-derived factors that have yet to be fully characterized, affecting the growth, development, and survival of various types of neuronal populations in the central nervous system.

It is further contemplated that by exploiting the full potential of the BDNF molecule, novel peptide fragments, derivatives, or BDNF mutants may be generated that act as antagonists of some or all of the BDNF biological functions. Such BDNF antagonists are useful for selective treatment of sensory neurons, for example in the treatment of chronic pain syndromes.

In yet other embodiments of the present invention, antibodies directed against the BDNF protein, or fragments or derivatives thereof, may be administered to patients with a variety of neurological diseases or disorders in need of such treatment. For example, such treatment is required for patients suffering from BDNF overproduction. Antibodies against BDNF can be used to eliminate aberrant regeneration of sensory neuroses (e.g., postoperatively) or as discussed above in the treatment of chronic pain syndromes. Because of the high levels of BDNF mRNA in neuroblastoma tissue, BDNF may be an autocrine tumor growth factor for neuroblastomas; accordingly, antibodies against BDNF may be administered for therapeutic purposes to cause tumor regression in a particular embodiment of the invention.

5.10. Pharmaceutical compositions

Active compositions according to the invention comprising all or part of the BDNF gene product, including a protein, peptide fragments or derivatives, or antibodies (or antibody fragments) directed against the BDNF protein or peptide fragments or derivatives thereof, or combining BDNF with a second reagent such as NGF or skeletal muscle extract may be administered in conjunction with any biocompatible pharmaceutical carrier including, e.g. but without being limited to, saline, saline buffer, dextrose and water.

The BDNF protein, peptide fragment or derivative thereof may have an amino acid sequence or a subset thereof substantially identical to that shown in FIG. 1 and 5; it may seem preferable to use a BDNF protein having specifically all or part of the amino acid sequence from about 134 amino acids to about 252 amino acids, as shown in FIG. 1, or a functionally equivalent sequence if that sequence contains a functional portion of the BDNF molecule. BDNF can be derived from sequences corresponding to BDNF genes from any suitable animal species, including, but not limited to, human, porcine, rat, chicken, cow, dog, sheep, goat, cat. rabbit, etc.

The amount of BDNF protein, peptide fragment, derivative or antibody that will be effective in treating a particular disorder or condition will depend on the origin of the disorder or condition and can be determined by standard clinical methods. Whenever possible, it is desirable to first determine the dose-response curve and the pharmaceutical composition of the invention in vitro, for example, in BDNF bio-experimental systems described above, and then using an animal model before testing the effect of the composition on humans. According to the data obtained in vitro, in a particular embodiment of the invention, a pharmaceutical composition effective to aid in the survival of sensory neurons can form a local BDNF protein concentration between about 5 and 25 ng / ml and preferably between 10 and 20 ng / ml. In a further particular embodiment of the invention, a pharmaceutical composition effective to enhance the growth and survival of dopaminergic or cholinergic neurons can form a local concentration of BDNF protein between about 10 and 100 ng / ml.

Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, venous, subcutaneous, or oral and nasal administration. Additionally, the pharmaceutical composition of the invention may be desirably administered to the central nervous system by any suitable route, including intraventricular or intrathecal injection; intraventricular injection may be facilitated by introducing into the stomach a catheter attached, for example, to a reservoir such as the Ommaya reservoir.

Further, the pharmaceutical composition of the invention may need to be administered topically to the area to be treated; this may be done, for example, but is not limited to topical infusion during surgery, catheter injection or implantation, and said implant may be porous or non-porous or made of a gelatinous material such as a sialastic membrane or fiber.

Pharmaceutical compositions containing BDNF protein, peptide fragments or derivatives which are administered by liposomes, microparticles or microcapsules can be obtained by the invention. In various embodiments of the invention, such compositions are used to maintain a constant release rate of BDNF or related product.

It is envisaged that cells producing BDNF and related substances, BDNF antagonists, or antibodies to BDNF may be introduced into areas where BDNF levels need to be increased or decreased.

⁴⁹ LT 4011 B

6th Example: Molecular cloning and characterization of porcine brain neurotrophic factor

Because BDNF is extremely rare in tissues, conventional methods for cloning the BDNF gene cannot be applied. Instead, a limited number of protein sequences are expanded using amplified DNA technology as follows:

(I) kilograms of pig brain were purified per microgram of BDNF.

(II) Purified BDNF was analyzed by protein micro-sequencing technology and the amino acid sequence of a fragment of 36 amino acid residues was determined.

(III) Using the established amino acid sequence (II), oligonucleotides are synthesized and used as primers in the polymerase chain reaction using the porcine superior colliculus cDNA as a template to amplify the DNA encoding the corresponding amino acid fragment.

(IV) The DNA reaction product (III) is sequenced, and (V) The corresponding oligonucleotide primers are synthesized and used in the polymerase chain reaction with porcine superior colliculus cDNA to generate overlapping DNA fragments corresponding to BDNF mRNA above and below the coding sequences. an initial fragment of 36 sequences. Thus, the entire region encoding porcine BDNF was cloned in two overlapping fragments.

A detailed description of each of the steps, as well as further description of the BDNF gene, is given below.

6.1. Materials and Methods

6.1.1. Purification of BDNF from pig brain

Purification of BDNF from porcine brain was performed using a procedure substantially similar to that described by Hafer and Barde (Hafer and Barde 1988, Nature 331: 261-262).

Six kilograms of porcine brain were homogenized in an UltraTurrax homogenizer, 0.2 M sodium phosphate buffer, pH 6, containing 1 mM EDTR and freshly added phenylmethanesulfonyl fluoride (1 mM) at a ratio of 1 kg brain to two liters of buffer. The pH of the supernatant was adjusted to 4 with 1 M HCl and stirred for 2 h. at 4 ° C. After 25 min. Supernatants (200 μg centrifugation) were combined (adjusted to pH 6 with 1 M NaOH) corresponding to 3 kilograms of brain for 2 hours. with 1 liter of pre-swollen carboxymethylcellulose and stabilized to pH 6 with 0.1 M sodium phosphate. After two washes with a total of 20 liters of 0.1 M sodium phosphate at pH 6, the resulting pellet was poured into a column and morning washes in the same buffer containing 0.13 M NaCl. The active fractions were washed with phosphate buffer containing 0.5 M NaCl followed by dialysis against 2x5 L of 5 mM potassium phosphate at pH 6.8. The dialyzed fractions obtained by treatment with 2x3 kg of starting material are placed in a 130 ml layer of a hydroxyapatite column which is pre-stabilized with 5 mM potassium phosphate at pH 6.8. The column was then flushed in a linear potassium phosphate gradient from 500 mL 5 mM to 500 mL 700 mM at pH

6.8, and BDNF activity was further eluted at 500 mM potassium phosphate (see, Barde et al., 1982, EMBO, 1: 549-559). After addition of 1 M potassium phosphate, the resulting active fractions are brought to a concentration of 700 mM potassium phosphate and placed in a 5 ml phenyl-sepharose column stabilized with 700 mM potassium phosphate at pH 6.8. After washing with 40 ml of the same buffer, the BDNF activity was washed with 0.1 M potassium phosphate at pH 6.8, dialyzed in distilled water, and lyophilized. The lyophilized material is transferred to a buffer containing 0.1% SDS but without mercaptoethanol prior to SDS gel electrophoresis as described by Barde et al. (1982, EMBO, 1: 549-553) and then transferred to an SDS gel containing acrylamide with a linear (not exponential) gradient of 10 to 45%. After completion of the electrophoretic separation, the gel was briefly stained (10 min) with Koomassi blue, bleached for 20 min, and the band migrating at cytochrome c level was cut and washed from the gel, SDS removed as described by Barde et al. (1982, EMBO, 1: 549-553). The purification procedure, which was eventually followed by sequencing, was modified by Hofer and Barde (1988, Nature 331: 261-262) and did not use gel electrophoresis in its final step.

6.1.2. Protein sequencing

The BDNF sequence is determined directly (55 pmol as defined by amino acid analysis with an initial yield of 40 pmol for the amino terminus histidine) or cleaved as follows: 5-10 µg BDNF (from five different blanks) is cleaved according to this procedure. V8: g 5. aureus V8 is added to 5 µg BDNF with 0.2 NH4CO3 pH 8 containing 10% acetonitrile (total volume 50 µl) and incubated until morning at room temperature. Trypsin: 1 µg TPOK-treated trypsin (bovine pancreas, Sigma, type ΧΠΙ) is added to 8 µg BDNF in Tris-HCl (0.1 M, pH 8) containing 10 mM CaCl2 (total volume 40 µl) and incubated until morning at 37 ° C. Cyanogen bromide (CNBr): incubate 10 µl BDNF for 3 hours. at room temperature (total volume 60 µl) with 10% (w / v) CNBr in 70% (v / v) formic acid solution (final concentrations). After the addition of 500 µl H2O at the end of the reaction, the sample is concentrated to 500 µl using a high-speed vacuum evaporator. 50 µl Tris-HCl (1.0 M at pH 8) is added along with 5 µl beta-mercaptoethanol and the sample is incubated until 37 ° C in the morning. After addition of 5 µl iodomethane, the sample is evaporated to dryness using a high-speed vacuum evaporator. It has been found that after CNBr digestion it is necessary to perform BDNF recovery and alkylation as demonstrated by SDS gel electrophoresis by Swank-Munkres and HPLC (1971, Anai. Biochem 39: 462-477), no fragments are obtained without reconstruction. This indicates that BDNF has disulfide bridges arranged such that no cleavage releases peptides. After each digestion, the dried samples are resuspended in 0.1% 3-fluoroacetic acid (TFA) and loaded onto a reverse phase HPLC column (Applied Biosystems) and the peptides are washed for 60 min at 0.1 mL / min with a linear gradient of 0-60% acetonitrile. in 0.1% TFA. Detection was performed at 214 nm using a Waters 441 UV detector. The sequences of these peptides were determined using automated Edman degradation by a gaseous micro-sequencer (Model 47 A, Applied Biosystems) according to Hewick (Hewick 1981, J. Biol. Chem. 256: 7990-7997) and Hunkapillar (1983, Methods Enzymol 91: 227-236). ). PTH amino acid detection was performed according to Lottspeich (Lottspeich, J. Chromatogr 326: 321-327).

6.1.3. Preparation of DNA arrays

Pig genomic DNA was isolated according to the method of Herrmann and Frischlauf (1987, Metchods Enzymol 152: 180-183).

For preparation of cDNA, total RNA is extracted from 6 grams of the superior colliculus pig brain. Tissue samples were collected by dissection and frozen in liquid nitrogen at a local slaughterhouse. Standard methods were used for RNA extraction (Okayama et al., 1987, Methods Enzymol 154: 3-20). 80 pg total RNA was transcribed using reverse transcriptase from murine leukemia virus Moloney (BRL, according to manufacturer's instructions, except for addition of 1 μNR RNA syn and no actinomycin D) using primer mix CGGATCCGAATTCTGCAGl'l ΓΓΓΙ ΓΙ ΓΓΓΓ with A, C or G as the terminal 3 'nucleotides (oligo 3 engineered for compatibility with the 3' poly-A moiety and having Bam HI, ScoRI and Pst I recognition sites).

6.1.4. Polymerase chain reaction

The polymerase chain reaction was performed according to the method described by Saiki et al. (Saiki 1985, Science 230: 1350-1354).

6.2. Results and Discussion

6.2.1. Protein sequencing results

The results of protein sequencing are shown in Table IV.

Table IV

The peptide sequence of BDNF was experimentally determined

N-terminal

V8

V8

CNbr

CNbr

Trypsin

Trypsin

HSDPARRGELSV

XVTAADKKTAVD

KVPVSKGQLKQYFYE XGGTVTVLEKVP (V) (S) GYTKEGXRGIXRGI (T) AVDMSGGTVTVLEK ALTMDSK

Joined sequence:

VTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYE, underlined amino acids encoded by the oligonucleotide primers used in the PCR reaction. above j 6.1.2. section. Four contiguous segments of the amino acid sequence were identified using Edman degradation, which could be used to isolate 36 amino acids, representing approximately one third of the total amino acid sequence.

6.2.2. Oligonucleotide synthesis and use of PCR reaction to obtain the DNA encoding the amino acid fragment

The two fully degenerate 17-mer oligonucleotide primers were chemically synthesized using sequences encoding two segments of the six amino acids, which are the 36 amino acid residue fragment described above.

6.2.1. section, at the amino and carboxyl terminus, respectively. Specifically, two separate mixtures of 17-nucleotides (aligned with all possible codons and generated by oligo 1 and oligo 2) were chemically synthesized, based on the sequence AADKKT (sense) and KQYFYE (anti-sense) (underlined in Table ΠΙ) and 130 pmol each primer. added to 1 pg of porcine genomic template DNA. 35 cycles of amplification using PCR reactions were performed according to the manufacturer's instructions (Gene Amp TM, perkin - Elmer Cetus). The denaturation lasted one minute at 94 ° C, the primer was renatured for two minutes at 45 ° C and expanded for two minutes at 72 ° C. A DNA band of predicted size (101 bp) was excised from 3% agar gel (stained with ethidium bromide) and asymmetrically amplified as described by Innis et al. (Innis, 1988, Proc. Natl. Acad. Sci. USA, 85: 9436-9440) with a 100-fold excess of either oligo I or oligo 2.

6.2.3. nucleotide sequence of a cDNA fragment

Nucleotide sequences of the resulting sense and anti-sense DNA fragments were determined by the deoxynucleotide chain termination method (Sanger et al., 1979, Proc. Natl. Acad. Sci. USA, 72: 3918-3921) using oligonucleotides 1 and 2 as primers. at the ends with 32p. The resulting nucleotide sequence had only an open reading frame uninterrupted by a strand termination codon. The deduced amino acid sequence from this open reading frame was in complete agreement with the complementary amino acid sequence found in the analogous region of porcine BDNF.

6.2.4. Cloning of whole pig BDNF cDNA

The entire region encoding porcine BDNF was molecularly cloned in two overlapping fragments. To obtain the 5 'region of the BDNF cDNA (with respect to the sense strand), a 50-mer oligonucleotide primer containing 21 bases of the exact sequence of the region described in the BDNF sense strand was synthesized for use as a sense primer. Nucleotide (underlined sequence corresponding to the sense BDNF strand of Fig. 1, positions 643 to 663) encoding the Thr-Ala-Val-Asp-Met-Ser-Gly (the first two bases of the Gly codon) of this primer. 9 additional nucleotides were introduced to create cleavage sites for restriction endonucleases due to Spe I and Sal I using molecular cloning. A triple-degenerate 31-stranded oligonucleotide primer complementary to poly-A was introduced at any nucleotide (T, C, or G) cDNA sense strand to provide recognition sites for Bam HI, Eco RI and PstI restriction nucleases. the sequence of the antispasmodic primer (oligo 3) was (5¹) CGGATCCGAATTCTGCAGITTTTTITTTrrK (3), where x = A, C, or G. Synthetic oligonucleotide primers were used to amplify sequences from porcine superior colliculus cDNA prepared as described above by PCR. Specifically, 3 ′ of amplified DNA was obtained using 10 liters of reverse transcription reaction mixture, 150 pmol of the sense primer (oligo 4) and 150 pmol (oligo 3) as the anti-sense primer in the PCR reaction. Southern blot analysis was performed on amplified DNA products; and the band giving the hybridization signal with the 32P-tagged end oligonucleotide AAGGATCCTGCAGTTGGCCTTTCGAGAGGC (oligo 5 used as an anti-sense primer in the 5 'reaction described below and having Bam HI and Pst I recognition sites) was cleaved, extracted by extraction, RI and Sali, were cloned

Bluescript SK⁺ plasmid (Stratogen)) and nucleotide sequence analysis.

To obtain the remaining BDNF coding sequence (above or 5 '), the cRNA was prepared as described above and ligated to the 5' end of the poly-A using a terminal deoxynucleotide transferase. The same mixture of 31-dimensional oligonucleotides, each containing the above section of 12 consecutive T residues, was used to obtain a primer complementary to the attached poly-A tails. A single 30-strand oligonucleotide primer containing 17 bases corresponding to the complementary strand with the BDNF coding sequence and BamHI and Pstl recognition sites for the restriction endonucleases was synthesized. The primer had the following sequence: (5 ')

AAGGATCCTGCAGTTGGCCTTTCGAGACGG (3 ') (oligo 5' see above), underlined region complementary to the coding sequence for BDNF from position 709 to position 693 (Fig. 1). This sequence corresponds to the segment encoding the amino acid sequence Pro (last two codon bases) Val-Ser-Lys-Gly-Gln. These primers were added to the DNA with poly-A tails and, as above, amplified by PCR reactions. Reaction products were excised with Pstl and cloned and blotted using a Bluescript vector using a deoxynucleotide chain termination method.

6.2.5. The nucleotide sequence of porcine BDNF cDNA

The linked nucleotide sequence determined from porcine BDNF cDNA fragments is shown in Fig. 1 together with the deduced amino acid sequence. This sequence has an open reading frame for a polypeptide of 252 amino acids. The initiating Met codon (ATG) is identified by detecting two contiguous sequence termination codons (TAG-TGA) in the same scan frame, starting 36 base pairs above. The amino terminal of porcine BDNF, as determined by direct protein sequence analysis, corresponds to the polypeptide residue His 134. Thus, nucleotide sequence data indicate that mature BDNF is obtained by a process polypeptide. Just before His 134 residue is the sequence Arg-ValArg-Arg. Such sequences, with one major amino acid residue followed by a neutral residue and then two other major residues, are linkage targets for the proteolytic processing of polypeptide precursors. The derived amino acid sequence of mature BDNF precipitates a protein with 119 amino acids (molecular weight 13,311 daltons) with an alkaline charge (pi = 9.99) and has properties consistent with the preliminary characterization of BDNF as assessed by biologically active factor by two-dimensional gel electrophoresis. The amino acid sequence of the BDNF moieties obtained by protein analysis of the amino acid sequences (64 amino acid residues in total) was completely identical to the amino acid sequence derived from the nucleotide sequences of the cDNA clones (Fig. 1, underlined). This polypeptide precursor sequence is compatible with BDNF processing in at least two steps: first, the signal peptide, most likely 18 residues, must be cleaved from the amino-terminal and then cleaved between Arg 133 and His 134 to release the mature polypeptide. If this model is correct, the precursor should be labeled BDNF.

7th Example: The BDNF gene differs from the NGF gene in various vertebrate species

7.1. Materials and Methods

7.1.1. Preparation of NGF and BDNF DNA probes

A plasmid was obtained from British biotechnology Ltd with a synthetic gene encoding mature human NGF and no sequence encoding normal human NGF, having several conserved codon changes, for the introduction of convenient restriction sites for endonuclease digestion. Two 18-mer oligonucleotide primers were synthesized by PCR to amplify the 270 bp segment of this gene encoding the amino acid residues 9 to 111. To obtain the labeled DNA probe, 10 cycles of PCR reactions with 32p_dOTP were performed. The BDNF probe was obtained using similar procedures except that amplification was performed using porcine genomic DNA as the starting material and the amplified segment corresponded to the region encoding amino acids 28-111 of mature BDNF. The complementary strand (antiprasmin) primer corresponding to amino acid region 106-111 was synthesized with the sequence (5 ') ACATACACAGGAAGTGTC (3'). The sense strand oligonucleotide primer was made from amino acid residues 28-33: (5 ') GCAGTGGACATGTCGGGT (3'). These two probes were subjected to Southern blot hybridization (Southern, 1975, J. Mol. Biol. 98: 508-517) under stringent conditions at 2 x SSC at 68 ° C.

7.1.2. Detection of nucleotide sequences of BDNF genes in various animals

The same two described above 18-mer oligonucleotides comprising the mature porcine BDNF region encoding amino acids 28-111 were used as primers under standard PCR conditions to amplify 252 bp of porcine, rat, chicken and human DNA and the resulting DNA reaction product nucleotides. sequence was determined by the deoxynucleotide chain termination method (Sanger et al., 1979, Proc. Natl. Acad. Sci. USA, 72: 39183921). In some cases, the amplified DNA band was excised and extracted after agar gel electrophoresis and re-amplified prior to nucleotide sequencing. In other cases, amplification is not required.

7.2. Results and Discussion

Prior to the present invention, the BDNF protein was purified exclusively from pig. A major need was the demonstration that BDNF is not merely a porcine nerve growth factor beta subunit (beta-NGF, or simply NGF, as mentioned herein), the purification and molecular cloning of which has not yet been described. This was particularly important as it was previously reported that the physical properties of porcine BDNF are in fact identical to those of the beta-NGF monomer in many animal species. In the past, it has been difficult to determine the exact relationship between BDNF and NGF because of the lack of neutralizing antibodies to porcine BDNF and the unknown amino acid or nucleotide sequence of porcine BDNF. It could be read that differences in biological activity observed between BDNF and NGF may, for example, simply reflect differences in NGF between pig and other animal species (e.g., mice), or may be due to differential modification of NGF protein molecule in various tissues (e.g., pig brain). and in the mouse salivary gland), or due to protein modifications accidentally made in one of the stages (or stages) of swine brain cleaning.

If BDNF were found to be distinct from NGF, it would be important to determine if the BDNF gene is present in other animals, especially humans. Prior to the present invention, there was no information on this subject since BDNF was purified from only one species. The presence of neurotrophic activity in most crude extracts and conditioned media, which is probably different from NGF, does not mean that there is a substance that is identical or substantially equivalent to porcine BDNF.

Comparison of the deduced amino acid sequence of the derived porcine BDNF with the known NGF sequences of several animal species (human, cow, guinea pig, mouse, chicken, and snake) showed that BDNF is much less bound to any vertebrate NGF than to various species of NGF (Fig.2). . An unexpected feature of mature BDNF parent structure is the similarity to NGF structure, with only three flaws introduced for compatibility optimization, there is a 51 overlap common for different NGF (snake to human) and BDNF (Fig.2). Importantly, there are six cysteine ​​residues between these overlaps. Although the exact disposition of BDNF disulfides is not yet known, it is clear that such bridges are present (Table 3, explanation). The 3 tryptophan and 2 phenylalanine residues detected in BDNF molecules are in the same positions in the NGF molecule. It should be noted that 6 aspartic acid residues (out of 7 in BDNF) and 7 valine (out of 9) are in the same positions in both mammalian NGF and BDNF. These five amino acids make up about half of the overlapping amino acids between the two proteins. In contrast, there are some striking differences between BDNF and total NGF. Additionally, in addition to the three faults mentioned above, there are 21 positions where the amino acids overlap in all NGFs but differ in the BDNF molecule.

Most of the precursor sequence is not related to the NGF sequence with two exceptions: the putative BDNF secretory signaling sequence has 5 overlapping amino acids (out of 13 amino acids) and is generally remarkably similar to the mouse NGF signaling sequence, which has been shown to undergo cleavage position on methionine inducing translation (Edvads et al., 1983, Mol. Cell. Biol.8: 2456-2464). It appears possible that alanine, which is also in position 18 of the BDNF molecule, is a potential cleavage site for deletion of the BDNF signal sequence. Another similarity with NGF begins at the only consensus N-glycosylation sequence (underlined twice in Fig. 1), corresponding to asparagine 126. This asparagine is located eight amino acids upstream of the cleavage site initiating mature BDNF. The same structure was found in several NGFs; as well as the Arg-X-Basic-Arg sequence forming the last four amino acids in the precursor (Schvarts et al., 1989, J. Neurochem. 52: 1203-1209).

Evidence that NGF and BDNF are encoded by different genes in different animals was obtained by preparation of DNA probes from molecularly cloned human NGF and porcine BDNF and Southern blot hybridization with genomic DNA digested with restriction endonuclease EcoRI. Genomic DNA was analyzed in the following species: human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast. DNA was digested with EcoRI and analyzed by Southern blotting in double filters with human NGF and porcine BDNF Labeled probe. For each probe, a single band was detected in all organisms except the yeast. In most cases, bands hybridizing to NGF and BDNF probes in any organism had different motility during electrophoresis, although in some cases (e.g., mouse DNA) the EcoRI fragments hybridized with NGF and BDNF probes were approximately the same size and could not be isolated by electrophoresis. conditions (Fig.3).

Part of the sequence encoding mature BDNF was amplified by PCR from genomic DNA of pig, chicken, rat and human, and the nucleotide sequence was determined. Sequence analysis of the amplified region of porcine genomic DNA accurately confirmed the sequence obtained by molecular cloning of porcine brain cDNA. Genomic sequences of rat, human, and chicken BDNF in this 252 br segment were also determined (Fig. 5). It should be noted that the resulting rat and human amino acid sequence is at least amino acid 28-111 identical to that of porcine BDNF, although several different nucleotides have been detected in different species (e.g., conservative alterations at the third codon position). The chicken had the only amino acid substitution in this zone, with 61 mature protein residues in the chicken being lysine compared to methionine in mammalian BDNF. Sequence analysis data, together with experimental data from southern bloth hybridization, unequivocally demonstrate that BDNF is encoded by a highly conserved gene that is distinct from the gene encoding NGF.

8th Example: BDNF RNA is expressed in nerve and other tissues

8.1. Materials and Methods

8.1.1. Preparation of RNA

Total RNA was extracted from adult female mice according to Okayama et al. (1987, Methods Enzymol 154: 3-20). Briefly, frozen tissues were homogenized in 5.5 M guanidintiocyanate, centrifuged to remove debris, and brought to a density of 1.51 µg / ml by transferring supernatant onto cesium trifluoroacetate. After 24 or. centrifugation at 125,000 g in a rotor SW-27 (Bekman), the RNA was resuspended and precipitated with ethanol and 8 M ammonium acetate and stored at -70 ° C. Electrophoresis was performed according to Lehrach et al. (Lehrach 1977, Bilchemistry 16: 4745-4751) 1.3% on agar-formaldehyde gels. RNA was transferred onto nylon membranes (Hybond-N Amerscham) and hybridin until morning in 1 ml 2X SSC in 50% formamide with mouse BDNF³²P-cRNA probe (10 ^ cpm see below) at 62 ° C. The flushing lasted 60 minutes. at 65 ° C in 0.1 x SSC solution. After rinsing, incubate for 60 min. at room temperature with 0.1 µg / ml RNAsease A (Farmacia) and film exposed for 48 h. at -70 ° C (with intensifying screen).

8.1.2. DNA probe preparation

Mouse brain hDNA was screened with two independent BDNF oligonucleotides. Double positive clones were isolated and subcloned into Bluescript SK⁺ plasmid (Stratagen) EcoRI site. A nucleotide sequence corresponding to porcine 350-828 of the porcine sequence was determined (see Figure 1). Only four amino acid differences between mouse and porcine BDNF were detected in this sequence, indicating that this porcine and mouse BDNF domain is highly conserved. The single stranded RNA probe was prepared using this template and T3 polymerase (Promega). The specific activity of this probe was 10 ^ cpm / pg.

8.2. Results and Discussion

Northern blot analysis was used to compare BDNF mRNA expression from nerve tissue to other tissues. Northern blot analysis was performed using mouse tissues allowing for faster RNA extraction than pig tissues. The 32P-cDNA probe detected a signal at approximately 1.45 kb in the brain (Fig. 4) and in the spinal cord (data not shown). Importantly, no signal was detected in any of the other tissues, including kidney, intestine, lung, liver, bile, heart, and muscle (Fig. 4). Assuming that the size of the porcine mRNA is similar to that of the murine mRNA, the cDNA sequence shown in Figure 2 represents more than 80% of the complete mRNA sequence.

An important observation for BDNF physiology transdifferentiation is that the mRNA encoding this protein is only found in the central nervous system and not detected in any of the seven non-neural tissues, BDNF mRNA was detected not only in the brain but also in the spinal cord and superior colliculus (sequence shown). Fig. 1, obtained from a superior colliculus cDNA template). This supports the idea that BDNF is a neurotrophic factor of target origin, and that BDNF-responsive neurons are either CNS-dependent or CNS-related. In fact, all neurons known to respond to BDNF or enter the CNS, like dorsal root or sensory ganglion neurons (Lindsay, 1985, Chem. Biol. 112: 310-328; Davies, 1986, J. Neuroscience, 6: 1897-1904) or are CNS neurons as retinal ganglion cells (Jonson, 1986. J. Neuros., 6: 3031-3038). Further research is needed to more accurately determine the distribution of BDNF synthesis sites in the central nervous system, but it is already clear that BDNF mRNA distribution is significantly different from NGF mRNA found in most non-CNS tissues (Choiman, 1984, EMPO 3: 31833189; Schelton 1984, Proc Natl Acad Sci USA 81: 7951-7955).

9th Example: Molecular cloning and characterization of human and rat BDNF genes

9.1. Materials and Methods

9.1.1. Genetic DNA and cDNA libraries

Adult human retinal cDNA and lambda-ZAPII library were obtained from Stratagene. The human placental genomic DNA library at EMBL3 / SP6 / T7 was obtained from Klontex. The human fetal brain cDNA library in ZgtII was obtained from Klontex. The rat genomic DNA library at EMBL3 / SP6 / T7 was obtained from Klontex. Both genomic libraries were prepared by partial digestion of genomic DNA with restriction nuclease Sau3A and insertion into the vector BamHI site. The rat brain cDNA library for lambda-ZAPII was obtained from Stratagene.

9.1.2. Preparation of BDNF DNA probes

32P-labeled BDNF DNA probes were prepared using the same oligonucleotide primers described above in 7.1.1. section, using a PCR reaction with human genomic DNA to amplify the regions encoding the amino acid residues 28-111 of human BDNF. In parallel, a specific rat BDNF probe labeled with 32P was obtained using rat genomic DNA as a template in the PCR reaction.

9.1.3. Library screening

Screening of lambda-phage libraries was performed by standard methods (Benton and Davies, 1977, Sci. 156: 180-182; Maniatis et al., 1978 Cell. 15: 687701) by hybridization with 50% formaldehyde in dextransulfate and Denhart's solution at 42 ° C. . Filters were pre-hybridized with 50% formamide at 42 ° C with 5xSSCPE 10% Denchardt, 0.5 mg / ml salmon sperm DNA, 0.1% SDS and 10% dextransulfate. Hybridization was performed in the same buffer with only 2% Denchardt solution, salmon sperm DNA was 0.1 mg / mL, and SDS and dextrin sulfate were absent. After hybridization, the filters were washed at 68 ° C. The human BDNF probe was used to screen human genomic libraries and retinal cDNA libraries. The rat BDNF probe was used to screen the rat genomic library and cDNA library. Human and rat NGF probes prepared as described above were used for library screening

7.1.1. section.

9.2. Results and Discussion

At least 670000 spots from each library were re-selected. Positive human clones were those that crossed with the human BDNF probe but did not cross with the human NGF probe described above. BDNF genomic clones were obtained from human and rat libraries with frequency, one copy of the BDNF gene for each haploid gene. About one million spots were reselected in human and rat libraries. Three positives were obtained from the rat genomic library and one from a human. Positive clones from human retinal cDNA and rat brain libraries were obtained at a frequency compatible with very low levels of gene expression; out of 670000 rat brain cDNA clones, only two positive clones were identified; out of 670000 human retinal cDNA clones, only one positive clone was identified. No positive clones of 670000 were found in the DNA library prepared from the human fetal brain. Nucleotide sequences of human BDNF cDNA and genomic clones were performed using synthetic oligonucleotide primers that were exact copies of the sequences encoding human and rat BDNF as described above in 7.2.1. section. The longest human BDNF clone obtained had an insert of approximately 1.6 to 1.8 kbr and, as expected, had the exact sequence of a portion of human BDNF detected after direct amplification from human genomic cDNA as described above in 7.2. section. Detailed sequence analysis of this cDNA clone (Figure 5) revealed that it contained an open reading frame encoding a polypeptide of 247 amino acids, similar but not identical to the full-length precursor of porcine BDNF. Inside the region corresponding to the mature BDNF polypeptide (e.g., from the His 134 codon to the terminal end codon) no difference was detected in the deduced amino acid sequence. All nucleotide changes between human and porcine were conservative in coding sequence. The residue of the polypeptide, a putative precursor of BDNF, had some differences in the human and porcine amino acid sequences, with the most striking difference being the lack of five consecutive Ser codons in the porcine human codon of the BDNF gene (247 instead of 252 ar).

Libraries prepared in the EMBL3 vector have foreign inserts of 10-25 kbr. The exact size of the insert in a finely studied human genomic BDNF clone has not been determined. However, this clone containing the only Eco RI fragment of restriction endonuclease, approximately 4 kb in size, which was cross-linked with the labeled BDNF probe, was used for library screening.

This fragment has the expected length derived from Southern blot hybridization of human genomic DNA with porcine BDNF probe as described above. Nucleotide sequence analysis was performed on a human clone using synthetic oligonucleotides, which are cDNA sequences that act as primers for DNA synthesis from bacteriophage template DNA. The nucleotide sequence encoding the putative human BDNF precursor was found to be identical to that of the human cDNA clone, except for the single prepro-substitution of nucleotide 785 corresponding to valine (GTG) substitution of the amino acid methionine (ATG). Figure 5 This change may reflect polymorphism in the human genome. As with human NGF, no nitron was detected inside the sequence encoding the putative human BDNF. Rat cDNA sequence data are also shown in Figure 5

10th Example: Expression of recombinant BDNF

10.1 Materials and Methods

10.1.1. Prepare the vector for BDNF expression

The sequence corresponding to porcine prepro BDNF was prepared using oligonucleotide primers (150 pmol each):

ATAATCTAGATGACCATCCTTTTCCTT (Seam)

ATAATCTAGACTATCTTCCCCTCTTAAT (anti-spasm thread)

PCR reaction and using 1 pg of porcine genomic template (each primer has an additional link). The amplification reaction was performed as described, except that the renaturation temperature was 50 ° C. After digestion with Xbal, the amplified DNA was inserted into the Xbal site in pCMV to produce pCMV'-pBDNF'1 (-1 represents the sense orientation in Figure 6 and corresponds to COS + V in Table) and the plasmid was introduced into the bacterium by XL-1 electroporation.

10.1.2. BDNF expression in COS cells pCMVI-pBDNF DNA from positive clones (cross-checked with oligo 5, Fig.2) was excised from Xbal and Pst I. The size of the resulting products allowed to determine the orientation of the inserts, and both plasmids were used for transfection of COS cells by the calcium phosphate pathway. (Chen et al., 1987 Mol. Biol. 7: 2745-2752) and harvested the culture medium after 24 hours. BDNF activity was assayed by bioassays with chicken embryo dorsal root nodules detailed above.

10.2. Results and Discussion

E8 chicken spinal cord sensory neurons were placed in a vessel maintained at 6000 wells, incubated for 24 hours, and counted (Lindsay et al., 1985, Develop Biol. 112: 319-328). The mean of three calculations ± standard deviation was evaluated. When administered, BDNF and NGF concentrations were in lng / ml at which maximal survival was observed with each of the factors. COS + refers to cells transfected with plasmids containing BDNF inserts in a sense orientation, whereas COS + refers to cells transfected with plasmids containing BDNF inserts in reverse orientation. COS belongs to untransfected cells. At a dilution of more than 1:20, no increase in survival was observed compared to controls in COS and conditioned COS media. In all experiments without NGF, monoclonal antibodies against NGF (Korsching et al., 1988, Proc. Natl. Acad. Sci. USA 80: 35133516) at concentrations of 1 ng / ml were used. As shown in Table V, only medium from COS cell cultures carrying pOMVI-pBDNF plasmids with a sense orientation showed an increase in chicken sensory neuron survival compared to controls. It follows that recombinant BDNF is biologically active. Further, the addition of BDNF isolated from porcine brain did not significantly increase the level of sensory neuron survival achieved by recombinant BDNF alone, indicating that recombinant BDNF is capable of saturating BDNF receptors. It has also been found that recombinant BDNF, when used in combination with NGF, can act in a complementary and synergistic manner to improve the survival of chick sensory neurons.

Table V.

Survival of cultured chicken sensory neurons

COS medium (final dilution) COS +

1:20

1:50

2,510±263

1:200

2,833 ± 171 cosCOS

211±16

250±87

BDNF + COS +

NGF + COS + BDNF only

2,516±209

5,770±72

2,718±424

11th Example: Generation of antibodies against BDNF

11.1. Materials and Methods

A polyclonal antiserum specific for BDNF molecules designated sera4 was generated by NZ white rabbit by immunization with a synthetic peptide

11.1.1. Synthesis of peptides and their coupling to a carrier

A peptide consisting of 34 amino acid residues, designated B5, was synthesized by conventional methods. This peptide has the amino acid sequence shown below, which corresponds to 33 residues of mature BDNF (residues 153-185 of the full-length porcine prepro BDNF sequence shown in Fig. 1) with an additional cysteine ​​residue at the amino terminus (shown in written font) for binding to the protein carrier. m-maleimidobenzoic acid -N-hydroxysuccinimide (MBS): as desired: Cys-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser-GlyGly-Thr-Val-Thr-Val-Leu-Glu-Lys-Val-Pro-Val- Ser-Lys-Gly-Gln-Leu-Lys-GlnTyr. The B5 peptide was bound to bovine serum albumin using bisdiazobenzidine (BDB). Fresh BDB was prepared by dissolving 46 mg of benzidine-HCl. 35 mg of NaNC&gt; 2 are dissolved in 1 ml of water and added to the benzidine solution with stirring for 1 hour at 4 ° C. 21 mg of BSA was dissolved in 3 mL of 0.16 M borate, 0.13 M NaCl, pH 9. About 15 mg of B5 peptide was dissolved in 1.5 mL of borate-NaCl buffer, pH 6. The peptide solution was added to BSA solution and placed on ice, 1 mL of BDB was added to BSA peptide solution, and the reaction mixture was incubated for 2 hours at 4 ° C; at that time the pH was monitored and maintained at level 9 by the addition of small amounts of 0.5 M NaOH, if necessary. The reaction was terminated by the addition of 0.2 ml of a 1% buffered phenol solution. Excess reagents were removed by dialysis in phosphate-buffered saline (PBS).

11.1.2. Immunization

A total of six rabbits were immunized according to the following procedures:

Rabbits using peptides 1 and 4 with B5 attached at their O terminus to BSA

BDB;

Rabbits 2 and 3 using B5 peptide linked to their N terminus with BSA

MBS;

Rabbits 5 and 6 with B5 peptide mixed with powdered nitrocellulose.

In all cases, 1 ml of immunogen (100pg B5 / 500pg nitrocellulose 5 and 6 rabbits) in 0.5 ml PBS + 0.5 ml Freind's complete adjuvant was used for the first immunization. The mixture was injected subcutaneously at several sites in the back. The second immunization was performed 3 weeks later and was identical to the first, except that a partial adjuvant was used in place of complete Freud's adjuvant. Subsequent injections were given every four to six weeks. Rabbits were bled 1 week after immunization and generally tested for the ability of the antiserum to bind pure B5 peptide in an enzyme linked immunosorbent assay (ELISA).

11.1.3. Detection of antibody binding to BDNF

100 The pg of antigen (B5 peptide) in water was filled into the wells of a microtiter dish and left to dry in the morning, then briefly rinsed with water and blocked with 100 pg of 1% gelatin for 30 min at room temperature. The wells were washed 3 times with distilled water and then added with 100 pg of antiserum and left at 4 ° C until morning. The wells were then rinsed three times with PBS / 0.5% triton λ-10, followed by the addition of 100pg of peroxidase-labeled antitrust immunoassay (1: 1000 dilution) and incubation at room temperature for 3 hours. The wells were rinsed twice and added with 100 pg ABTS (10 mg ABTS Sigma dissolved in 10 ml 0.1 M sodium citrate, pH 4, + 10 pg FOCC) and incubated for approximately 5 min. until the color appeared. The reaction was terminated by the addition of 10 pg of 1% NaN3- The samples were diluted 1: 5 with water and measured at 415 nm.

11.2. Results and Discussion

Antiserum from rabbit 4 (Lega 4) showed the highest titer (Fig.7c) and was used in further experiments. Antibodies from serum 4 were partially isolated by ammonium sulfate precipitation; and an equal volume of saturated ammonium sulfate was slowly added to a portion of the antiserum with stirring, and the solution was stirred for an additional 15 min and then centrifuged at 2000 g. The precipitate is washed twice with 50% saturated ammonium sulphate and then redissolved in a volume of PBS corresponding to the initial volume of serum. Ammonium sulfate was removed by dialysis with several changes in PBS. The dialyzed solution was dispensed into 1 ml volumes and lyophilized using a high-speed vacuum evaporator. An antibody sample from serum 4 was resuspended in 0.5 ml water and assayed for reactivity to the B5 peptide by ELISA. The reaction was detected at a 1: 4000 dilution.

Polyclonal antibodies against synthetic peptide (B5) corresponding to the porcine BDNF fragment of 33 amino acids were generated by immunization in rabbits as described above. Serum 4, showing the highest titre against the synthetic peptide, demonstrated reactivity against purified BDNF from porcine brain by ELISA (Fig. 7b). Weak reactivity was also detected by immunoblotting (data not shown). However, antiserum failed to block BDNF activity in bioassays with sensory neurons of the dorsal root of the chicken embryo.

12th Example: New biological effects of BDNF

These results indicate that BDNF can:

(I) maintaining survival and eliciting a fully differentiated state in dopaminergic CNS neurons;

(II) maintaining survival of cholinergic CNS neurons;

(III) Inhibit the proliferation of astroglial cells. These biological effects of BDNF have not been previously described. Because dopaminergic neurons, cholinergic neurons, and astroglial cells may be associated with neural diseases or disorders, BDNF may be useful in the treatment of neuropathologies involving these cell populations.

12.1. Materials and Methods

12.1.1. Methods for culturing Suhstantia nigra dopaminergic neurons

The ventral midbrain was removed by dissection from the rat embryonic brain of all ages, from day 13 embryonic development (E13) to day 15 embryonic. Usually, two replicates were used in each experiment. The dissection solution had the following composition: NaCl, 138.8 mM; KCl, 2.7 mM; Na 2 HPO 71-120, 80 mM; KH2PO4, 1.5 mM glucose, 6 mg / ml; BS A 0.1 mg / ml, pH 7.4. After preparation, this solution was sterilized through a 0.2 µm filter. The dissection was performed under non-sterile conditions. When the tissues were extracted from the entire brain, the remaining procedures were performed under sterile conditions. Fabric fragments were placed in a 35 mm culture dish and shredded with small scissors. Subsequently, 2 ml of culture medium F-12 containing 0.125% trypsin was added to the tissue and incubated at 37 ° C. At the end of this incubation period, DNA HI is added to the suspension to a final concentration of 80 ng / ml. Another identical incubation was performed and the tissue suspension was then added to 8 ml of growth medium consisting of minimal medium (MEM) with 2 mM glutamine, 6 mg / ml glucose, 5 units / ml streptomycin and 7.5% fetal calf serum (FCS). The sample is centrifuged on a centrifuge table at room temperature at 300 rpm for 5 minutes. The medium was aspirated and 2 ml of growth medium added to the cell pellet. Cells were triturated eight times with a pipette having a 1 mm opening. The remaining cell fragments were allowed to settle under the influence of earth's gravity and a larger sample of the supernatant was counted, using a hemocytometer, for quantification of the cells. After determining the cell density, these cells were transferred to culture plates, maintaining a density of 50,000 cells per square centimeter.

Culturing dishes are prepared the day before dissection. Tissue dishes (24 wells, 2 cm2 for each well) were pre-coated with polyiornithine (molecular weight 30000-70000 g / M) at 0.5 mg / ml for 3 hours at room temperature. Plates were rinsed extensively with water and then treated with mouse laminin, 5 mg / ml at room temperature for 3 hours. The dishes were then rinsed with water as above and incubated in growth medium until 37 ° C in the morning in a humidified atmosphere of 5% CCU, 95% air. The medium was removed from the vessels the following day and replaced with fresh growth medium. When cells were placed in growth dishes, they were transferred to an incubator at 37 ° C and 5% CCU (95% air) for 24 hours. Growth medium was changed to 1: 1 (v / v) sulfur free (SFM) Bass eagle Medium medium and F-12 medium with glucose (33 mM), glutamine (2 mM), NaHCO₃ (15 mM), HEPES (10 mM) with the addition of insulin (25 µg / ml), transferrin (100 µg / ml), putresin (60 µg / ml), progesterone (20 nM), sodium selenite (30 nM), penicillin (5 units / ml), streptomycin (5 mg / ml), and T₃ (30 nM). In some experiments, purified NGF and BDNF were added to the cultures after two days of culture change.

Solutions used for dopaminergic neuronal growth were prepared using water from the Milli-Q reagent water system. The tissue culture medium mixture was obtained from Gibco Laboratory, Santa Clara, California, as well as calf lethal serum (lot number 43N1086) and mouse laminin. All other media components were purchased from Sigma Chemical (St. Louis, MO) and were of the cell culture reagent type tested. Poliornithine and DNAH I were also obtained from Sigma. Trypsin was obtained from Worthington (Freehold, NJ), lot number 3667. Commercial chemicals were of analytical purity and purchased from Baker Chemical (Philipsburg, NJ). The BDNF used in the experiments was isolated from pig brain by dr. Barde, according to his own methodology (YA Barde, 1982, see above).

12.1.2. Immunocytochromic staining of cultures from ventral midbrain

Fresh fixative solutions were prepared for each experiment. When stained with tyrosine hydroxylase (TH), the fixative was 4% paraformaldehyde in phosphate Sorenson buffer. Sorenson buffer was prepared by adding 0.2 M KH2PO4 solution to 0.2 M NasPCU until pH 7.3. Subsequently, paraformaldehyde was added to the solution and briefly heated to dissolve and cooled to room temperature before use.

For the start of the procedure, the culture medium was carefully aspirated from the culture vessels and carefully added with the appropriate fixative solution. Incubate for 20 minutes at room temperature. This was followed by 3 rinses in Sorenson's phosphate buffer, 5 min each. each, easily turning. The cells were then incubated for 30 min in quenching solution at room temperature with gentle agitation. Extinguishing solution for culture staining with TH, containing Sorenson phosphate buffer containing 2% normal horse serum. The cultures were then incubated in permeation buffer for 30 min. at room temperature with gentle stirring. For cultures stained with TH, the solution was composed of Sorenson buffer with 0.2% saponin and 1.5% normal horse serum. Following the permeation step, cultures were incubated with primary antibodies until 4 in the morning. Antibodies to rat TH are murine monoclonal antibodies of the IgG2a isotype. Their concentration was 40μ g / ml in 10 mM Na₃PO₄, 50 mM NaCl in 0.2% saponin solution at pH 7.5. After incubation with primary antibodies, the cultures were washed three times for 15 min. each time in the respective increasing bandwidth buffer. The cultures were then incubated with secondary antibodies conjugated to biotype, namely, biotyped equine anti-mouse IgG. This incubation was for 2 hours. with gentle stirring at room temperature. Next, washings as described above were followed, followed by incubation of the culture with a preformed horseradish peroxidase x-biotyped complex (reagent ABC, Vector Laboratory, Burlingance, CA) prepared according to the manufacturer's instructions. After 30 minutes incubations at room temperature with gentle mixing were followed by washing the cultures as described previously. The cultures were then incubated with 55 mM Tris-HCl containing 0.5 mg / ml diaminobenzidine and 0.01% hydrogen peroxide, pH 7.5. The reaction product was allowed to develop for 2-5 minutes and then the solution was removed and the cultures washed several times with ice-cold PBS. Then determine the number of positive cells per square centimeter.

Paraformaldehyde and glutaraldehyde were obtained from FLUKA Chemical. Vectastain kits containing normal antiserum (used as a blocking reagent), biotyped affinity purified anti-immunohemoglobulin, avidin DH, and biotyped HRP-H were purchased from Vector. Diaminobenzidine was obtained from BRL (Gaithersberg, MD).

12.1.3. Methods used to determine ^ H-dopamine intake in the ventral midbrain.

Studies of intake of ^ H-dopamine (^ H-DA) were performed according to Dal Togo et al. (1989, J. Neurosci 8: 733-745) with minor modifications. The buffer for this purpose was composed of: NaCl 136.8 mM, KCl 2.7 mM, Na2HPO₄* 7H2O8mM, KH2PO4 1.5mM, glucose 5.0mM, CaCl2 1.0mM, MgSO4₄ 1.0 mM, ascorbic acid 0.1 mM, pargyline 0.1 mM, pH 7.4. 5.0 μΜ BZT (benzotropin mesylate) is added to this buffer as necessary.

Cells were rinsed once with preheated buffer (37 ° C) and poured into 2 cm² wells of 0.4 ml of this buffer. The cultures were then allowed to pre-incubate for 5 min at 37 ° C. At the end of this pre-incubation, 0.1 ml was added to the buffer (230 nm, 40 ° C (mM)).³H-DA so that the final concentration of H-DA in the buffer is 50 nM. The culture was incubated for 15 min. at 37 ° C and then washed 4 times with 0.5 ml of the described buffer at 4 ° C. Two additional washes were performed with ice-cold PBS (10 mM NaaPCL, 150 mM NaCl, pH 7.6).

After the final wash, 0.2 ml of 0.2 M NaOH was added to the cells in the 2 cm wells and allowed to stand at room temperature for 2 hours. Thereafter, NaOH extract was collected and counted in a scintillation counter (Pakkard, LS 500 TD) with 10 ml of Ultimagold scintillation fluid. Specific consumption calculation was performed by discontinuing consumption with 5 μ 5 BZT. Typically, this accounted for 70-90% of consumption.

^ H-DA was obtained from NEN (Boston, MA). Ascorbate, pargyline, BZT, and glucose were obtained from Sigma (St. Louis, MO). Ultragold Scintillation fluid was purchased from Pakkard (Seterling, VA).

12.1.4. Methods for obtaining cultures of the basal terminal brain cholinergic neuron

Primary cultures of basal terminal brain cholinergic neurons were obtained from 17-day-old rat embryos. Specifically, the cholinergic neurons used in the present study were derived from the medial septal nucleus and the diagonal Brock ribbon nucleus. This neural population first penetrates the ammonium horn. Dissociators received mixed cultures (neurons and glia) in this way.

The septum was released by dissection from the surrounding tissue and removed from the fetal brain. The tissue pieces were then harvested, scissored and treated with 12.5% ​​trypsin for 20 min. at 37 ° C. Trypsin was inactivated by dilution in vessel medium (Dulbeck's modified MEM medium (DMEM), containing 1% penicillin and streptomycin, 5% horse serum, and 1% N5-hormone supplement). The cell suspension (alone) was obtained by triturating the fragmented tissue fragments with a Pasteur pipette. Dissociated cells were counted using a hemocytometer and distributed into vessels of appropriate density for the culture medium. 3-6 hours later. after seeding, Ugandan test cultures were added to the cultures, and the cells were then grown in vitro for 10 days by changing the smear every 3 days.

12.1.5. Cholinacetyltransferase assays

After treatment, the cells were either used in experiments with CAT enzyme (cholinacetyltransferase) or stained immunologically with CAT according to this protocol. Monoclonal antibodies to CAT were purchased from Beringer Manngeim Biochemical Co. At the end of the experiment, the cells were washed twice with DMEM. The culture was fixed in two steps. 50 μΐ of 4% paraformaldehyde was added to 50 μΐ of DMEM and incubated for 10 min. This solution was removed and replaced with 100 μΐ 4% formaldehyde and incubated for 30 min. at room temperature. After fixation, the cells were washed 3 times with PBS buffered with phosphate and made permeable by incubation with 0.5 mg / ml saponin for 30 minutes. Detergent was removed by three washes with PBS and added blocking 5% rabbit normal serum solution for 30 minutes. After removal of the blocking solution, primary antibodies diluted 1: 3 in 1% rabbit normal serum were added and the culture was incubated at 4 ° C until morning. This solution containing the primary antibody was removed by washing with PBS. Bound immunoglobulin was detected by the Vectastain ABC method. Diaminobenzidine tetrahydrochloride (DAB) was used as a substrate for the peroxidase reaction, which was usually carried out for 1-5 minutes. The reaction was terminated by rinsing the cultures twice with 0.1 M Tris-HCl pH 7.2. The culture was stored in 30 mM Tris containing 0.15 M NaCl, 42% glycerol, and 0.15% Zephiran (Pierce Chemical Co., Rockvills, IL) at 4 ° C, pH 7.6.

12.1.6. A method for generating pure cultures from astroglial cells

Pure glial cell cultures were typically prepared by the method of McCarthy, KD and DeVellis (McCarthy, KD and DeVelles, J. 1980, J. Cell. Biol. 85: 890-902) from the first or second postnatal day-old rat ammonium horn. Ammonium horns were removed by 5 pups and shredded with scissors. The tissue pieces are then exposed to 2 ml of 0.125% trypsin for 20 min at 37 ° C. Protease was inactivated by dilution in medium (10% calf fetal serum (Gibco), DME, 0.5% penicillin (3000 mcg / ml), and 0.5% glutamine). The only cell suspension was obtained by passing digested tissue fragments through a Pasteur pipette with an overlay. Cells were separated by centrifugation for 5 min. at 900 rpm, resuspended in plate medium and counted on a hemocytometer. The single cell suspension was divided into three portions, each in a tissue culture dish containing 75 cm 2 area and cultured to approximately 80% confluency. The cells were then re-cultured using a trypsinization method similar to that just described. Glial cells were counted and placed in dishes, maintaining a density of 10,000 cells per 0.9 cm 2.

12.2. Results

12.2.1. Effect of BDNF on tyrosine hydroxylase in ventral midbrain cultures

Immunocytochemical staining as described in 12.1.2 above. , used to evaluate the effect of BDNF on cells having a positive response to tyrosine hydroxylase (TH) (Fig. 8). Maximum increase of more than 200% relative to control was detected on day 8 in ventral midbrain cell cultures stimulated with BDNF. Earlier on day 3, only very slight increases were observed in BDNF-stimulated cultures.

12.2.2. Effect of BDNF on dopamine intake in secondary midbrain cultures²H-dopamine (²H-DA) consumption was measured by Dal Taco et al. (1983, J. Neurosci 8: 733-745) with minor alterations as described in 12.1.3 above. section. A slight increase in dopamine uptake was detected on day 8 of cultivation in ventral midbrain cultures stimulated with BDNF (Fig. 9).

12.2.3. Effect of BDNF on terminal brain cholinergic neuron expression of cholinacetyltransferase

FIG. Figure 10 a shows the effect of BDNF on the number of CAT-positive cells after 12 days of in vitro cultivation. A 5.9-fold increase in CAT cells was detected with the addition of 100 ng / ml BDNF, and the calculated EC50 value was 10 rtg / ml. Cultures having a density of 26000 (black band) or 150000 (dotted band) cells per well, treated in the same manner but with NGF only, were used as positive controls (Fig.10 b). The density, 260000 cells / well, corresponds to the density used for the BDNF assay. This increase in the number of CAT-immuno-positive cells is similar to the increase reported previously. The potential ability of BDNF to act on cholinergic neurons was also tested by measuring CAT-enzymatic activity (F. Fonnum; I. Neurochemistry, 1975, 24: 407-409). Figure 11 shows the CAT lesions obtained with BDNF treatment. In this case, a 1.8-fold increase was obtained with 100 ng / ml BDNF and the calculated EC50 was 61 ng / ml.

12.2.4. Effect of BDNF and EGF on astroglial cell cultures

Type II astrocytes have been shown to have high affinity for receptors from the set of neurotransmitters and neuropeptides. Yes, astrocytes can respond to signals of neuronal origin. For this reason, and because type II astrocytes are the cellular component of primary cultures, the direct effect of BDNF on glial cells was investigated. Prior to addition of growth factor, these cells were kept for 4 days in vitro to reach 60% confluency. followed by 42 hours of processing. with NGF and BDNF. After 18 or. incubation in medium added [²H] methylthymidine. The effect of EGF is shown in Figs. 12th century As announced above, EGF was found to be a mitogenic factor in astrocytes. Maximum response was detected in the presence of 10 ng / ml EGF which gave a 5: 2-fold uptake of pFI] methylimidimidine. FIG. 12 b. shows the effect of BDNF on pH] methylimidimidine uptake. The response to BDNF was biphasic: very low doses (0.1 ng / ml) resulted in a slight increase in thymidine uptake, and doses greater than 1 ng / ml BDNF retained pH] methyl thymidine uptake. A 5 ng / ml dose of BDNF gave 24% inhibition while increasing the glial cell proliferation rate during the treatment period.

12.3. Discussion

These in vitro experiments clearly demonstrate that BDNF maintains survival or induces complete differentiation in dopaminergic neurons from developing mouse black matter, as shown by tyrosine hydroxylase staining and changes in dopamine uptake in cultures of rat embryonic midbrain. Because it is these neurons that degenerate in Parkinson's disease, it is highly likely that BDNF may have therapeutic potential in treating Parkinson's disease either by reducing neuronal loss, or by increasing tyrosine hydroxylase (an enzyme that limits dopamine synthesis levels), or possibly both.

In addition, like nerve growth factor NGF, BDNF acts on the survival of rat basal hindbrain cholinergic neurons, as shown by increased CAT staining, increased CAT activity, and enhanced acetylcholinesterase staining in rat embryonic medial septal nucleus and diagonal Brook Belt nuclear cultures. Accordingly, BDNF itself or in combination with NGF may be useful in the treatment of diseases and disorders associated with basal terminal brain cholinergic neurons, including Alzheimer's disease.

13th Example: Identification of a new member of the BDNF / NGF genetic family

The method of identifying new members of the BDNF / NGF genetic family by PCR with degenerate oligonucleotides based on the amino acid conserved between NGF and BDNF segments (Boxes 1-4, see Section 5.8) was initially tested to determine whether both pairs of such primers could be used for amplification of both. The NGF gene contains both the BDNF gene from several species of animal genomic DNA. This method was subsequently used to identify a novel gene that has homology to NGF and BDNF in all of the four boxes mentioned above.

13.1. Materials and Methods

13.1.1. Polymerase chain reaction

The PCR reaction was performed essentially as described in section 6 above.

13.2. Results

13.2.1. Amplification of NGF and BDNF sequences from genomic DNA

The congenital synthetic oligonucleotide primers were synthesized by the boxes 1 and box 2 segments conserved between NGF and BDNF amino acid sequences (see section 5.8 above) and were used in the PCR reaction with rat genomic DNA as a template. The exact primer sequences were as follows (degenerate positions with a mixture of two or more bases activated at the oligonucleotide synthesis stage, shown in parentheses; underlined tails with restriction endonuclease cleavage multiple sites facilitating incorporation into vector j in the subsequent cloning step; A = adenine, G C = cytosine, T = thymine and N = mixture of A, C, G, T):

Boxing 1 (Sense), Primer IB:

5-GACTCGAGTCGACTCGGTGTG (C, T) GACAG (C, T) (AG)

T (C, T, A) AG-3 'boxing 2 (anti-spasmodic), primer 2C:

5-CCAAGCTTCTAGAATTCA (C, T) TT (N) GT (C, T)

TC (A, G) (A, T) A (A, G) AA (A, G) TA

300 ng of the mixture with each degenerate primer is added to 300 ng of rat genomic DNA in 10 μϊ standard PCR reaction mixture. 35 cycles of 1 min each were performed. incubation at 94 ° C, 2 min at 43 ° C and 2 min at 72 ° C. The expected product size for BDNF and NGF gene amplification by PCR using these primers was 175 base pairs, including two 17-tail (underlined) tails turned on for convenience for further cloning steps. Electrophoresis of the reaction mixture on 8% polyacrylamide (5% glycerol) gave the backbone of the amplified DNA with the expected size, 175 pairs of bases.

13.2.2. Detection of sequences complementary to BDNF / NGF probe from various animal basement DNA

175 The bp band was removed from the gel by electroelution and further amplified, in a second PCR reaction, under a seven-cycle reaction condition identical to that of the first PCR except that the concentrations of dGTP, dATP, and TTP were reduced to 50 μΜ, and . ^ ορρ. The radiolabeled DNA product was isolated from the reaction mixture by chromatography on an even column. This probe, labeled with RIB / 2C (rat DNA amplified from primer IB and 2C), was then used to detect complementary sequences of various animal genomic DNA (results obtained in rat, mouse and chicken are shown in Fig. 13), after digestion with Eco RI. restriction endonuclease and blotting with nitrocellulose by Southern hybridization with genomic DNA digested with EcoRI as shown in Figs. 13th

The sizes of EcoRI fragments from genomic DNA containing NGF and BDNF sequences were determined in controls in parallel blots using radiolabeled human NGF and BDNF probes obtained from the cloned genes by PCR. The positions of NGF and BDNF genomic EcoRI fragments are shown in Figs. 13 as N and B, respectively. Results of similar analysis are shown in Fig.3 and equivalent results were obtained with the human probe as with the porcine BDNF probe Fig.3 As shown above, NGF and BDNF probes crossed with a single EcoRI fragment of various vertebrate genomic DNA. For example, the rat DNA BDNF test detected a band of approximately 8.8 kb, whereas the NGF test detected a band of about 10.4 kb.

As shown in Figs. 13 for all species tested (chicken, mouse, and rat data), the RIB / 2C probe crossed with the DNA strand indistinguishable from the NGF probe as well as the DNA band indistinguishable with the BDNF probe (mouse NGF and BDNF genomic). EcoRI fragments have the same electrophoretic motility (approximately 11.3-12.0 kb). This indicates that the abnormal oligonucleotide primers IB and 2C can be used to amplify sequences from both NGF and BDNF genes. It should be noted that in some cases additional bands have been observed in the genomic Southernblotting hybridized with the IB / 2C probe. For example, mouse genomic DNA cleaved with EcoRI contained at least two additional bands (labeled XI and X2, approximately 10.0 kb and 1.5 kb, respectively) that did not correspond to either NGF or BDNF. Similarly, at least two additional bands were detected by hybridization with rat DNA (XI, X2 approximately 7.3 and 1.2 kb, respectively), and at least one was chicken DNA (X, approximately 2.6 kb). In some cases, additional bands not marked in this image were also detected. Detection of such bands, which are neither NGF nor BDNF, indicates the possible existence of an additional member (s) of the genetic family. Similarly, using other sets of primer pairs and genomic DNA arrays (data not shown), detected bands clearly distinct from known bands belonging to the BDNF and NGF gene sequences.

13.2.3. Identification of a novel gene, cognate BDNF and NGF

Specific testing of the hypothesis that a novel gene linked to NGF and BDNF can be identified by PCR using abnormal oligonucleotide primers was performed using box 3 and box 4 primers (see section 5.8 above) and mouse genomic DNA, as a matrix. The synthesized degenerate primers had the following sequences:

boxing (meaningful):

5'-GGGGATCCGCGGITG (T, C) (C, A) GIGGIAT (T, C, A) GA-3 Boxing (Anti-Prismatic)

5′-TCGAATTCTAGAIIC (T, G) IAT (AG) AAIC (T, G) ICCA-3 ′ wherein Gguanidine, A-adenine, C-cytosine, T-thymine, and I-inosine; mixtures of more than one base per position are shown in brackets. It should be noted that inosine is used in some positions corresponding to the third (unstable) base in the codon to enable the genetic code to degenerate. It is also possible to use a mixture of four conventional DNA bases instead of inosine, and essentially overlapping results were obtained with such primers.

Using a degenerate boxing 3 / box 4 primer pair, PCR with genomic NGF and BDNF sequences from mouse DNA was expected to amplify the segments at approximately 30 bp. Using the primers shown above, the PCR reaction was run for 4 cycles at 45 ° C and then 31 cycles at 49 ° C. The products were analyzed by gel electrophoresis, and the main band of expected size was observed. The mouse NGF gene has a restriction endonuclease Hind II cleavage site in the box between 3 and box 4, while the BDNF has an ApaI cleavage site in this region. Therefore, it was expected that cleavage of the PCR amplification product with HindIII and ApaI could remove the NGF and BDNF sequences from the parent product band. However, when the PCR product was completely cleaved by these restrictionases, it remained in the amplified DNA. This suggests that at least one new gene has been amplified in addition to NGF and BDNF genes.

13.2.4. Characterization of a New Genetic Family Member

The cleavage-resistant PCR product was gel-washed and used as a template in asymmetric PCR reactions in which one of the original degenerate primers was 10-100 times higher in molar concentration than the other primers. This asymmetric amplification allowed the production of single-stranded DNA arrays suitable for nucleotide sequencing by strand termination. Sequence analysis of the novel gene (designated herein as No 3/4 but also referred to as neurotrophin-3) was further expanded by PCR amplifying between the exact primer located between box 3 and 4 and the poly-A sequence at the 3 'end of the transcript, using the strategy for rapid cDNA amplification (RAGE) described by MA Frahman, MK Dush, and GR Martin, Proc. Natl. Acad. Sci. USA, 85: 8998-9002 (1988). DNA nucleotide sequencing revealed that a novel gene with an open reading frame capable of encoding a polypeptide different from NGF and BDNF but highly related to its amino acid sequence was amplified (Fig. 14).

Initial confirmation that this novel gene encodes a neurotrophic factor was obtained by determining its paternity of expression in rat tissues by northern81 blot hybridization. This analysis showed that this new gene is expressed much more strongly in brain tissue than in any other tissue examined.

13.3. Discussion

As shown above (see Fig. 13), the DNA probe obtained by PCR amplification using boxing primers 1 and 2 with rat genomic DNA as a template (RIB / 2C) crossed with new bands known to contains NGF and BDNF gene sequences from each of the animal species studied, digested with Eco RI. A similar assay was performed using a radiolabeled new gene probe amplified using primers Box 3 and 4 with mouse genomic DNA (M5 / 4). In each case, the M5 / 4 probe hybridized to the single major strand of genomic DNA cleaved with EcoRI and different from the band containing NGF and BDNF sequences. It should be noted that EcoRI fragments derived from mouse, rat, and chicken genomic DNA, whose hybridization with the M3 / 4 probe was monitored, always coincided with one of the new bands obtained by crossing with RIB / 2C. These were the fragments: a 19.0 kb EcoIR fragment from rat DNA (XI from Fig. 14), a 7.3 kb fragment from rat DNA (XI from Fig. 14), a 2.6 kb ribbon from chicken DNA (X from Fig. 14). 14). This suggests that portions of the same gene were amplified from rat DNA using primer pair IB / 2C and from mouse DNA with primers 3 and 4. So at least one new gene has homology to NGF and BDNF in all four homology boxes, described above.

The idea of ​​homologous boxing between NGF, BDNF, and an additional member of the genetic family (e.g., M3 / 4, also known as NT-3) has been expressed herein by means of primary amino acid sequences and by novel methods of genetic identification of the family. However, it is important to keep in mind that the secondary and tertiary structure of these neutron factors, their interaction with specific factors, is likely to be additionally included in order to rationally construct new molecules with potential therapeutic value.

For example, NGF has 6 cysteine ​​residues, all of which are shown to be involved in the formation of disulfide bridges. Numbering them from the N-terminus to the C-terminus as cisl-cis 6 gives disulfide bridges eis l-cis-4, eis 5 and eis 3- eis 6. All six cysteine ​​residues are conserved between NGF and BDNF, and three cysteine ​​residues in segment M3 / 4 positioned exactly as NGF and BDNF will go 4, go 5 and go 6. This suggests that there is a close relationship between the secondary structure of all members of the ghetto family, which is mainly determined by the preserved cysteine ​​residues. It should be noted that the aforementioned NGF and BDNF homology boxes contain 5 of the 6 cysteine ​​residues (will go in box 1 1, will go in box 2 2 will go in 3 box 3 and will go 5 and will go in 6 box 4). This supports the idea that the remains and their immediate neighbors play an important role in the overall structure of the neurotrophic factor. These structural determinants, which determine the high affinity for specific interaction with receptors believed to be involved in each neurotrophic factor, are located at unique sites in the molecule.

Accordingly, the novel chimeric genes may be obtained by recombination between family members (e.g., by in vitro recombination or direct gene synthesis) in any of the four homology boxes already described, or in any other molecule. Such chimeric proteins appear to have similar secondary structure due to conserved eis residues and other amino acid residues, but may have novel biological properties. For example, BDNF / NGF chimeric proteins may be bifunctional in terms of interaction with BDNF and NGF receptors. Chimeric proteins may also differ from the parent molecule (or molecules) in their two-dimensional structure and other physico-chemical properties. Chimeric proteins can also function as antagonists of any of their parent molecules.

Active BDNF / NGF fragments can be used to build other family members based on knowledge of critical core regions for appropriate folding, plus information on what areas are required for specific receptor interaction. Comparison of new family members with existing ones can be used to detect new homology boxes , which can assist in the search for additional members of the BDNF / NHF genetic family. For example, comparison of M3 / 4 with BDNF allowed the detection of some useful homology boxes. One of them, particularly interesting, has a fourth conserved ghost remnant, the only one not found in the 1-4 boxes described above. The segment containing that eis residue is actually a sufficiently long identity segment or segment with the conserved amino acid sequence of BDNF and M3 / 4, namely: His Trp Asn Ser Gln Cys / Arg or Lys / - Thr / Thr or Ser / - Gln / Ser or Thr-Tyr-ValArg-Ala-Leu-Thr. Within this segment, at least two homology boxes can be selected useful for the synthesis of degenerate oligonucleotides (e.g., His-Trp-Asp-SerGln-Cys requires 36-fold degeneration for 18-primer or 48-fold for 17-fold, a useful box would also be useful). and Tyr-Val-Arg-Ala-Leu-Thr).

14th Example: Increased BDNF expression in neuroblastase cells

14.1. Materials and Methods

14.1.1. Cell linen

CHP100, CHP126, CHP134, CHP234, LANI, LAN5, NB9, SV5V, V79, FO1, BU2, HO1, HL60 and COL 320 are cell lines maintained in the laboratory by dr. Fred Alt, who gave the DNA for Northern blotting (Fig. 15). All cell lines are from human tumors. CHP100 cell line from neuroepithelioma, CHP126, CHP134, CHP234, LANI, LAN5, NB9 and SV5V - neuroblastoma cell line, V79 - retinoblastoma cell line, FO1, BU2, HO1 melanoma cell line, HL60 - promyelocytic leukemia cell line, COL 320 - colon cell line of neuroendocrine carcinoma of the gut.

14.1.2. Preparation of RNA

RNA preparation and Northern blotting were essentially the same as in 8.1.1. see chapter. above using a full-length human cDNA probe. 10 µg of total RNA was used in each gel track for Northern blotting (Figure 15), except that RNA was less loaded on the LANI track and 1 µg of poly (A) was used for SV5V.⁺ RNA.

14.2. Results

FIG. Figure 15 shows the results of northern blot analysis of a BDNF probe cross-linked with a panel of RNA samples from multiple human cell lines. High levels of RNA crossed with the BDNF probe were detected in CHP234 and LAN5 cell lines, and smaller amounts were detected in CHP126 and CHP134. All positive lines were derived from human neuroblastoma tumors.

15th Example: Rat ammonium horn BDNF and NGF RNA regulation, which is activity dependent, in the presence of non-NMDA glutamate receptors

15.1. Materials and Methods

15.1.1. Treatment of rats with kaolinic acid

90 minutes after depression of locomotor activity, rats were usually given diacepam (valium) after administration of cinic acid (12 mg / kg body weight) to the abdomen. As shown in Fig.19 valium administered after cainic acid did not affect the subsequent increase in BDNF and NGF mRNA levels. Rat ammonium rage, 3 hours later. after administration of cinic acid, and without Valium, had similar increases in BDNF and NGF mRNA levels compared to animals receiving cinic acid followed by valium.

15.1.2. Preparation of ammonium horn cell cultures

Ammonium horn brains from rat E17 embryos were removed by dissection and incubated for 20 min. at 37 ° C in PBS in PBS phosphate buffer, free from calcium and magnesium ions containing 10 mM glucose, 1 mg / ml albumin, 6 µg / ml DNA 'and 1 mg / ml papain. After flushing with papain-free solution, ammonium horn cells were carefully dissociated with a drained Pasteur pipette. Cells were harvested by low speed centrifugation, resuspended in DMEM solution, added 10% calf fetal serum, and seeded in plastic culture plates at a density of 0.5 x 10 4 cells in 35 mL pre-coated with poly-DL-ornithine (0.5 mg / mL) and laminin (5 g / ml). After 3 hours. after sowing, the medium was changed to a sulfur-free medium which had additives as described by Brewer and Cotman (Brain Res. 497: 65, 1989) but without glutamate. Neurons remained viable in culture for up to 3 weeks and were typically used in experiments 7 days after seeding.

15.1.3. RNA amplification

Total cellular RNA was extracted as described by Chomcynsky and Sacci (Anai. Biochem 1987, 162: 156-159) from 0.5 x 10 4 cells after addition of a truncated RNA repair standard (FIG. 30). NGF mRNA and cRNA were amplified together in a reverse transcription reaction / polymerase chain reaction (RT / PCR) pool containing 1/5 extracted RNA, 1xRT / PCR buffer (10mM Tris-HCl pH 8.5, 10 mM KCl, 1.5 mM MgCO, 0.1 mg / ml gelatin 0.1% triton X-100), 0.25 mM dNTP, 0.1 µg of each of the 5 'and 3' primers, 5 units. RNAse (Promed), 3.2 pcs. AMU reverse transcriptase (Life Science) and 2 pc. Taq polymerase (Genefit) in a total volume of 25 ml. This mixture was coated with mineral grease and incubated for 30 min at 41 ° C, heated to 92 ° C for 60 seconds, and renatured primers at 35 ° C for 60 sec. and expanded the primers at 72 ° C for 60 sec. Amplification products (203 bp from NGF mRNA and 153 bp from repair standard) separated on 3% Nusieve / Agarose 3: 1 gel (FMC Bioproducts), alkaline blotting with Hybond N-flow membrane (Amersham), and cross-linked as described (Haumann and Thoenen). 1986, J. Biol. Chem. 261: 9246; Lindhold et al., 1988, J. Biol. Chem. 263: 16348). For absolute quantification, known amounts of transcribed in vitro mRNA and recovery standard were co-amplified in parallel reactions, as recently described by Wang et al. (PNAS 86: 9717-9721, 1989).

15.2. Results and Discussion

BDNF and NGF are members of a genomic family with approximately 50% identical amino acid sequence (Leibrock et al., 1989, Nature 341-149). These molecules have tightly conserved domains. The 6 cysteine ​​residues inside these domains are likely involved in the stabilization of the three-dimensional structure of these molecules, which is necessary for their biological activity. However, NGF and BDNF also have different domains that determine their different neuronal specificity (Lindsay et al., 1985, Dev. Biol. 112: 319; Jonson 1986, J. Neurosci 6: 3031; Hafer and Barde 1988, Nature 331: 261; Rodriguez - Tebar et al., 1989, Dev. Biol. 136: 296). Furthermore, the difference between these neurotrophic molecules is confirmed by their sites of synthesis, NGF is expressed both in the periphery (Korsching, S and Thoenen, H. 1983, Proc. Natl. Acad. Sci. USA, 80: 3513; Ebendal et al., 1983, Exp. Cell Res 148: 311; Heimann et al., 1984, EMBO, 3: 3183; Dhelton and Richardt, 1984, Proc. Natl. Acad. Sci. USA, 81: 7951), as well as in the central nervous system (Korsching et al., 1985, EMBO, 4: 1389; Shelton and Reichardt, 1986, Proc. Natl. Acad. Sci. USA, 83: 2714; Whittemore, 1986). Proc Natl Acad Sci USA 83: 817; Large et al. 1986 Science 234: 352). Innervation density in neurons responsive to NGF reflects the amount of NGF present in the respective tissue tissues (Korsching and Thoenen 1983, Proc. Natl. Acad. Sci. USA 80: 3513; Ebendal 1983, Exp. Geli. Res. 148: 311; Heimann et al., 1984, EMBO, 3: 3183; Dhelton and Richardt, 1984, Proc. Natl. Acad. Sci. USA, 81: 7951; Korsching et al., 1985, EMBO 4: 1389, Schelton and Reichardt, 1986, Proc. Natl. Acad. Sci. USA 83: 2714, Whittemore et al., 1986, Proc. Natl. Acad. Sci. USA 83: 817; Larg et al., 1986, Science, 234: 352). Unlike NGF, BDNF is predominantly expressed only in CNS neurons and contains BDNF mRNA levels much higher than NGF mRNA levels, for example, approximately 50-fold in ammonium horn. In the peripheral nervous system, NGF is synthesized by various types of nerve cells (Bandtlow et al., 1987, EMBO, 6: 891), whereas in the brain, NGF is predominantly localized in neurons, as demonstrated by in situ crossover (Rennert and Heinrich 1986, Biochem. Biophys. Res. Commun 138: 813; AyerLezievre et al., 1988, Science 240: 1339; Whittmore et al., 1988, J. Neurosci. Res. 20: 403). However, cultured astrocytes of type 1 as shown also produce significant amounts of NGF (Lindsay 1979, Nature 282: 80; Furukava et al. 1987, Biochem. Biophys. Res. Com. 142: 395). The relative contribution of neurons and astrocytes to NGF synthesized in the brain has not yet been elucidated. Considering the central expression of BDNF and NGF mRNAs in the central nervous system, the following questions were investigated: whether neuronal activity can influence the levels of these two RNAs and, if so, which transmitter (s) may be involved in regulation. In the first set of experiments, neuronal cultures were prepared from the ammonium horn of rat E17 embryos. As shown in Figs. 16a. depolarization of ammonium horn neurons induced by high (50 mM) potassium increased BDNF mRNA levels. Peak levels were reached between 3 and 6 hours after potassium elevation. The potassium-induced increase in BDNF mRNA could be suppressed by removal of potassium ions from the medium and reduced by inhibiting calcium entry by nifodipine, which blocks the transport pathways (Fig. (Fig.16b). 16b).

Because the human ammonium horn cultures used were composed of a mixed population of neurons with different transmitter and receptor expression paternas, the effect of different physiological and synthetic receptor agonists on BDNF and NGF expression was investigated here. The results, shown in Table VI, indicate that of all the substances tested, cinic acid, a glutamate receptor agonist (Managhan et al. 1989, Annu. Rev Pharmacol Toxicol 29: 365), gave the highest BDNF mRNA increase in ammonium horn neurons to date. In contrast, other substances, such as carbaxol (a muscarinic receptor agonist) and, to a lesser extent, histamine and bradykinin, only slightly, although appreciably, increased BDNF mRNA (Table VI). Because NGF mRNA levels in ammonium horn neurons are very low, a quantitative polymerase chain reaction method was developed here to detect NGF mRNA abnormalities. Using this method, BDNF mRNA levels, as well as NGF mRNA levels, were increased in ammonium horn neurons with potassium and cinic acid (Fig. 17).

As shown in Figs. 18, maximal BDNF mRNA increase in ammonium horn neurons was obtained with cinic acid at a concentration of 25 μΜ. Further elevation of its concentration reduced BDNF mRNA levels, which probably reflects the toxic effect of high concentrations of cinic acid on ammonium horn neurons (Fig. 18). Neurotoxicity mediated by the glutamate receptor has previously been reported (Chai et al., 1978, J. Neurosci. 7: 357; Rothman, and Olney, 1987, Trends Neurosci, 10: 299, Chai 1988, Neurosci, 1: 623) for various CNS neurons after the use of analogues of excitatory amino acid glutamate. However, the cinic acid concentrations required to enhance BDNF and NGF mRNA expression in ammonium horn neurons can be accurately distinguished from those causing neurotoxicity.

In order to further investigate the action of cainic acid, it was investigated here whether blocking the increase in BDNF mRNA levels by any of the known glutamate receptor antagonists (Managhan et al., 1989, Annu. Rev. Pharmacol. Toxicol 29: 565). Kinurenic acid, a glutamate-receptor broad-spectrum antagonist, as well as CNQX, a competitive non-NDA receptor inhibitor (Managhan et al., 1989 Annu. Rev. Pharmacol. Toxical.29: 365), completely blocked the cinic acid-induced increase in BDNF mRNA in ammonium horn neurons (Table VII). On the other hand, MK-801, which specifically blocks MNDA receptors, could not block BDNF mRNA increase in these neurons. In addition, the NMDA itself did not alter BDNF mRNA levels (Table VI). This means that cinic acid acts directly through its receptors and that this effect is not due to the release of endogenous glutamate (which also acts on NMDA receptors). This suggests that kaolinic acid raises BDNF mRNA levels in vitro by utilizing non-NMDA receptors.

Table VI

Effects of various receptor agonists on BDNF mRNA expression in neuronal cultures

Supplements nothing carbaxol (50μΜ) carbaxol (50μΜ) + atropine (ΙΟμΜ) nicotine (ΙΟΟμΜ) histamine (50μΜ) serotonin (ΙΟΟμΜ) dopamine (ΙΟΟμΜ) norepinephrine (25 μΜ) substance M (1 μΜ) somatostatin (1 μΜ) bradostatin (1 μΜ) μΜ) fatty acid (25 μΜ)

NMDA (25 μΜ)% from control

100±5

220+25

85±15

110±7

150±12

95±6

115±7

75±10

85±9

110±8

155±15

1365±70

105±70

Table VII

Effect of various glutamate receptor antagonists on the price of acid

<td colspan="2">induced BDNF mRNA expression</td>

<td>Accessories</td><td>% of control</td>

<td>nothing</td><td> 100±60</td>

<td>cinic acid (25 μΜ)</td><td> 1250±60</td>

<td>kynurenic acid (1 μΜ)</td><td> 70±6</td>

<td>kynurenic acid (1 μΜ) + price</td><td> 84±7</td>

<td>acid (25 μΜ)</td><td></td>

<td>CNGX (10 μΜ)</td><td> 109±6</td>

<td>CNQX (10 μΜ) -t-cinic acid (25 μ</td><td> 103±7</td>

<td>M)</td><td></td>

<td>MK-801 (5 μΜ)</td><td> 96±5</td>

<td>MK-801 (5 μΜ) + Fatty Acid (25 μ</td><td> 1130±80</td>

<td>M)</td><td></td>

To assess the physiological relevance of these in vitro studies, similar mechanisms have been investigated in vivo. Adult rats of both sexes were treated with Wistar, weighing 180-200 g, with 12 mg / kg cost acid. After various time periods, changes in BDNF and NGF mRNA levels in the ammonium ridge and cortex were determined by Northern blot analysis. Figure 19 shows that cainic acid induces an increase in BDNF and NGF mRNA levels in both brain areas. The increase in BDNF mRNA was significantly greater than the increase in NGF mRNA. In addition, mRNA levels remained elevated 24 hours after the addition of cinic acid. Variation in BDNF and NGF mRNA over time indicated that the maximal increase in ammonium rage was reached 3 hours after cinic acid administration (Fig.19). It is interesting to note that prior to the increase in BDNF and NGF mRNA levels, S-fos mRNA levels increased: these two phenomena may overlap randomly, as recently shown for sciatic nerve injury (Hengerer et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 385). In addition, there is a delay in the increase of both BDNF and NGF mRNAs in the cortex relative to an increase in ammonium horn, indicating that signals that induce an increase in BDNF and NGF expression spread from the ammonium horn to other areas of the brain. These data are in agreement with previously published data (Morgan et al., 1987, Science 237: 192), indicating that the increase in c-fos induced by convulsant metrazole occurs first in the ammonium rage and then in the cortex.

Previous studies have shown that NMDA receptors such as NGFI-A (Cole et al., 1989, Hattge, 340: 474) are involved in the regulation of ammonium horn mRNA, thus investigating whether NMDA receptor antagonists MK-801 and ketamine can block BDNF. and an increase in NGF mRNA in vivo. However, confirming the result obtained in vitro, MK-801 did not inhibit mountainous acid-induced increase in BDNF mRNA in ammonium rage, although it effectively suppressed seizures due to NMDA receptor activation (Fig.20). Most likely, this NMDA activation of the receptors results from endogenous glutamate released by cainic acid (Bizer and Koil, Neuroscience, 1988, Lett 8: 303; et al., 1973, Brain Res. 139: 381). Similarly, elevation of NGF mRNA in the ammonium horn after its treatment with kaolinic acid was not terminated by either MK-801 or ketamine. In a previous study, Gali and Isachson (1989, Science 243: 758) showed that electrolytic lesions that induce limbic seizures elevate NGF mRNA levels in rat ammonium horn. However, these results obtained with MK-801 and kaolinic acid showed the need to clearly distinguish between the increase in seizures and the increase in BDNF and NGF expression. Treatment of rats with diacepam (Valium) before injection of cainic acid completely blocked the increase of BDNF and NGF mRNA in rat ammonium rage (Fig.20). The blocking effect of diacepam on BDNF and NGF mRNA increase is likely due to inhibition of neuronal activity by the inhibitory GABA-ergic system. However, after 30 minutes. after the administration of the price acid (ie approximately 30 min. from the onset of convulsions), BDNF and NGF mRNA elevation could not be further blocked by diacepam, indicating that a relatively short period of increase in activity may be sufficient to initiate a cascade of events leading to an increase in expression of two neurotrophic factors.

This study suggested that non-NMDA receptors could be involved in the regulation of BDNF and NGF mRNAs in the ammonium horn, thereby distinguishing this regulatory mechanism from Cole et al., Previously described. (1989, Nature 340: 474), who reads that genes encoding ammonium horn mRNA are likely regulated by NMDA-subtypes of glutamate receptors. It has been reported that some of the effects of cainic acid on neurons can also be mediated by the quisqualate-type glutamate receptor (Monaghan et al., 1989, Annu. Rev. Pharmacal. Toxical. 29: 365).

Finally, the present invention showed that neuronal activity regulates the level of mRNA encoding the neurotrophic factors BDNF and NGF in rat ammonium horn and cortex. Of all the substances tested, only cinnamic acid, through its non-NMDA glutamate receptors, induced an increase in BDNF and NGF mRNA in both in vivo and ammonium horn neuronal cultures. In contrast, there was no evidence that peripheral NGF synthesis in non-neuronal cells is regulated by normal transmitter substances or neuropeptides released by innervating neurons (responsive to NGF) (Barde et al., 1984, J. Cell Biol. 99: 839; Hellweg et al., 1988, Exp. Cell. Res. 17:18). BDNF is predominantly expressed in the central nervous system and there is at least a partial overlap between the central pathways utilizing glutamate as a neurotransmitter and regions shown to be rich in BDNF and NGF mRNA (Hafer et al., EMBO) - /; Monaghan et al., 1989, Annu. Rev. Pharmacol. Toxical 29: 365). Specifically, there is a surprising similarity between the distribution of NGF mRNA as shown by in situ crossover (Haf EMBO, in press) and cainic acid receptors expressed by autoradiography in the ammonium horn, the cerebral cortex, and the cerebellum (Monaghan et al., 1989, Annu. Rev. Pharm .Toxical .29: 365). These data are consistent with the hypothesis that glutamate is a physiological transmitter that regulates BDNF and NGF mRNA in the central nervous system.

16th Example: Brain neurotrophic factor increases survival and function differentiation in rat septal cholinerginin neuronal cultures.

16.1. Materials and Methods

16.1.1. Obtaining dissociated cells and cell growth conditions

The septal area is released from the surrounding tissues in rats (Sprague - Dawley) after 17 days of gestation (day 17 of embryo E17). Tissue fragments were harvested, washed 3 times with Ham F-10 and then transferred to a 35 mm plate for tissue culture and shredded. Single cell suspension was made by incubating tissue with 0.25% trypsin for 20 min at 37 ° C. After inactivation of trypsin for 5 min. incubated at room temperature in culture medium (see. below) containing 50 µg / ml DNAse type I (Sigma) and the pieces were dissociated by periodically passing the fragments through the narrowed tip of a Pasteur pipette. The dissociated cells were then centrifuged at 300 g for 45 sec, the supernatant removed, and centrifuged again. The free cell pellet was resuspended and pooled in growth medium and the cell yield determined using a hemocytometer. Finally, these cells were seeded in 6 mm wells coated with polyiornithine (10 µg / ml) and laminin (10 µg / ml). Cell viability was checked after 24 hours. in growth culture, determining the ability of cells not to miss blue trypan.

Normal growth medium for 5HS / N3 cultures composed of neurons and glia contained: 5% (v / v) horse serum (Gibco), 1% additive No3 (v / v) (Romijin et al., 1982, DW. Brain Res. .2: 583-589), 0.5% (v / v) glutamine (200 mM, Gibco) and 0.25% (v / v) penicillin-streptomycin (10,000 units / ml and 10,000 pg / ml, respectively). ) (Gibco) in Modified Dulbeck Media (DMEM).

Neuronal enriched cultures were prepared by changing the growth medium 5-6 hours after inoculation with DMEM containing: 1% No3 additives, 0.5% glutamine and 0.25% penicillin and streptomycin. In both conditions, treatment with cytosine-arabinose (1 μΜ / 24 h) was used to limit glial cell proliferation.

16.1.2. Assay for Cholin-Aethyltransferase (CAT) Activity

Growth medium was removed from the culture by washing the cells twice with 100 μΐ PBS. Cells were lysed in one cycle of freeze-thaw and incubated for 15 minutes with 50 mM KITjPO at pH 6.7 containing 200 mM NaCl and 0.15% (v / v) Triton - 100. Two microliters of cell lysate was taken and assayed for CAT activity according to the procedure. micro-fonum (Fonnum 1975, J. Neurochem, 24: 407-409). The final mixture of materials was: 0.2 mM [¹⁴C] Acetyl-CoA / NEN 54.4 µCi / mole /, 300 mM NaCl, 8 mM choline bromide, 20 mM ethylenediaminetetraacetic acid and 0.1 mM neostigmine in 50 mM NaH 2 PO 4 (pH 7.4) ) buffer. At these enzyme and substrate concentrations, the enzymatic reaction remained linear for 90-120 minutes. The specificity of the induction of choline-ethyl transferase was tested by the addition of a specific inhibitor of CAT activity, Nhydroxyethyl-4- (l-naphthylvinyl) pyridinium (HNP), during an experiment (White, HL and Cavallito, CY, 1970, J. Neurochem 17: 1579-1589).

16.1.3. Acetylcholinesterase (AChE) activity assays

Levels of AChE in lysates were measured by Potter (Potter. 1967, J. Pharmacology and Experimental Therapeutics 156: 500-506) and Johnson and Russel (Johnson and Russel 1975, Anai. Biochem. 64: 229-238) with minor modifications.

Lysates were prepared in 50 mM KH2PO4 buffer, pH 6.7 containing 200 mM NaCl and 0.25 (v / v) Triton Χ-100, and coupled with [^ Hyacetylcholine iodide (NEN, NEN-113, 73.3 m Ci / mmole). /, Etopropavin (0.1 mM)%) at 50 mM at pH 7.0. After 15 minutes After incubation at 37 ° C, the reaction was terminated by the addition of 100 μl of stop buffer containing: 1 M chloroacetic acid, 0.5 M NaOH and 2.0 M NaCl. Specific AChE activity was determined by the addition of 1.0 (10'6) M neostigmine.

16.1.4. Measurement of high affinity uptake of choline

Choline uptake via high affinity, Well⁺ The dependent mechanism has been studied by the method of Vaca and Pilar (Vaca and Pilar 1979, J. Gen. Physiol. 73: 605-628). Cells were rinsed once with 100 μΐ F10 (Ham) followed by washing with 100 μΐ Tyrodes solution. 10 minutes after depolarization induced by Tyrodes solution containing 25 mM potassium, the cells were incubated for 20 min.

at room temperature with pH] choline [NEN, NEN109, 0.11 μΜ, 86.7 Ci (mmole) in normal Na^-* · 'Or containing Li⁺ ion, in Tyrodes solution. The uptake reaction was terminated by double washing the cells with PBS. Accumulated choline was extracted by incubating the cultures for at least 20 min. 100 μΐ of cold acetone containing 1 M formic acid. The soluble fraction was then removed and counted. Specific choline uptake is the difference between total and Na + -dependent choline uptake.

16.1.5. Histochemical staining with acetylcholinesterase

Cholinergic cells were identified by histochemical staining with acetylcholinesterase using a modified Geneser-Jensen and Blackstadt method (1971, Z. Zellfordt 114: 460-481). After fixation of the cultures in 4% paraformaldehyde, these cells were incubated for 5 to 6 days at 4 ° C in an AChE substrate solution containing 4 mM acetyl-lithium-choline iodine, 2 mM copper sulfate, 10 mM glycine and 10 µg / well. ml of gelatin in 50 mM acetate buffer pH 5.0. Visualization of the reaction product was carried out as described previously (Hartikka, Hefti 1988, J. Neurosci 8: 2967-2985).

16.1.6. Immunohistochemical staining for NGF-receptor

The cultures were washed twice with DMEM and then fixed with 4% paraformaldehyde. The cells were then treated for 3-4 hours in sodium phosphate buffer, pH 7.4, containing 5% bovine serum albumin, 0.02% Triton X100, and 5% sucrose (Hartikka 1988, J. Neurosci 8: 2567-2585). NGF-R was detected using the monoclonal antibody 192-IgG (Taniuchi and Johnson 1985, J. Cell. Biol. 101: 1100-1106; Chandler et al., 1984, J. Biol. Chem. 259: 68826889) and diluted 1: 1000 in buffer. containing 5% horse serum. Cultures were incubated with the diluted antibody for 15-18 hours at 4 ° C. Bound murine immunoglobulin was detected with biotinized equine anti-mouse IgG (1: 200 Vector). Immunoreactive cells were identified using 3 ', 3'-diaminobenzidine as substrate for bound peroxidase.

16.1.7. Purification of BDNF and NGF

Purification of BDNF from porcine brain and NGF from murine male forearm was performed according to previously published procedures (Barde et al., 1982, EMBO, 1: 549-553; Lindsay et al., 1985, Dev. Biol. 112: 313-328). ). Recombinant BDNF used in some of these studies has been previously described (Maisonpierre et al., 1990, Science 247: 1446-1451).

16.2. Results

Partition cell cultures in wells coated with polyiornitinulinamine for 24 hours. axons grow and many cells have a characteristic phase-bright soma (Fig. 21 a and b). Continuous axon growth in vitro resulted in an increase in cellular contact at day 2-4 (compare Fig. 21 c and d with 21 e and f). Even after 4 days, the culture contained very few non-neuronal cells, as no phased dark cells were found. The histochemical staining with AChE and immunohistochemical staining with AChE receptors were used to evaluate the effect of BDNF on junctional cholinergic neuron survival cultures. A typical morphology of AChE-positive cells is shown in Fig.21 g, h. Several NGF receptor-positive neurons are shown in Fig. 21 i. BDNF gave a 2.4-fold increase in AChE-positive cells in low density (defined as cell density between 66700-133300 cells per cell in rat embryo septal cell cultures, as shown in Fig. 22a). The effect of NGF at the same cell density was slightly less ( We found that BDNF, like NGF, can lead to an increase in AChE-positive neurons in septal cultures, prompting us to investigate whether these two factors affect similar or different populations of neurons. As shown in the third panel of FIG. At the 22nd century, adding BDNF and NGF to saturated levels did not result in a greater increase in AChE-positive neurons than that obtained with each of the factors alone. In terms of neuronal survival, these results indicate that cholinergic neurons in rat septum cultures responsive to BDNF and NGF must be located in closely overlapping populations.

The effect of BDNF on the survival of AChE-positive neurons depended on the density at which the cells grew (Fig. 22a). At high crop densities (from

200,000 up to 266,000 cells into cNA, neither BDNF itself nor NGF produced an increase in AChE-expressing neurons. This may be due to increased levels of endogenous neurotrophic activity or, on the other hand, due to increased extracellular (cell-to-cell) contact resulting from high cell densities that promote survival.

At low cell densities, AChE positive cells increased in proportion to BDNF concentration, with maximal response observed at a dose of 10 ng / ml, with increases in wells of AChE positive cells from control values ​​of 163.2 ± 12.9 to 388 + 26.2 in treated cells. in cultures (2.5 fold increase). For comparison, the maximal response to NGF was observed at 30 ng / ml and gave an increase in the number of AChE positive cells from 121.0 ± 7.6 to 231.7 + 12.9 (1.9-fold increase). The horizontal area on the (plateau) curve graphically depicting the cellular response appears to be stable for BDNF and NGF, in the sense that there is no decrease in AChE-positive neurons at 100 ng / ml.

It is clear that in cultures with low cell density, both BDNF and NGF are able to increase the number of cells detected by AChE histochemistry. This increase may be due to salvage cholinergic cells that die in culture due to growth factor deficiency, supplementation of pre-existing cells, or the induction of a cholinergic marker, AChE. In an attempt to differentiate between these possibilities, additional experiments (Fig. 23) were subsequently performed with a series of cultures maintained for a total of 12 days. One group of cultures treated 5-6 hours later. after sowing, maintained with BDNF and NGF for a full 12 days. The second culture group was maintained for 5 days without growth factors and then treated with BDNF or NGF for the remaining 7 days (-5 / + 7). Comparison of the two groups of cultures shows that the response to BDNF and NGF added after 5 days is essentially the same as that seen when cells were continuously growing in the presence of these factors (compare solid and dotted bars in Figure 23). However, in the third group of cultures in which BDNF and NGF were present only for the last 5 days (7 / + 5), the results were surprisingly different. Under these conditions, neither NGF nor BDNF were able to increase the amount of AChE-positive cells. In other experiments, exposure of septal neurons to NGF or for 3-4 days was found

BDNF is insufficient to significantly increase the enzymatic activity of AChE and, at the same time, to detect cells by AChE staining. No increase in AChE-positive neurons was detected in -7 / + 5 cultures because the growth factor did not have sufficient exposure time to induce AChE-positive cells. In vivo has been shown (Monotero and Hefti 1988, J. Neurosci 8: 2986-2999; Higgins et al. 1989, Neurosci. 3: 247-256), as well as in vitro, that NGF is able to regulate its own receptor, both at mRNA and protein levels. Because BDNF was found to show similar effects to induction of cholinergic phenotype and cell survival of NGF rearrangement cultures, the potential for BDNF to regulate NGF receptor levels as defined by IgG192 immunosuppressed cells was examined. At 10 ng / ml, BDNF increased 3-fold the number of NGF receptor-immuno-positive cells as shown in Figs. 24. Interestingly, at higher concentrations (25-100 ng / ml), BDNF exposure progressively decreased. Thus, the dose response, in this case, was significantly different from the observed effect of BDNF in these cultures on other parameters such as AChE positive cells. For positive control, these cells were treated with 50 ng / ml NGF, which also resulted in a threefold increase in positive cells. Therefore, based on IgG staining, BDNF exerts approximately the same effect as NGF in regulating the increase in NGF receptor expression.

In addition to its effects on cell survival, the potential of BDNF to promote features of the cell cholinergic phenotype was investigated. A linear increase in CAT activity was detected in septal cultures grown with increasing amounts of BDNF up to 50 ng / ml when the reaction saturated at a 1.8-fold increase compared to controls, as shown in Figs. 25th Induction of CAT activity in parallel cultures treated with NGF reached a plateau at 25 ng / ml, a 3.4-fold increase compared to controls. The response to BDNF or NGF did not show a significant decrease to three times the concentration sufficient to produce a saturation effect. Induction of CAT activity by BDNF or NGF did not affect the level of HNP - independent transferase activity (HNP = N-hydroxyethyl-4- (1-naphthylvinyl / pyridium)).

When BDNF and NGF were found to increase CAT activity in septal cultures, the cellular response to the combined effects of both factors was investigated (Table VIII). Two BDNF concentrations, 5 and 25 ng / ml, were used in these experiments, which increased CAT activity by 1.4 and 2.6-fold, respectively. When NGF (50 ng / ml) was added to BDNF, which itself increased CAT activity by 2.8-fold, the level of CAT activity reflected at least an additive effect (Table VIII). For the NGF concentrations used, the purely additive effect of NGF resulted in an increase of 4.2 and 5.4 times the control values, respectively. In fact, the observed values ​​were slightly higher than the simple additional effect.

Table VIII

Effect on CAT activity with simultaneous addition of BDNF and NGF

<td>Concentration</td><td>CAT-activity</td><td>Control (%)</td>

<td>(ng / ml)</td><td>(pmol (HR) well)</td><td></td>

<td>Control</td><td> 561,3±34,8</td><td></td>

<td> (50)</td><td> 1546,3±89,7</td><td> 280</td>

<td>BDNF (5)</td><td> 768,0+25,4</td><td> 240</td>

<td>BDNF (25)</td><td>1470, l ± 66.2</td><td> 260</td>

<td>NGF (50) + BDNF</td><td> 2767,6±177,2</td><td> 490</td>

<td> (5)</td><td></td><td></td>

<td>NGF (50) -t-BDNF</td><td> 3742,0±669,3</td><td> 670</td>

<td> (50)</td><td></td><td></td>

<td>Partitioning cells</td><td>were reared 12</td><td>days on 5HS / N3 at</td>

for the indicated trophic factor concentrations, CAT activity was determined as described. Values ​​are mean ± SEM of 3-5 replicates.

The biological reaction observed with BDNF treatment due to BDNF-dependent secretion of endogenous NGF was tested. To investigate this issue, monoclonal antibodies against NGF (27/21; Korsching and Thoenen 1983) were used to block any NGF-related reaction. As shown in Table IX, the effect of NGF on CAT activity was significantly reduced by anti-NGF antibodies, whereas the BDNF-induced reaction remained essentially unchanged. It should be noted that the basal level of CAT activity was reduced by approximately 20% in cultures pretreated with NGF compared to untreated cultures (control). However, the observed increase in CAT activity in septal cultures induced by BDNF appears to be a direct effect without requiring a mediator in the form of endogenous NGF increase.

Table IX

Effect of anti-NGF on the ability of NGF and BDNF to induce enzymatic CAT activity

Concentration of CAT activity

Control

BDNF (25) NGF (50) Control + AntiNGF pmol (HR) well

517.53±3,2

1021.6 ± 106, l

1139.0 ± 0.99, l

418.7 + 27.0% from control

197

220

BDNF + Anti-NGF 819.2 ± 68.8

NGF + Anti-NGF 551.0 + 27.0

158

107

After 12 days of growth in 5HS / N3 medium at the indicated trophic factor concentrations, the septal cells (seeded at 2.3 (10 ^) cells per cm ^) were harvested. CAT activity was determined as described in the Experimental Methods section, data are mean ± SEM of six replicates.

The possibility that AChE activity is similarly regulated as CAT activity by BDNF or NGF was also investigated (Fig. 26). Unlike CAT activity, AChE dose-dependence on BDNF or NGF appears to increase linearly to 50 ng / ml. At this concentration, NGF and BDNF increased AChE activity by 274% and 237%, respectively, compared to untreated controls. The time-dependent increase in BDNF- and NGF-induced CAT activity was investigated (Fig. 27). In cultures,

100 treated with 50 ng / ml BDNF, increased CAT activity to 170% of control values ​​within three days. This increase continued until day 6, when a 2.5-fold increase in plateau was achieved compared to control values ​​throughout the remainder of the study. In contrast, CAT response to NGF (50 ng / ml) was faster, reaching 2.5 times the control values ​​on day 3. Next, CAT activity increased linearly, reaching 3.2 times the control values ​​at 12 days.

Astrocytes cultured for some time in culture have been shown to synthesize various neurotrophic activities in addition to NGF (Lindsay 1979, Nature 282: 80-82; Lindsay et al. 1982, Brain Res 243: 329-343; Aldsay et al. 1989, Dev. Brain Res. 48: 225-241). Although we have assured that BDNF effects in this study cannot occur through elevation of NGF levels, it is clear that BDNF may act indirectly through regulation of expression of other neurotrophic factors, particularly in glial cells. To evaluate the influence of non-neuronal cells on the effect of BDNF on CAT activity in septal cultures, responses of cholinergic neurons to BDNF in mixed glial-neuron cultures and neuronal enriched cultures were compared (Fig. 28). In cultures enriched with neurons, the response of CAT activity to BDNF is described by a bell-shaped curve; maximal increases were obtained at a dose of 5 ng / ml and a significant decrease in enzymatic activity (p&gt; 0.001 compared to 15-25 ng / ml BDNF) was detected at 25 ng / ml. Similar to that shown in Figs. As a result, CAT response to BDNF in the glial cell fusion layer was linear to the highest dose tested at 25 ng / ml BDNF.

In the presence of non-neuronal cells, higher levels of BDNF were required, equivalent to an increase in CAT activity compared to that found in analogously treated cultures enriched with neurons. However, the ability of BDNF to maximize CAT activity in septal cholinergic neurons was essentially the same for both glial and non-glial cells.

Based on the activity of CAT and AChE, it is now clear that BDNF may act similarly to NGF in amplifying cholinergic phenotypic markers. To further investigate this, the effect of BDNF on Na⁺-dependent, high affinity choline

101 uptake was compared with that of NGF (Fig. 29). Cells were grown for 12 days with 30 ng / ml BDNF and accumulated choline to 3.0 times the control. In parallel cultures, NGF (25 ng / ml) induced a 2.3-fold increase in accumulated choline.

16.3. Discussion

Compared with established effects in peripheral neurons (Barde et al., 1982, EMBO J., 1: 549-553; Lindsay et al., 1985, Dev. Biol., 112: 319-328; Davies et al., 1986; J Neurosci 6: 1897-1904; Hofer and Barde 1988, Nature 331: 262-263) The neural specificity of BDNF in the central nervous system is poorly characterized. To date, the only known responsive neurons belong to a small subpopulation, Thr-1-positive retinal ganglion cells, primarily identified in rat E17 embryonic retinal cultures (Johnson et al., 1986, J. Neurosci 6: 3031-3038). Further studies also found that BDNF also affects adult retinal ganglion cells in explant cultures (Thanos et al., 1989, Eur. J. Neuro 1. 19-26).

In this study, BDNF is shown to improve survival in cholinergic neuronal cultures from rat embryo septum, as shown by AChE histochemical staining, increased expression of various cholinergic phenotypic markers, i.e. AChE and CAT activity, and high affinity choline uptake. Additionally, BDNF was found to increase NGF receptor expression in septal cultures.

In initial experiments, BDNF was found to increase the amount of AChE-positive neurons in E17 septal cultures by approximately double. In the case of NGF (Hartikka and Hefti, 1988, J. Neurosci 8: 2967-2985), this effect is most pronounced at low cell densities. Interestingly, the simultaneous addition of BDNF and NGF did not result in a further increase in AChE-positive cells beyond the levels obtained with each of the growth factors alone. These data confirm that BDNF and NGF affect essentially the same population of septal cholinergic neurons.

As previously reported on NGF (Hefti et al., 1985, Neuroscience 1: 55-58), BDNF did not induce cholinergic neuronal survival in cultures developed with

102 at relatively high cell densities. It has been found that cholinergic neuron survival, expressed as a ratio of inoculated cells, is better at higher cell densities than at low cell densities even without the addition of any neurotrophic factor. There are several possible explanations for this, including increased cellular contact, beneficial effects of increased levels of non-neuronal cells, or steeper increases in endogenous neurotrophic activity. As a last resort, this did not detect a significant increase in endogenous NGF levels, as the addition of excess anti-NGF to untreated high density cultures reduced the basic CAT activity by only 20%. In such cultures, endogenous NGF could increase CAT activity 3.4-fold.

As detected at the protein level and at the mRNA level, many reports indicate that NGF can regulate upregulation of its own receptor, both in neurons, cultured cultures, and in vitro. In the present invention, the monoclonal antibody IgG-192 was used to detect NGF receptors. has been described in both sensory rat neurons and rat brain (Taninchi and Johnson 1985, J. Cell. Biol. 101: 1100-1106). After confirming the phenomenon found by Hartikka and Hefti that NGF can increase its own receptor level in septal cultures, as observed by an increase in IgG-192 immunosuppressive cells, we additionally found that BDNF increased IgG-192 immunosuppressive cells up to 3-fold. The response to BDNF was biphasic, characterized by the fact that at high concentrations in the range of 25-100 ng / ml, BDNF did not produce such a significant increase in positive cell numbers as at 10 ng / ml. Interestingly, this type of reaction is completely different from how BDNF induced an increase in AChE-positive neurons. In the latter case, no decrease in the maximal effect due to BDNF was observed at high BDNF concentrations. It should be noted that low affinity NGF receptor has also recently been shown to be associated with similar affinity, suggesting that low affinity NGF and BDNF receptors may overlap (RodriguezTebar et al., 1990, Binding of Brain-Derived Neurotrophic Factor to the Nerve Growth Factor Receptor, Neuron, 4, 487-492).

103

A possible similarity between BDNF and NGF activity was suspected when BDNF, like NGF, induced AChE activity. Although BDNF was also found to improve cholinergic neuron survival to a similar degree as NGF, the effect of BDNF on CAT activity was not as great as that of NGF. In several experiments, the maximum induction of CAT by BDNF was in the range of 1.8 to 2.6 fold increase, whereas in NGF, an increase of more than 3 fold was usually observed. Thus, while there may be some similarity between the mechanisms of action of BDNF and NGF, it is quite possible that subtle differences exist in the expression and regulation of their respective receptors, their binding to a secondary mediator (s) necessary for induction of CAT activity.

Studies of temporal variations have confirmed that differences in the level of BDNF and NGF-induced CAT activity may indicate different regulatory pathways. Neurons in septal cultures responded to BDNF with an increase in CAT activity for up to 6 days when the reaction went plateau. In contrast, NGF-induced CAT activity was tilted linearly from day 3 to day 12. The interruption of the induced rise in CAT activity on day 6 in cultures treated with BDNF may indicate alterations in expression development either in the BDNF receptor or in the component required for the reaction. Given that the amount of cholinergic neurons (AChE-positive neurons) detected in high-density cultures on day 12 is independent of exogenous BDNF or NGF, it seems unlikely that differential nerve cell death is due to differences in time-varying induction. CAT activity with these two growth factors.

Initial experiments involving basal terminal brain cholinergic cells exposed to BDNF did not suggest primary cell types, neurons, or glia responding to these factors. Although measurements have been made in highly enriched peripheral neuronal cultures, BDNF has been shown to exert its influence directly on neurons (Lindsay et al., Dev. Biol. 112: 319328; Lindsay, 1988, J. Neurosci 7: 2354-2405), it can be predicted that BDNF-induced neural effects in the present study may have been caused by the primary effect of BDNF on astroglial or other neuronal cells. However, it was discovered that

104

BDNF is equally effective in inducing CAT activity in the presence of a confluent monolayer, predominantly of astroglial cells, or of glial cells that are significantly removed from cultures by treatment with a mitotic inhibitor, cytosine-arabinose. An interesting observation made in these experiments is that the shape of the BDNF dose-response curves was clearly different in the two cases. In the presence of a glial monolayer, the response to BDNF was linear growth with increasing BDNF concentration. In cultures enriched with neurons, the response to BDNF moved to the left in a dose-dependent manner. One possible explanation for these results would be that glia can itself express the BDNF receptor, thereby reducing the effective concentration of the ligand for neuronal localized receptors.

On the other hand, the inhibitory effect of astrocytes may have been direct, independent of the effect on ligand concentration or receptor expression, and transmitted, for example, by significantly enhanced BDNF degradation. Similar results have been reported by Hartikka and Hofti (1988, J. Neurosci 8: 2567-2985), as well as by Honeger and Lenua (1982, Dev. Brain Res. 3,225-238). It has been shown that glial cells from various brain regions can significantly influence the morphology of neurons bound to them (Prochiantz et al., 1979, Proc. Natl. Acad. Sci. USA, 76: 5387-5391; Prochiantz et al., 1981). Nature 293: 570-572). Thus, septal glial may have either membrane or secretory properties that may act to inhibit expression of the cholinergic phenotype. Under similar experimental conditions, the regulatory properties of ammonium horn astrocytes have now been investigated.

From the data presented, it is clear that both BDNF and NGF are capable of enhancing the function of septal cholinergic neurons. It is interesting to assess the likelihood that two highly related neurotrophic factors affect a single neuronal population. There have been some assumptions that BDNF and NGF activity overlaps in the early stages of peripheral neuronal development, particularly in the subpopulation of dorsal root node neurons and their precursor, and subsequently releases and functions independently in different populations (Lindsay et al., 1985, Dev. Biol. 112: 319-328: Esberger and Rohrer, 1988, Dev. Biol., 126: 420-432; Barde 1989, Neuron 2: 1525-1534).

105

However, this has not been accurately determined using appropriate markers to detect subpopulations of sensory neurons. Because this study is concentrated in embryonic septal cultures, the only time of development, E17, this separation is likely to occur at later stages of septal development. In this way, it may be of interest to investigate the temporal and spatial differences in the responses of septal cholinergic neurons to BDNF and NGF. Now, it would be interesting to investigate the effects of BDNF on cholinergic basal maxillary neurons in vivo, as it is currently reported that NGF from the ammonium horn is involved in the normal development and maintenance of terminal cholinergic neurons in vivo. It will be interesting to determine whether or not a BDNF-like role is played in vivo. The recent discovery that BDNF mRNA is particularly abundant in ammonium rage supports this role for BDNF.

17th Example: enzymatic conversion of BDNF to active and mature BDNF

17.1. Materials and Methods

Commercial endoproteinase Arg-C (purified from mouse forearm) was used to enzymatically convert large, primary form hBDNF (prepro BDNF) into a biologically active mature protein. This enzymatic digestion was performed by in vitro reaction. The substrate for this enzymatic reaction was prepro BDNF synthesized with CHO-DG from 44 cells which were isolated in cell culture medium and harvested. The culture medium was composed of F-12 (Ham) nucleotide-free medium supplemented with 1% calf fetal serum (FBS) and 1% penicillin and streptomycin. The reaction was run at 37 ° C for 3 min. using 5 units of enzyme and 50 μΐ cellular CHO-GD44 cellular supernatant containing prepro BDNF. Cleavage of prepro BDNF to mature hBDNF was performed under these conditions.

17.2. Results and Discussion

Prepro-form completely converted the recombinant hBDNF (approximately 31000 daltons) to the mature bioactive form of hBDNF (approximately 12000 daltons) in vitro using the endoproteinase Arg-C. Recombinant human brain neurotrophic factor was successfully produced in mammalian cells (CHO106

DG44). The hBDNF gene was stably integrated into the CHO cell genome and amplified using a methotrexate amplification strategy. Recombinant hBDNF was secreted into the medium and showed biological activity. Metabolic labeling with subsequent electrophoresis on SDS polyacrylamide gel (15%) showed that most of the synthesized [²²The S] -labeled hBDNF product secreted into the medium migrated in prepro-form with a molecular weight of 31000 (Fig. 30. Lane 2). [²²S] - The tagged protein secreted from CHO-DG44 wild-type cells is shown in Figs. 30., lane 1. To produce the most mature form of hBDNF (molecular weight 12,000), a specific in vitro strategy for the enzymatic cleavage of the prepro-form hBDNF was developed.

The trypsin cleavage products are shown in Figs. 30., Lanes 3-6. Bovine pancreatic trypsin (purchased from Vortingten), free from chemotrypsin, was dissolved in saline phosphate buffer and added to the solution with 50 μΐ of CHO cellular hBDNF sample. Concentrations of 25, 50, 75 and 100 μg / ml trypsin were used. [²²S] - Labeled reaction products are shown in Figs. 30. Lanes 3-6, respectively. The enzymatic digestion reaction was carried out at 37 ° C for 5 minutes. The gradual decrease in the intensity of the 31000-prepro-hBDNF tag was accompanied by a corresponding increase in its 12500-product. The mature form of hBDNF (molecular weight 12,000) did not generate under these conditions. FIG. 30. Track 7 shows [²²S] -labeled reaction products after enzymatic cleavage of CHO - cellular hBDNF with endogenous Arg-C, isolated from mouse forearm and obtained from Beringer Mangeim Chemicals, Ine. 100 pieces were restored. of this enzyme in lyophilized form with 100 nl of water in Milli-Q and stored at -20 ° C. 5 units were used for enzymatic cleavage of CHO cells with hBDNF. (5 nl) enzyme and 50 nl S] -labeled protein from CHO cell supernatant containing prepro hBDNF (Figure 30. lane 2). As can be seen from Figs. 30th 7th tako, 5 pcs. endoproteinase-C was able to convert the prepro-form hBDNF (31000 ms) to the mature form hBDNF (12000 ms) within 5 minutes at 37 ° C.

Enzymatic treatment of supernatant in CHO-cells expressing hBDNF with endoproteinase Arg-C increased the biological activity of BDNF

107 with crude supernatant. Supernatants of CHO-DG44 cells expressing hBDNF were treated with endoproteinase Arg-C for 3 minutes at 37 ° C. 200 nl of supernatant was treated with 20 units. enzyme and investigated the physiological activity of both treated and untreated hBDNF using explants from the dorsal root node of the E8 chicken. 24 hours after the addition of hBDNF, a qualitative evaluation of axonal regrowth was performed. As noted above, BDNF-treated samples with endoproteinase had a significantly higher bioactivity of hBDNF than control (untreated) samples. The concentration curve for these data is shown in Figs. 31st

Finally, purified endoproteinase Arg-C can be used to obtain a mature bioactive form of recombinant neurotrophic factor from the human brain from incompletely processed molecule precursors (i.e., pro-BDNF). This new approach will allow the development of efficient, large-scale production of bioactive human BDNF from mammalian cell cultures. For example, it is possible to immobilize Arg-C endoproteinase on a solid matrix and activate the step by efficient column chromatography.

18th Example: Brain neurotrophic factor aids in survival and maturation of rat ventral midbrain dopaminergic neurons

18.1. Materials and Methods

18.1.1. Cell cultures

Dissociated cultures were generated by mechanical and enzymatic digestion of ventral midbrain tissues extracted from rat embryos by dissection of ages E14-E15. Typically, tissue collected from two or three rat littermates embryos from simultaneously fertilized Sprague-Dawley rats was treated with trypsin (0.123%, Vortington) in F-12 (Gibco) medium for 20 minutes at 37 °.

C. After washing with growth medium (MEM containing the following additives: Glutamine 2 mM, Glucose 6 mg / ml, Penicillin G 0.3 units / ml, Streptomycin 5 µg / ml, Fetal calf serum 7.5%). The fabric was briefly centrifuged at low speed for 5 minutes and the sediment triturated. After one to two minutes for the non-dispersed cells to settle, the homogeneous cell suspension was seeded at

108 mm plates (pre-coated with polyiornitine and laminin Lindsay et al., 1985, Dev. Biol. 112: 315) containing growth media at a density of 5 x 10 4 cells per sq. cm. After incubation in growth medium until morning, allowing the cells to anchor, cells were grown with and without BDNF in a specific medium free of serum as described by Bottenstein and Shato (Bottenstein and Shato 1979, Proc. Natl. Acad. Sci USA 76: 514) except the fact that the insulin was 20 ng / ml. For dopaminergic cell visualization, cultures were fixed with 4% paraformaldehyde, washed extensively, made permeable to 0.02% saponin in Sorensen buffer with 1.5% horse serum, and stained with monoclonal antibody to rat TH (firm Boehringer, Mangheim). Bound primary antibody was visualized using the Vectsastasin ABC (Vector Labs) kit.

18.1.2. Measurement of dopamine uptake

Cultures were prepared as described above 18.1.1. and raised for the specified number of days with or without porcine BDNF 50ng / ml. Dopamine uptake was determined in triplicate cultures at the indicated time points. Cells were pre-washed in a special buffer of the following composition: 136.8 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4 7H2O, 1.3 mM KH2PO4, 5 mM glucose, 1 mM CaCO3, 1 mM MgSO4, 0.1 mM of ascorbate and 0.1 mM pergiline at pH 7.4. Samples investigating non-neuronal uptake of 1 H-dopamine were stored in the indicated benzotropin buffer (BT) at a concentration of 5 μΜ. After washing, the cells were preheated to 37 ° C for 5 minutes in fresh buffer and 3H-dopamine was added (NEN, 40). ° C (mmole)) to a final concentration of 50 nM. The cultures were then incubated at 37 ° C for 15 minutes, the solution removed, and the cells transferred to ice, washed 4 times with ice buffer as described above. Cells were harvested by addition of 0.5 M NaOH to culture plates and cells counted in a Bekman scintillation counter with 15 micron UltimaGold (scintillation fluid). Specific neuronal uptake of dopamine was determined on the basis that uptake (cpm) observed in the absence of BZT was lower than uptake in the presence of BZT. Typically, between 70 and 90% of total uptake could be inhibited by BZT. In replicated cultures, the amount of TP + neurons at each time point

109 was determined by immunocytochemical staining and the results shown are the uptake normalized to TH + base.

18.1.3. Temporal expression of BDNF

COS M5 cells were transfected with the vector CDM8 containing the sequence encoding human BDNF. 72 hours after transfection, 50 ml of culture supernatant was harvested and 6 M urea was dialyzed. The dialyzed supernatant is combined with ampholines (pH 3.3-10 BioRad) for 4.3 hours. Fractions were collected, dialyzed against 25 mM NaPO 4 in buffer pH 7.6, pre-rinsed with BSA (0.5 mg / ml) to prevent nonspecific adsorption. Fraction activity promoting axonal growth in E8 explant cultures from the dorsal root node of the chick was tested. The active fractions were collected in a tank. Activity analysis of the fractions in the reservoir by SDS PAGE followed by silver staining (Wraey et al., 1981, Ann. Biochem. 118: 157) on 8.18% gel showed a weak band of 68 kD corresponding to BSA and a single band of approximately 12 kD corresponding to that previously detected with porcine BDNF (Barde et al., 1982, EMBO, 1: 549). (data not shown). Comparative bioassays performed with explants from chicken dorsal root nodule and sympathetic nodules to determine specific activity and neuronal specificity of purified recombinant human BDNF were similar to those obtained with purified porcine BDNF.

18.1.4. Production of hBDNF from transfected CHO cells

CFIO-DG44 cells were kindly donated by dr. L.Chasin (Columbia University, USA). These cells are low in dihydrofolate reductase (dhfr) (Urlaub and Chasin 1980, Proc. Natl. Acad. Sci. USA 77: 4216-4220). CHO - DG44 cells were maintained in F-12 (Ham) medium with 10% calf fetal serum, 1% penicillin and streptomycin, and 2 mM glutamine. Cells were inspected twice weekly and usually screened for mycoplasma infection. Prior to transfection, all plasmid DNA was purified by double cesium chloride electrophoresis. The human brain neurotrophic factor gene was subcloned into the mammalian expression vector pCDM8 to produce pC8hB as

110 described previously (Maisonpierre et al., Science 247, 1446-1451). The attenuated dihydrofolat reductase gene, p410, was kindly provided by dr. L.Chasin. These two genetic constructs were co-transfected into CHO-DG44 cells by electroporation (investigated by Shigekawa and Dawer 1988, Biotechnologies 6: 742-751). Approximately 1 x 106 cells were transfected with 20ng pC8hB and 0.2 ng p410. 48 hours after transfection, CHO-DG44 cells cleaved in selective medium consisting of nucleotide-free F-12 (Ham) medium, free of thymidine and hypoxanthine; -HT, plus 10% dialysis calf fetal serum and 1% penicillin and streptomycin.

Individual colonies were isolated approximately 10 days after cell insertion into nucleotide-free selective media. Individually selected colonies resistant to growth medium F-12 (-HT) were treated with 0.02 or 0.05 or 0.1 μΜ of methotrexate without nucleotides to initiate gene amplification (Alt et al., 1978, J. BIOL. Chem 253: 1357-1370). Colonies resistant to ΜΤΧ (methotrexate) were grown in the fields and further amplified with 1.5 or 2.0 or 2.5 μΜ. A single colony resistant to 2.5 μΜ ΜΤΧ was isolated and selected for large-scale cultivation and hBDNF production. Analysis of this clone by Southern blot (clone DGZ 1000-B-2.5) showed 50-fold amplification of the BDNF gene compared to wild-type CHO-DG44 cells. Bioassays with explants of the dorsal root node (DRG) of the chicken E8 embryo showed that this clone produced approximately 9.5 ng / ml hBDNF (not shown). Recombinant hBDNF was produced on a large scale by culturing DGZ 1000-B-3-2.5 cells in 400 cm3 of rollerllacons. The medium was harvested approximately 4 days after cell fusion. Purification of BDNF from the culture supernatant was performed as above for the COS supernatant.

18.2. Results and Discussion

The description of the molecular signals controlling neuronal survival and target innervation specificity is fundamental not only because it helps to understand how the nervous system is constructed, but also because it is likely to provide a better understanding of the mechanisms that maintain and restore mature neurons in certain synaptic pathways. in configurations. Whereas it is widely recognized that the survival and maintenance of neurons depends on specific neurotrophic factors of target origin (Levi-Montalcini and Angeletti, 1969, Physiol. Rev 48: 534; Thoenen and Barde 1980, Physiol. Rev. 60: 1284; Purves 1988, Body and Brain, HaRevard University Press, Cambridg Barde, 1989, Neuron 2: 1525; Lindsay 1988, The Making of Nervous Systems, Oxford, p.148), to date very few such molecules have been fully characterized. To date, only 2 neurotrophic factors, nerve growth factor (NGF) and brain neurotrophic factor (BDNF), have been shown to selectively support a variety of neuronal populations in vivo (Levi-Montalcini and Angeletti, 1968, Physiol. Rev 48: 534; Barde 1989, Neuron). 2: 1525; Leibrock et al., 1989, Nature 341: 149).

Neuronal cell cultures are widely used in bioassays for the identification of neurotrophic activity in cells and tissue extracts (Levi-Montalcini and Angeletti, 1968, Physiol. Rev. 50: 1284; Lindsay 1988, The Making of the Nervous System, Oxford, Varon, and Adler 1981, Adv. Cell Neurofid. 2: 118) and for the determination of the neuronal specificity (or specificities) of purified neurotrophic factors (Ebendal, 1989, Nera Growth Factors, John Wiley et al., 1982, EMBO J. 1: 549; Davies et al., 1986, J. Neurosci 6: 1897; Lindsay et al., 1985 Dev. Biol. 112: 319).

To date, many studies have focused on the neurotrophic needs of different classes of neurons in the peripheral nervous system. The most well-characterized neurotrophic factor was NGF due to its availability: it has been shown that in vitro and in vivo NGF is essential for the survival and maintenance of neuronal subpopulations in both the peripheral and central nervous systems (LeviMontalcini and Angeletti, 1968, Physiol. Rev. 48 : 534; Thoenen and Barde, 1980, Physiol. Rev., 60: 1284; Whittemore and Seiger, 1987, Brain Res. Rev. 12: 439, Snider and Johnson, 1989, Ann. Neurol. 26: 489; Heffi et al., 1989, Neurobiology of Aging 10: 515; Martinez et al., 1987, Brain Res. 412: 295; Takei et al., 1988, J. Neurochem. 51: 111, Hartikka and Heffi, 1988, J. Neurosci. 8: 2967). Difficulties in identifying neurotrophic growth factors for nonspecific central nervous system neurons were due to limited access to purified neurotrophic factor preparations different from NGF, and to difficulties in homogeneous preparation.

112 preparations of specific populations of neurons. Two recent observations prompted us to investigate the effect of BDNF on the survival of dopaminergic neurons in the CNS (black matter). First, it is now clear that the BDNF gene is expressed in the central nervous system, and at relatively higher levels than the NGF gene (Leibrock et al., 1989, Nature 341: 149). Second, it has been reported that a protein purified from the bovine striatum, a target of black matter dopaminergic neurons and having characteristics that are apparently similar to those of BDNF, can enhance in vitro survival of dopaminergic neurons prepared from bovine midbrain (Dal Toso et al., 1988 , J. Neurosci 8: 733).

Fig.32a. show a typical image of dissociated ventral midbrain cells after 9 days in culture. Virtually all of these cells had phasoroidal perikarya and long growths. Several cells had a clear fibroblastic morphology, but no astroglial cells were detected when the culture was stained with an antibody against the astroglial marker, glial fibrillar acidic protein (GFAP; Bignami et al., 1072, Brain Res. 43: 429). Immunocytochemical staining with monoclonal antibodies to TH was performed to visualize dopaminergic neurons in these cultures. As expected, the amount of ΊΉ + neurons in the culture was small (arrows, Fig.32 b, c) and varied depending on the growth time, between 0.1 and 0.5% of the seeded cells. Among TH + cells were neurons with different morphologies, as shown in Fig.33.

In all cultures, a gradual decrease in TH + cell count (Fig. 34a, Fig. 35) was observed in all cases after the first 3-4 days in vitro. On the 8th day of culture, for example, TH + cells in control cultures represented only 25% of the amount found in similar cultures on the second day (Fig. 35). However, in cultures treated with BDNF, the loss of TH + cells was significantly reduced compared to control. At all time points studied, 8 days in vitro, TH + cells were higher in BDNF-treated cultures than in untreated control cultures, for example 1.8-fold after single BDNF addition (Fig. 34a) and 3-fold after multiple addition (Fig. 34a). 35). Even when added only once to cultures maintained for 8 days, BDNF activity is dose-dependent (Fig. 34b). Confirming previous messages (Dal

113

Toso et al., 1988, J. Neurosci 8: 733; Knusel et al., 1990, J. Neurosci 10: 558), NGF (50 ng / ml) had no effect on TH + cell content (Fig. 34c).

Further examining the effects of BDNF on dopaminergic neurons in these cultures investigated the ability of TH + cells to absorb ^ H-dopamine. As shown in Table X, the ability to uptake dopamine (normalized to ΤΉ + neuron) increased significantly after 8 days in culture, but declined thereafter. However, BDNF was found not to alter the ability of TH + cells to uptake dopamine, since the same temporal characteristics and similar values ​​were found with and without BDNF (Table X). Based on this result, it was hypothesized that BDNF is likely to act directly by improving the survival of dopaminergic neurons in midbrain cultures, rather than inducing the expression of a dopaminergic phenotype in neurons that do not necessarily require BDNF for its survival. To further investigate this, experiments were conducted in which TH + neurons were detected in cultures where BDNF addition was initially delayed for several days. It was found that when BDNF addition was delayed to day 5 and TH + cell counts at days 6, 8 and 10, these cultures had significantly less dopaminergic cells compared to parallel cultures in which BDNF was added on day 2 (Fig. 36). ). Addition of BDNF, although delayed, clearly increased TH + neurons compared to control, but at equivalent time points, it was found that delayed addition never rescued as many neurons as BDNF on Day 2.

Taken together, these results contradict the hypothesis that BDNF acts by increasing the expression of the ΤΉ gene in a fixed number of cells and supports the hypothesis that BDNF acts by increasing the survival of dopaminergic neurons that would otherwise be lost if this factor were not added to the cultures early. Several publications have been published on the stimulation of the maturation of midbrain dopaminergic neurons and enhancement of survival in vitro using either tissue extracts or various neurotrophic factors (Dal Toso et al., 1988, J. Neurosci. 8: 733; Knusel et al., 1990). J. Neurosci 10: 558; Prochiantz et al., 1979, Proc. Natl. Acad. Sci. USA, 76: 5387; Di Porzio et al., 1980, Nature,

114

288: 370; Prochiantz et al., 1981, Nature 293: 570; Denis-Donin et al., 1983, J. Neurosci 3: 2292). However, to date there have been no publications on the direct or selective maintenance of TH + neurons in complete absence of glial cells or cell division (as observed, for example, with IGF-1 or FGF, Dal Toso et al., 1988, J . Neurosci 8: 733). Consistent with previous publications using mouse early embryonic tissue (Dal Toso et al., 1988, J. Neurosci 8: 733), E14 cells from rat ventral midbrain were cultured in a culture-free medium, as it turned out to be essentially free of astrocytes and fibroblasts. The use of relatively low cell density in these assays, 30,000 cells per cm in a row, reduced the effects of any possible endogenous neurotrophic activity and allowed for a more accurate cell count. Based on the results presented, it appears that the neurotrophic factor, Dal Toso et al. (1988, J. Neurosci 8: 733) Partially purified from the bovine striatum is probably BDNF. From this it can be assumed that BDNF is produced in the striatum and assimilated by dopaminergic neuronal outgrowth from the black matter. However, to date, northern blot analysis has not detected increased levels of BDNF (or other members of the neurotrophin family) in striatum tissue.

It is clear that even multiple additions of BDNF cannot preserve all of the black matter dopaminergic neurons in the studied growing period. This phenomenon has already been observed in an experiment with similar conditions by Dal Toso et al. (1988, J. Neurosci 8: 733), which showed that this death can be linked to the cell density used; at higher cell densities (4x higher than those used here), high levels of catecholaminofluorescent cells could be detected and the spontaneous disappearance of these cells decreased. Intercellular contact may be necessary for the long-term survival of these cells. Further studies, particularly on the effects of BDNF on the development and maintenance of ventral midbrain dopaminergic neurons and in vivo, are necessary to understand the physiological implications of the BDNF activities described herein. Given the specific death of black matter dopaminergic neurons in Parkinson's disease, it is particularly desirable to detect a neurotrophic factor that selectively affects these

115 cells. Therefore, the discovery here that BDNF supports the survival of these neurons responsive to NGF encourages further animal experiments that will determine whether BDNF can protect dopaminergic neurons from the neurotoxic effects of 6-hydroxydopamine or MPTP (1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine). Such research may lead to a new therapeutic treatment for Parkinsonism.

Table X.

BDNF does not directly increase dopamine uptake in ventral midbrain cultures ^ H-dopamine uptake (cpm (TH) -l- neuron / 15 min)

<td>days of cultivation</td><td>control</td><td>exposed to BDNF</td>

<td> 3</td><td>0.51 ± 0.1</td><td> 0,95+0,2</td>

<td> 6</td><td>4.5 ± 0.3</td><td> 3,1+0,6</td>

<td> 8</td><td> 18,5+5,3</td><td> 27,3±1,2</td>

<td>IO</td><td> 3,37±0,24</td><td> 2,21+0,18</td>

19th Example: BDNF inhibits the uptake of gamma-butyric acid in a dose-dependent manner

19.1. Materials and Methods

19.1.1. Ammonium horn cell cultures

Ammonium horn was removed by dissection from the embryo of an E18-E19 rat, SpragueDawley species, and harvested in F10 medium. Tissues were chopped, washed twice in F10 (Gibco) and trypsinized with 0.25% trypsin (Gibco) for 20 min. at 37 ° C. Pepsin was inactivated by the addition of serum medium consisting of Minimum Essential Medium (MEM) with calf fetal serum (FCS 10%), Glutamine (2 mM), Penicillin (25 units / ml) and Streptomycin (25 units / ml). Dissociated cells were harvested by gentle trituration, harvesting, and centrifugation at 500 rpm for 30 seconds. The centrifugation was repeated twice and the cell derby was resuspended in serum medium. The cells were then left in 6 mm wells or 35 mm

116 plates coated with polyiornithine (10 µg / ml) and laminin (10 µg / ml). In most experiments, cells were seeded at low density, approximately 71,000 cells / cn. Five to six hours after cell inoculation, the medium was replaced with another serum-free medium containing 1% N3 and penicillin-streptomycin (25 units / ml and 25 µg / ml, respectively), and BDNF was added. The medium was changed every three to four days with the addition of BDNF each time.

19.1.2. Measurement of high affinity uptake of gamma-aminobutyric acid (GABA)

High affinity GABA uptake was measured according to the improved Tamazavva and Appel methodology (1986, Brain Res. 399: 111-124). Cells were washed in a special buffer containing 140 mM NaCl, 2.6 mM KCl, 1 mM KH2PO4, 1 mM Na2HPO4, 6 mg / ml glucose, 1 mM MgCl2, 1 mM CaCl2, and 0.1% BSA. After washing, cells were incubated in the indicated buffer for 5 min. at 37 ° C. After that added³H-GABA (NEN, ΝΕΤ-19ΙΧ, ΠΙ.4 Ci (mmol)) to a final concentration of 12 mM and incubated for 10 min at 37 ° C.

The cells were then kept on ice and washed three times with the indicated buffer. Cells were incubated with 0.14 NaOH for 2 h. at room temperature and calculated³The amount of HGABA in the extract. It was found that³H-GABA uptake was linear for at least 30 minutes. GABA uptake in non-neuronal cells was inhibited by the addition of 2 mM R-alanine, whereas neuronal specific uptake was verified by inhibition with 1 mM nipecotinic acid.

19.2. Results and Discussion

Recently, we detected signs of high levels of BDNF in ammonium horn cultures enriched in neurons but not in ammonium horn astrocytes. These data confirm that BDNF is localized in ammonium horn neurons. To test the effect of BDNF on ammonium horn neurons in culture, cells were treated with various concentrations of BDNF (rotor-purified from COS supernatants). After eight days of treatment, they measured high affinity GABA uptake in neurons. As shown in Figs. 37., BDNF inhibited GABA uptake in a dose-dependent manner. Thus BDNF may influence the survival and / or phenotypic expression of GABAergic neurons. BDNF did nothing

117 affecting neurons containing glutamate (as measured by measuring glutamate uptake) nor neurofilament protein levels. Thus, the action of BDNF in ammonium horn cultures is specifically targeted to GABA-ergic neurons.

Example: BDNF protects against toxic MPP + effects

20.1. Materials and Methods

20.1.1. Measurement of the neurotrophic effects of factors on SH-SV5V cells treated with MPP +

SH-SV5V cells were plated in 24 wells at a density of 1 x 10 3 cells per well. 24 hours after seeding, cells were treated with neurotrophic factors. 24 hours after treatment with neurotrophic factors, cells were treated with MPP + (10 μΜ). 48 hours after MPP + treatment, viable cells were counted using trypan blue removal. MNGF-COS (1: 5 dilution), hBDNF-COS (1: 5 dilution), purified rCNTF from E. coli (15 ng / ml), purified bFGF from bovine brain (25 ng / ml) were used as neurotrophic factors. rNT3-COS (1: 5 dilution) and purified hEGF (25 ng / ml).

20.1.2. Measurement of Neurotrophic Factor Effects on Ventral Middle Brain Cultures Treated with MPP +

Cultures of ventral midbrain dissociated cells were prepared from E14 rat embryos according to established procedures (see section 18 above). 48 hours after culturing, cultures were treated with appropriate neurotrophic factors as indicated. Among the neurotrophic factors were hBDNF (rotoformed from CHO-cells, &lt;50 ng / ml), purified mNGF (50 ng / ml), rNT-3 (COS supernatants diluted 1:50), bovine brain (10 ng / ml) and purified rCNTF from K coli (25 ng / ml). Twenty-four hours after the addition of neurotrophic factors, these cultures were treated with MPP + (1 μΜ). 48 hours after treatment, TH + neurons were identified by immunohistochemistry and counted. Averages were obtained from three independent experiments with three samples per experiment. ΤΉ + number of neurons after treatment with

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MPP + are represented by transverse broken lines. Open lines represent the amount of TPI + neurons without MPP + treatment.

20.2. Results and Discussion

When the ability of BDNF, NGF, CNTF, NT-3, bFGF, and EGF to protect cells from l-methyl-4-phenylpyridine (MPP +) toxicity was tested separately, experimental measurements by removal of blue trypan showed that only BDNF and NGF significantly protects against MPP + toxicity (Tables XI and XII).

Table XI

Protection of SH-SV5V cells from MPP + toxicity by BDNF and NGF

<td>help</td><td></td><td></td>

<td>Results:</td><td>The amount of viable cells</td><td></td>

<td></td><td>(% viable)</td><td></td>

<td>putative transfection</td><td></td><td></td>

<td>(COS-Mock) (1: 5)</td><td>0.4 x 10⁴</td><td> (5%)</td>

<td>BDNF-COS (1: 5)</td><td>1.2 x 105</td><td> (67%)</td>

<td>NGF -COS (1: 5)</td><td>1.7 x 105</td><td> (84%)</td>

<td>CNTF</td><td>0.6 x 10⁴</td><td> (14%)</td>

<td>NT-3</td><td>0.7 x 10⁴</td><td> (20%)</td>

<td>bFGF</td><td>0.4 x 10⁴</td><td> (11%)</td>

<td>EGF</td><td>0.1x10⁴</td><td> (5%)</td>

Table XII

Effect of BDNF and NGF on MPP + Toxicity Treatment

COS -MOCK (1: 2) (1: 5) (1:10) (1:20) (1:50) (1: 100)

Viable cells

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COS BDNF (1: 2) 73 (1: 5) 67 (1:10) 65 (1:20) 42 (1:50) 30 (1: 100) 25

COS NGF (1: 2) 88 (1: 5) 24 (1:10) 63 (1:20) 50 (1:50) 47 (1: 100) 34

The data show that BDNF exerts a marked defensive effect against MPP + toxicity in ventral midbrain cultures, whereas bFGF exerts a lesser effect (Fig. 38.). MPP + treatment was found to reduce 85% of tyrosine hydroxylase positive neurons compared to control cultures and only 40% in cultures pretreated with BDNF (prior to exposure to MPP +). MPP + is thought to be the active metabolite of MPTP toxin, which has been reported to induce Parkinson's syndrome in vivo, which is an accepted systemic model of Parkinson's disease. Thus, the protective effects of BDNF and NT3 suggest that these compounds can be used to treat Parkinson's disease or to prevent neurological damage following exposure to the toxin.

21st Deposition of microorganisms

1989 m. August 30 The following recombinant bacteriophage and plasmid DNA were deposited in the U.S. Culture Types Collection, 12301 Parklain Drive, Rockville, Maryland 20852:

ph BDNF -Cl 40648

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Xh BDNF -Gl 40649

This invention is not limited to the specific embodiments set forth in the description. Indeed, various improvements of the invention, in addition to the embodiments described, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such improvements include the following definition points. The various publications cited are incorporated by reference in their entirety.