WO2000053735A2

WO2000053735A2 - Methods for identifying modulators of synaptic plasticity and cell motility

Info

Publication number: WO2000053735A2
Application number: PCT/IB2000/000505
Authority: WO
Inventors: Stephen Morris; Richard Murphy; James Fawcett; Timothy Kennedy; Colleen Mannit
Original assignee: Mcgill University
Priority date: 1999-03-10
Filing date: 2000-03-09
Publication date: 2000-09-14
Also published as: WO2000053735A9; WO2000053735A3; AU3667600A

Abstract

The invention features a method for determining whether a candidate compound modulates the expression of a gene operably linked to a netrin promoter, including: a) providing a cell expressing a gene operably linked to a netrin promoter; b) contacting the cell with a candidate compound; c) detecting or measuring expression of the gene following contact of the cell with the candidate compound; and d) determining whether the candidate compound modulates the expression of the gene.

Description

METHODS FOR TDENTTFYTNG COMPOUNDS THAT MODULATE SYNAPTIC PLASTICTTY AND CELL MOTTLTTY

Background of the Tnvention

The invention relates to methods of altering synaptic structure and cellular motility.

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins. BDNF has been implicated in regulating the strength of synaptic connections through an effect on the structure of pre - and/or post-synaptic nerve endings. The precise mechanism by which BDNF exerts these effects has not been elucidated. Recent studies suggest that, in neurons, BDNF is transported anterogradely to the axon terminal. Thus, in addition to other possible sites of action, BDNF appears to be present and act at the synapse.

Neuronal production of BDNF increases in response to neuronal activity. This conclusion is supported by the observation that BDNF synthesis is upregulated in hippocampal dentate granule neurons of rodents treated with the epileptogenic agent kainic acid. In addition, BDNF protein can be detected in the presynaptic terminals of these neurons following seizure. The conditions under which BDNF is released in vivo and the mechanisms by which it alters synaptic structure have not been determined, however, some understanding of the method of signal transduction has been achieved.

The trk family of proto-oncogenes are receptors for the neurotrophin family of proteins. Neurotrophin signaling through trk receptors mediates cell survival and morphological differentiation of neurons. In addition, expression of trkB indicates a poor prognosis in neuroblastoma, and correlates with neurόblastoma tumor cell motility and the re-occurrence of tumor foci following surgical tumor removal. These observations are significant because BDNF preferentially binds to trkB. trkC, however, is also activated by BDNF. Thus, it seems likely that trkB and/or trkC could be involved in the mechanism through which BDNF exerts its effect on the synapse.

Netrins are a recently discovered family of secreted proteins that are essential chemotropic cues that guide the growth of developing axons in the brain and spinal cord. Netrins influence axon outgrowth through two classes of receptors: vertebrate homologs of the C. elegans gene unc-5, and the vertebrate homologs of the C. elegans gene unc-40: DCC and neogenin. Netrin function has been most thoroughly described in the developing spinal cord. Early in spinal cord development, netrin protein, secreted by floor plate cells in the ventral cord, appears to direct the growth of commissural axons originating from the cell bodies of spinal interneurons in the dorsal spinal cord. There is currently a need to understand the mechanisms of synaptic reorganization. It is believed that synaptic reorganization is a central component of numerous neurologically-based disorders, including drug addiction, epilepsy, and hyperalgesia. An understanding of the mechanism of reorganization would provide methods for developing rational approaches to treating these disorders.

Summary of the Tnvention

In general, the invention features a method for identifying a compound that modulates synaptic reorganization or cell migration.

In a first aspect, the invention features a method for determining whether a candidate compound decreases the expression of a gene operably linked to a netrin promoter, including: (a) providing a cell expressing a gene operably linked to a netrin promoter; (b) contacting the cell with a candidate compound; (c) detecting or measuring expression of the gene following contact of the cell with the candidate compound; and (d) determining whether the candidate compound decreases the expression of the gene.

In preferred embodiments, the cell is a neuron, a glial cell, such as an oHgodendrocyte, or is from a cell line, such as SN 48, PC12, NG108, 3T3, C6, PI 9, or a glioma cell line. In a more preferred embodiment, a decrease in the expression of the netrin gene inhibits synaptic reorganization by a neuron or inhibits migration of a glial cell. In another embodiment, the candidate compound also inhibits or decreases the cell's response to BDNF.

In a second aspect, the invention features a method for determining whether a candidate compound increases the expression of a gene operably linked to a netrin promoter, including: (a) providing a cell capable of expressing a gene operably linked to a netrin promoter; (b) contacting the cell with a candidate compound; (c) detecting or measuring expression of the gene following contact of the cell with the candidate compound; and (d) determining whether the candidate compound increases the expression of the gene.

In preferred embodiments, the cell is a neuron, a glial cell, such as an oHgodendrocyte, or is from a cell line, such as SN 48, PC 12, NG108, 3T3, C6, P19 or a glioma cell line. In a more preferred embodiment, an increase in the expression of the netrin gene promotes synaptic reorganization by a neuron or promotes migration of a glial cell. In another embodiment, the candidate compound also promotes or increases the cell's response to BDNF. In another preferred embodiment of the first or second aspect, the gene is a netrin gene, such as netrin- 1, or the gene is a reporter gene, such as gfp. In still another embodiment, the netrin promoter is the netrin- 1 promoter or a fragment or deletion of a netrin promoter. The gene expression may be measured by assaying the protein level of the expressed gene, or by assaying the RNA level of the expressed gene. In yet another preferred embodiment of the first or second aspect, the cell is in a mammal, such as a mouse. In one embodiment, the mammal is a transgenic mammal.

In a third aspect, the invention features a method for identifying a candidate compound that decreases synaptic reorganization, including: (a) providing a cell expressing recombinant netrin; (b) contacting the cell with a candidate compound; and (c) detecting or measuring synaptic reorganization following contact of the cell with the candidate compound.

In a preferred embodiment, the cell is a neuron, such as a septal neuron, a hippocampal neuron, a spinal cord neuron, or a dorsal root ganglion neuron. In another embodiment, the candidate compound also inhibits or decreases the cell's response to BDNF.

In a fourth aspect, the invention features a method for identifying a candidate compound that decreases cell migration, including: (a) providing a cell expressing recombinant netrin; (b) contacting the cell with a candidate compound; and (c) detecting or measuring cell migration following contact of the cell with the candidate compound.

In a preferred embodiment, the cell is a glial cell, such as an oHgodendrocyte, or is from a cell line such as an SN 48, PC12, NG108, 3T3,

C6, P19 or a glioma cell line. In another embodiment, the candidate compound also inhibits or decreases the cell's response to BDNF. In a fifth aspect, the invention features a method for inhibiting synaptic reorganization by a neuron by contacting the neuron with a compound that decreases netrin expression.

In a sixth aspect, the invention features a method for inhibiting migration by a tumor cell by contacting the tumor cell with a compound that decreases netrin expression.

In a seventh aspect, the invention features a method for identifying a gene which modulates netrin expression, including: (a) expressing in a cell (i) a first gene operably linked to a netrin gene promoter and (ii) a second candidate gene or a fragment thereof; and (b) monitoring the expression of the first gene, wherein a change in the expression identifies the candidate gene as a gene which modulates netrin expression.

In an eighth aspect, the invention features a method of isolating a gene that modulates synaptic plasticity or cell migration, including: (a) providing a cell expressing a first gene operably linked to a netrin gene promoter; (b) mutagenizing the cell; (c) measuring expression of the first gene, wherein an increase or decrease in the expression of the first gene identifies a mutation in a second gene; and (d) using the mutation as a marker for isolating the second gene, wherein the second gene modulates synaptic plasticity or cell migration. In a preferred embodiment of the seventh or eighth aspect, the cell is an

SN 48, PC 12, NG108, 3T3, C6, or PI 9, or is from a glioma cell line. In another embodiment, the first gene comprises a netrin gene, (e.g., netrin-1) or a reporter gene (e.g., gfp). In yet another embodiment, the netrin gene promoter is the netrin-1 gene promoter. The expression of the first gene may be measured by assaying the protein level of the expressed gene or the RNA level of the first gene. The methods of the first through eighth aspects can be performed in vitro or in vivo.

In a ninth aspect, the invention features a method for decreasing synaptic reorganization in a mammal, including administering a composition that decreases netrin signaling.

In a preferred embodiment, the mammal has epilepsy, a drug addiction, schizophrenia, chronic pain, or hyperalgesia. In one embodiment, the mammal is having a surgical procedure (e.g., an amputation); and the surgical procedure is accompanied by the administration of an addictive substance. In one preferred embodiment, the composition includes a netrin antisense RNA sequence. In another preferred embodiment, the composition includes an antibody direct to netrin.

In a tenth aspect, the invention features a method for decreasing cell migration in a mammal, including administering a composition that decreases netrin signaling. In a preferred embodiment, the mammal has a tumor (e.g., a glial tumor). In another preferred embodiment, the composition includes a netrin antisense RNA sequence. In yet another preferred embodiment, the composition includes an antibody directed to netrin.

In an eleventh aspect, the invention features a method for increasing cell migration in a mammal, including administering a composition that increases netrin signaling. In a preferred embodiment, the mammal has multiple sclerosis.

In a twelfth aspect, the invention features a netrin gene nucleic acid fragment or antisense RNA sequence for use in inhibiting synaptic reorganization or cell migration. Such nucleic acids of the invention and methods for using them may be identified according to a method involving: (a) providing a cell sample; (b) introducing by transformation into the cell sample a candidate netrin nucleic acid; (c) expressing the candidate netrin nucleic acid within the cell sample; and (d) determining whether the cell sample exhibits altered cell migration or synaptic reorganization. In a thirteenth aspect, the invention features a netrin polypeptide fragment for use in inhibiting synaptic reorganization or cell migration. Such polypeptides of the invention may be identified according to a method involving: (a) providing a cell sample; (b) contacting the cell sample with a candidate netrin polypeptide; and (c) determining whether the cell sample exhibits altered cell migration or synaptic reorganization.

By "synaptic reorganization" is meant the process in which neuronal connections are modified. Modifications include branching and sprouting of axonal or dendritic processes. In general, the same cellular processes which lead to synaptic reorganization in vivo also lead to synaptic reorganization in vitro. In vitro reorganization can be measured by qualitatively assessing an attribute such as neuronal branching or neurite number or length, or by quantitatively measuring any of the foregoing attributes. Additionally, the expression of several proteins, including, for example, GAP-43 synapsin, synaptophysin, CDC42, rac, rho, and the actin modifying enzyme NWASP, may be increased during synaptic reorganization. These proteins, or the mRNA encoding them, or reporter genes operably linked to the promoters of these genes, can be quantitated using any number of techniques known to those skilled in the art. Formation of actin stress fibers can be directly assessed using, for example, rhodamine- conjugated falloidin. Additionally, NMDA receptors cluster during synaptic reorganization.

A compound or condition that "inhibits" or "decreases" synaptic reorganization is one that causes a reduction in one or more of the above hallmarks of synaptic reorganization. Preferably, the reduction is at least 10%, more preferably, at least 25%, and most preferably at least 50%, when compared to the same cell in the absence of the compound or condition. A compound or condition that "promotes" or "increases" synaptic reorganization is one that causes an increase in one or more of the above hallmarks of synaptic reorganization. Preferably, the increase is at least 10%, more preferably, at least 25%, and most preferably at least 50%, when compared to the same cell in the absence of the compound or condition. By "migration" of a cell is meant the active movement of a cell from one location to another. Migration of glial cells in vitro can be readily assayed using any of a number of assays well-known in the art (e.g., Albini A. et al., Proc. Natl. Acad. Sci. USA 92:4838-4842, 1995).

Tumor cell invasion is considered herein to be a type of cell migration. Methods for specifically examining tumor cell migration in vitro, such as agar invasion assays, are also known in the art (e.g., Koochekpoor S. et al., Cancer Res. 57:5391-5398, 1997).

A compound that inhibits or decreases cell migration will reduce the number of cells that migrate in one or more of the foregoing assays, or will reduce the extent of cell migration. Preferably, the reduction is at least 10%>, more preferably, at least 25%, and most preferably at least 50%, when compared to the same cell in the absence of the compound.

A compound that "promotes" or "increases" cell migration will increase the number of cells that migrate in one or more of the foregoing assays, or will increase the extent of cell migration. Preferably, the increase is at least 10%, more preferably, at least 25%, and most preferably at least 50%, when compared to the same cell in the absence of the compound.

By a "reporter gene" is meant a DNA or RNA sequence that encodes a reporter protein that is capable of being readily detected either inside or outside a cell. Many different types of reporter proteins are known in the art. They frequently comprise proteins not normally found, or present in minor amounts, in some cells; they include enzymes that detoxify antimicrobial agents, such as aminoglycoside or aminocyclitol phosphotransferases or acetyltransferases, beta-lactamases or chloramphenicol acetyltransferase; enzymes of diverse origin that catalyze chromogenic, fluorogenic, or chemiluminescent reactions in the presence of exogenous substrates, such as beta-galactosidase, beta- glucuronidase, alkaline phosphatase, catechol 2,3-dioxygenase, or various peroxidases; enzymes that catalyze photoreactions, such as bacterial or firefly luciferases; enzymes, like glycosyl transferases, that generate nonproteinaceous structures easily detected by antibodies, lectins, or cognate binding proteins; proteins easily monitored upon cell surface expression or secretion such as surface or secreted antigens for which corresponding antibodies or recognition proteins are known; and proteins that catalyze the synthesis of, or stoichiometrically embody, fluorescent structures, without exogenous substrates, such as the jellyfish fluorescent proteins (e.g., GFP). By "operably linked" is meant that the gene for the reporter protein is positioned adjacent to a promoter that directs transcription of the gene and, ultimately, facilitates expression of the reporter protein.

By "promoter" is meant any minimal nucleic acid sequence sufficient to direct transcription of the reporter gene. Included in the invention are elements (e.g., enhancers or suppressors) that are sufficient to render spatially- or temporally-restricted gene expression either alone or in combination with a basal promoter, or elements that are inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the native gene or engineered into a transgene construct.

By "antisense," as used herein in reference to nucleic acids, is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand of a netrin gene. Preferably, the antisense nucleic acid is capable of inhibiting or decreasing synaptic reorganization or cell migration when present in a cell. Preferably, the decrease is at least 10%, relative to a control, more preferably 25%, and most preferably 1-fold or more. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Brief Description of the Drawings

Fig. 1A and IB are photomicrographs of frozen sections of the lateral septum of adult rats. Fluorescent immunohistochemical analysis revealed that BDNF (Fig. 1 A) and calbindin (Fig. IB) were co-expressed in a subset of neurons. Scale bar is 30 μm.

Fig. 2A and 2B are photomicrographs showing immunohistochemical analysis of frozen sections of adult rat lateral septum. Fluorescent immunohistochemical analysis revealed that TrkB (Fig. 2A) and calbindin (Fig. 2B) were co-expressed in a subset of neurons (arrows). Note, however, that TrkB can be expressed by a calbindin-negative cell (arrowhead).

Fig. 3A and 3B are photomicrographs showing immunohistochemical analysis of frozen sections of adult rat lateral septum. Calbindin (Fig. 3 A, arrow), and netrin (Fig. 3B, arrow) can be co-expressed within neurons. At least one cell within the same field is netrin positive (Fig. 3B, asterisk) and calbindin negative (Fig. 3 A, asterisk).

Fig. 4A and 4B are photomicrographs of rat lateral septal neurons, grown in culture for five days in the presence (Fig. 4A) or absence (Fig. 4B) of BDNF (50 ng/ml), immunostained for calbindin. Both the number and length of neurites increased following the addition of BDNF.

Fig. 4C and 4D are photomicrographs of rat lateral septal neurons, grown in culture for five days in the presence (Fig. 4A) or absence (Fig. 4B) of BDNF (50 ng/ml) and in the presence of 50 nM 5-fluorodeoxyuridine, immunostained for calbindin.

Fig. 4E is a series of illustrations that show that cultures receiving BDNF contain more than twice as many calbindin-positive septal neurons than cultures that were not exposed to BDNF.

Fig. 5 A is a series of photographs of immunoblots of protein from untreated and BDNF treated cultures of rat septal neurons illustrating that the expression of netrin is increased following exposure of the culture to BDNF.

Fig. 5B is a series of photographs of immunoblots of protein from untreated and BDNF treated cultures of rat hippocampal neurons illustrating that the expression of tubulin is unchanged following exposure of the culture to BDNF.

Fig. 5C is a series of photographs of immunoblots of protein from untreated and BDNF treated cultures of undifferentiated SN 48 cells illustrating that the expression of netrin is increased following exposure of the culture to BDNF. Fig. 6 is a series of photographs of immunoblots showing the induced increase of netrin expression following kainic acid induced seizure activity in rats.

Fig. 7 is a series of photomicrographs of rat hippocampal neurons in culture illustrating the inhibition of the elaboration of developing dendritic processes by an antibody that blocks netrin function. In support of netrin promoting the outgrowth of developing neurites, an antibody that blocks the function of endogenous netrin inhibits the elaboration of dendritic processes. Shown are mixed cultures derived from CA3 and dentate gyrus of the hippocampus of a post-natal day 1 rat. Both panels are neurons following four days in culture. Anti-netrin antibody was added at a concentration of 50 μg/ml. Addition of exogenous purified recombinant netrin protein blocks the effect of adding antibody and completely rescues the growth of processes, indicating that the antibody is not non-specifically poisoning cell growth.

Fig. 8 is a series of photomicrographs of astrocytes isolated from newborn rat hippocampus and grown in culture. Both netrin and the netrin receptor DCC were detected on the surface of developing astrocytes isolated from post-natal day 1 rat brain, suggesting a possible role for netrin in regulating astrocytic migration, activation, and/or differentiation. Astrocyte cell morphology was revealed with an antibody against the astrocytic marker glial fibrillary acidic protein(GFAP, Cy3 conjugated secondary antibody). Fig. 9 A is a schematic illustration of the expression of BDNF, netrin, and their corresponding receptors (trkB and DCC, respectively) on presynaptic and postsynaptic neurons.

Fig. 9B is a schematic illustration of how the simple wiring diagram of Fig. 9A would be applicable to the hippocampus. Detailed Description

We have discovered that netrin is a major effector of BDNF signaling through trkB activation and that, acting together, these molecules play a role in promoting synaptic rearrangements. Our conclusion is based upon the following observations: 1) BDNF is present in the terminals of nerve fibers that synapse on calbindin- containing neurons within the lateral septum of rats; 2) a population of calbindin-containing neurons co-express trkB and netrin; 3) when grown in culture in the presence, but not the absence of BDNF, calbindin-containing neurons undergo a significant morphological transformation characterized by increased numbers of cell processes and arborizations; 4) cultures of septal neurons increase their expression of netrin in the presence but not absence of BDNF; 5) kainic acid treatment, which induces maximal BDNF production in brain within 12 hrs (Fawcett J. et al., J. Biol. Chem. 272:8837-8840, 1997), also induces the production of netrin; and 6). Furthermore, kainic acid- induced increases in netrin synthesis occur approximately 12 hours after the maximal induction of BDNF synthesis, which is consistent with a cause and effect relationship.

Taken together, all of these data support the existence of a critical cascade involving BDNF and netrin. The steps of the cascade are as follows: First, neuronal activity promotes the secretion of BDNF from pre-synaptic neurons. BDNF activates trkB in post-synaptic cells, which in turn stimulates post-synaptic neurons to synthesize and secrete netrin. Netrin, acting on the pre- and/or post-synaptic cell, alters the structure of the synapse. Each of the observations is now described in greater detail. BDNF is present in the terminals of nerve fibers that synapse on calbindin-containing interneurons within the lateral septum of rats

For immunohistochemical analysis of expression of BDNF in brain tissue, adult rats were cardiac perfused with 2% formaldehyde with 0.2 M parabenzoquinone in 0.1 M phosphate buffer pH 7.2. Brains were removed and equilibrated with 30% sucrose in 0.1 M phosphate buffer pH 7.2. Brains were then embedded in Tissue Tek® (Sakura, Torrence, CA) and 40 μm sections cut using a cryostat.

Fig. 1A. and IB show that not all calbindin-containing neurons contain BDNF, suggesting that multiple populations of neurons exist within the lateral septum. Confocal microscopic analysis revealed that BDNF immunoreactivity was present in punctate structures surrounding calbindin-containing neurons. These BDNF-containing punctate structures have morphological characteristics consistent with them being presynaptic terminals.

A population of calbindin-containing neurons express trkB and netrin

Neurons in the lateral septum of adult rats were examined for netrin, trkB, and calbindin expression (Fig. 2 and Fig. 3). The method was as follows. Adult rats were euthanized with an overdose of sodium pentobarbital and cardiac perfused with 4% formaldehyde, 15% picric acid, in phosphate buffered saline (PBS) at a pH of 8.5. Brains were equilibrated in fixative overnight and then equilibrated with fixative in 30% sucrose. Brains were then embedded in Tissue Tek® and 40 μm sections cut on a cryostat. Antigenicity was enhanced by immersing sections in boiling PBS. Sections were processed with antibodies as follows. Primary antibodies were added at the following dilutions: rabbit anti-netrin 1 :500 (T.E. Kennedy lab); mouse anti-calbindin, 1 :5000 (Sigma Chemicals, St. Louis, MO); rabbit anti-trkB, 1 :1000 (Gift from Stuart C. Fienstein lab, UC Santa Barbara). The cells were incubated in the antibodies overnight at 4°C, then washed three times in phosphate buffer, and incubated with Cy3 goat anti-rabbit and/or Cy2 goat anti-mouse antibodies (Jackson Immunochemicals, West Grove, PA) at a final dilution of 1 : 1000 for 1 hour. The secondary antibody was removed and the tissue sections mounted in 50%) glycerol/PBS solution and coverslipped.

The results are shown in Fig. 2 and Fig. 3. trkB (Fig. 2 A) and calbindin (Fig. 2B) were co-expressed in a subset of neurons (arrows). Note, however, that trkB can be expressed in a calbindin-negative cell (arrowhead). Similarly, calbindin (Fig. 3A, arrow), and netrin (Fig. 3B, arrow) can also be co-expressed within neurons. At least one cell within the same field is netrin positive (Fig. 3B, asterisk) and calbindin negative (Fig. 3A, asterisk). Cells co- express netrin and trkB.

Cultured septal neurons undergo morphological transformation and increase their expression of both calbindin and netrin in response to BDNF

Using cultured septal neurons derived from embryonic day 16 rats, we tested the effect of BDNF on netrin and calbindin expression (Fig. 4). Neuronal cultures were prepared as follows (adapted from Mazzoni I.E. and Kenigsberg R.L., Neurosci. 45: 195-204, 1991). The septal region of the brain was removed from embryonic day 16 rats, incubated for 20 minutes at 37°C in 0.2% trypsin, dissociated in a pipette, and plated at a density of 1.8 x 10⁷ cells/ml in tissue culture dishes. The medium contained 500 μM glutamine, 25 μM glutamate with B27 and penicillin/streptomycin supplements. Fresh medium without glutamate was provided every two days. On the second day after plating, 50 ng/ml BDNF was added. For medium changes thereafter, 50% of the old medium was removed and supplemented with fresh medium.

After five days of BDNF treatment, cultures of septal neurons were washed three times with phosphate buffer (0.1M PB) and incubated in the same buffer with 4% formaldehyde for 20 minutes. The cultures were washed again three times and blocked in 10% normal goat serum, 0.1% Triton XI 00 in phosphate buffer for 20 minutes. Primary antibody was then added at the following dilutions: rabbit anti-netrin 1 :500; mouse anti-calbindin, 1 :5000; or rabbit anti-trkB, 1 : 1000. Double labeling was performed using mouse anti-calbindin with one of the other antibodies. The cells were incubated in antibody overnight at 4°C, washed three times in phosphate buffer, and then incubated with Cy3 goat anti-rabbit and/or Cy2 goat anti-mouse antibodies at a final dilution of 1 :1000 for 1 hour. The secondary antibody was removed and the cultures mounted in a 50% glycerol/PBS solution and coverslipped. In cultures of septal neurons, BDNF promoted the elaboration of large numbers of neurites (Fig. 4A) that were not apparent in cells deprived of BDNF (Fig. 4B). To establish a direct link between BDNF and morphogenesis, and rule out a possible confounding action through promotion of mitogenesis, the following experiment was performed. Cells were treated with 50 ng/ml BDNF in the presence of 10 nM 5-fluorodeoxyuridine, an inhibitor of cell mitosis. Even in the presence of this inhibitor, there was a marked increase in the number of neurites on BDNF-treated septal neurons (Fig. 4C) when compared to untreated controls (Fig. 4D). Quantification of calbindin-positive neurons and total cultured cells revealed an increase in the percentage of neurons that were calbindin-positive (Fig. 4E). This increase was not due to differential proliferation or death in the BDNF-treated cultures, as the total neurons/field was unchanged (Fig. 4E). We conclude from these data that BDNF was not promoting cell division, but rather promoted the survival and morphological differentiation of calbindin-containing post-mitotic neurons.

We demonstrated that BDNF treatment leads to increased netrin expression in cultures of neurons. Specifically, netrin expression was assayed by western blot analysis of cultured septal neurons, hippocampal neurons, and differentiated SN 48 cells (a cell line derived from a septal neuron- neuroblastoma fusion described by Lee H.J. et al., Devel. Brain Res. 52 219-228, 1990). Septal neurons were cultured as described above. Hippocampal neurons were cultured as described by Baranes D. et al., Proc. Natl. Acad. Sci. USA 93:4706-4711, 1996. SN 48 cells were cultured as described by Charest A. et al., J. Neurosci. 13:5164-5171, 1993. Five days after BDNF was first added to the medium, the medium was removed, and the cells washed in Tris-buffered saline (TBS) at 37°C, then treated with ice cold Tris lysis buffer. The lysis buffer contained 137 mM NaCl, 20mM Tris, pH 8.0, l%(v/v) NP40, 10% (v/v) glycerol, 1 mM PMSF, 10 μg aprotinin, and 0.2 μg leupeptin. After 20 minutes of rocking, the lysate was removed and centrifuged at 15,000 RPM for 15 minutes. The supernatant was removed and protein levels measured using the BCA kit (Pierce Biochemicals, Rockford, IL). Fifty micrograms of total protein extract was loaded per lane, separated using PAGE, and transferred to nitrocellulose. Blots were blocked for one hour in 3% normal goat serum, 5% milk containing 0.1% SDS, and 0.1 % Tween 20, all dissolved in TBS, pH 7.8. Blots were incubated in primary antibody overnight at 4°C. The antibody to netrin was used at a dilution of 1 : 1000, anti-tubulin (or anti-GAD) (Sigma, St. Louis MO) at 1 : 5000, and anti-BDNF (Santa Cruz Immunochemicals, Santa Cruz CA) at 1 : 5000. The blots were washed three times in TBST (Tris-buffered saline with 0.1% tween-20), incubated in secondary goat anti-rabbit HRP antibody (Boeringher Mannheim, Quebec, Canada) for 1.5 hours, washed, and developed using standard ECL protocols NEN Life Sciences, (Mandel Scientific, Quebec, Canada) according to the manufacturer's instructions.

Cultured septal neurons produced netrin in the presence but not the absence of BDNF (Fig. 5 A). The levels of expression of a control protein, tubulin, did not change following BDNF treatment. Similarly, BDNF induced a significant increase in netrin production above basal levels in primary cultures of hippocampal neurons (Fig. 5B) and in cultures of differentiated SN 48 cells (Fig. 5C). Together, these data demonstrate an increase in netrin expression following BDNF treatment.

Kainic acid treatment induces netrin expression in vivo

To determine whether endogenous production of BDNF in brain could be related to netrin synthesis, we monitored netrin expression following kainic acid treatment, conditions that we had shown previously to enhance the production of BDNF protein in cerebral cortex within 12 hours of induction (Fawcett, supra).

Adult female rats were injected intraperitoneally with kainic acid (10 mg/kg) and left to seize for periods ranging from two hours to three days. After appropriate seizure times, the brains were rapidly removed, the cortex dissected on ice and rapidly added to Tris lysis buffer (see above) (10% w/v) and prepared as outlined above. Netrin expression was strongly upregulated one day following kainic acid seizure, an increase maintained for at least three days (Fig. 6). This result is consistent with the in vitro data presented above showing that BDNF treatment promotes netrin production in cultures of calbindin-containing septal neurons. It is also consistent with our interpretation that BDNF, which is maximally produced in the cerebral cortex 12 hours following kainic acid treatment, induces the production of netrin, which is detectable in the same brain regions 24 hours following kainic acid treatment. The ability of BDNF to increase the number of neurites of cultured calbindin positive septal neurons can be blocked using reagents that interfere with netrin function. This was shown in the following experiment. Cultures of embryonic septal neurons were prepared as described above. Coincident with BDNF application, antibodies that block netrin function (50 μg/ml) were also applied. In the presence of blocking antibodies, the effect of BDNF was strongly attenuated.

The data presented herein suggest that BDNF released in an activity- dependent fashion by pre-synaptic neurons regulates the production of netrin in post-synaptic cells. Netrin produced by post-synaptic neurons, in turn, plays a key role in mediating the effects of BDNF on synaptic reorganization and neurite outgrowth. Netrin signaling is most likely through the netrin receptor DCC, which is expressed by the presynaptic neuron and present at the presynaptic terminal (see model, Fig. 9). We propose that BDNF, trkB activation, and coincident depolarization of the post-synaptic cell together promote the synthesis and release of netrin. Furthermore, we propose that netrin synthesis and release is also be promoted by activation of other trk receptor family members, activation of their downstream effectors, or by activation of other tyrosine kinases and their signaling pathways. While it has been shown that BDNF exposure leads to synaptic remodeling (Cabelli R.J. et al., Neuron 19:63-76, 1997), the action of netrin as an effector molecule regulated by this cascade is both crucial and novel. Additionally, we have demonstrated a new function for netrin, which may be assayed. Reagents that promote netrin function promote hippocampal neurite outgrowth and conversely, that reagents that inhibit netrin function inhibit hippocampal neurite outgrowth (Fig. 7).

Using recombinant netrin protein and antibodies that block netrin function, which act as gain-of -function and loss-of- function reagents, respectively, we have also demonstrated that netrin promotes astrocyte activation, implicating netrin in the control of glial cell motility and potentially in the control of glial tumor cell migration (Fig. 8). In the control condition, most cells have a flat morphology and express a low level of GFAP (left panel). In the presence of exogenously added purified recombinant netrin protein (170 ng/ml) cultured astrocytes exhibit well developed processes characteristic of an activated glial phenotype (center panel). In the presence of an antibody that blocks netrin function (50 μg/mL) astrocytes exhibit a flattened non-activated phenotype (right panel), the converse of that observed in the presence of recombinant purified netrin protein. These functions for netrin are novel findings.

The coordinated role of BDNF and netrin in regulating synaptic organization is observed in other brain regions. An example of the relationship between BDNF, trkB, DCC, and netrin may be seen in the developing hippocampus. We have found that netrin is expressed in multiple regions of the developing and adult rodent brain. This includes CA3 neurons of the developing hippocampus, which are the target cells of mossy fiber axons arising from dentate granule neurons. Epileptogenic agents, such as pilocarpine or kainic acid, increase the synthesis of netrin by CA3 neurons. Mossy fiber terminals that synapse on CA3 neurons contain the DCC receptor. Moreover, these terminals sprout new synaptic connections following seizure (Sloviter R.S., Ann. Neurol., 35:640-654, 1994). Granule cell mossy fibers projecting to CA3 neurons contain BDNF (Fawcett, supra) and the netrin receptor DCC, while CA3 neurons on which they synapse contain trkB (Yan Q. et al., J. Comp. Neurol. 378: 133-157, 1997) and netrin. The model we have developed suggests that BDNF released presynaptically from mossy fibers induces netrin production post-synaptically by CA3 neurons (Fig. 9B). We propose that post-synaptic release of netrin acts as a synaptic retrograde messenger that evokes a response presynaptically in the mossy fibers through DCC to alter synaptic structure.

This relationship between BDNF release, trkB activation, and increased netrin expression can be extended to other regions of the CNS. In the spinal cord the proximal axon of dorsal root ganglion (DRG) neurons projects into the upper lamina of the dorsal spinal cord. At the pre-synaptic nerve terminal, there is an accumulation of BDNF containing vesicles (Michael G.J. et al., J. Neurosci. 17:8476-8490, 1997). In this region netrin protein is distributed on the dendrites of post-synaptic neurons. Again, in this context trkB is present post-synaptically, again overlapping with netrin expression, adding support to the relationship between neurotrophin signaling and netrin expression (Yan, supra). Netrin-1 mRNA and protein is expressed in oligodendrocytes

We have determined that netrin-1 mRNA and netrin protein is present in cells with an oligodendroglial morphology in the adult rat and mouse spinal cord. We have confirmed the identity of these cells using a transgenic mouse line in which the oHgodendrocyte lineage is marked through expression of the lacZ reporter gene operably linked to the myelin basic protein promoter. This finding is the first indication that the extracellular signals that guide the extension of developing axons may also be used to promote oHgodendrocyte migration, direct oligodendrocytes or their processes to their axonal targets, and/or promote oligodendroglial-axonal interactions. Regulation of netrin expression in the oligodendroglial lineage is likely to be important in promoting oligodendroglial maturation, migration, myelination, and/or oligodendroglial-axonal interactions following demyelinating damage such as that characteristic of multiple sclerosis. The invention described herein allows for a screen for genes or compounds that modify the action of downstream components of tyrosine kinase signaling and hence affect netrin expression. This may be performed, for example, in cell lines or primary neuronal or glial cell cultures that express receptor tyrosine kinases such as trk family members. In these cells, netrin protein and mRNA levels are assayed using standard techniques, such as northern blots, western blots, and ELISAs, described in, for example, Ausubel, supra.

One can also search for the gene products that control netrin expression by isolating and characterizing the genomic control elements contained in the netrin promoter. Identification of these sequences provide reporter elements useful in drug screens for compounds that modify netrin expression. Thus far, we have isolated a >100 kilobases (kb) bacterial artificial chromosome containing and including the coding region and putative gene regulatory sequences for mouse netrin. Furthermore, we have isolated a series of restriction fragments containing netrin coding sequence and 5 -prime upstream sequence. Using standard techniques known to those skilled in the art, such as those described in Ausubel et al. et al., 1997, Current Protocols in Molecular Biology, Wiley Interscience, New York, these fragments are subcloned into plasmid vectors containing reporter genes. The expression of these reporter genes in these vectors will be regulated by the netrin regulatory sequences. A reporter cell or animal, containing the reporter gene operably linked to the netrin promoter, is then constructed for use in screening assays for genes or compounds.

Another approach is to use transgenic animals, such as the lacZ -netrin fusion mice previously described (Serafini et al.(1996), supra). Reporter gene expression is then measured following treatment of animals with candidate compounds. Alternatively, primary cell cultures, or cell lines, derived from these animals are used in screening assays as described herein.

Libraries of genes and compounds can each be screened using the methods described herein. In one approach, candidate genes are screened for the ability to modulate synaptic rearrangement or migration of tumor cells, oligodendrocytes, or oHgodendrocyte precursors by monitoring expression from a netrin promoter. For example, the netrin promoter can be fused to a gene that confers a growth advantage when gene expression is increased. For example, cells with mutations in one of a variety of cell cycle genes display growth inhibition. The mutated gene can be placed under control of the netrin promoter. A library is then screened for genes or compounds that increase expression from the netrin promoter and, thus, allow for cell growth. Those skilled in the art will recognize that other positive selection systems will also work in this system.

In another example, gene fusions that link the netrin promoter to a gene product that confers a growth disadvantage (or, in the most extreme case, death) can be used to select for genes and compounds that down-regulate netrin expression. In this case, any gene or compound that decreases and/or eliminates netrin expression will alleviate the growth disadvantage and allow cells to grow. One example of a negative selection system is described by Shiau A.L. et al, Gene Ther. 5: 1571-1574, 1998, in which the expression of the E. coli cytosine deaminase in the presence of 5-fluorocytosine results in cell death. Those skilled in the art will recognize that other negative selection systems will also work in this system.

While the systems described above allow for the selection for or against netrin expression, similar technologies can be used to screen for changes in netrin expression. Other examples of useful promoter-reporter gene fusions that can be quantified include, but are not limited to, those encoding enzymatic or fluorescent reporters such as beta-galactosidase, chloramphenicol acetyltransferase, luciferase, and green fluorescent protein (GFP). Cells expressing a reporter gene from the netrin promoter can be mutagenized; cells that have altered reporter gene expression are likely to contain a mutation in a gene that regulates netrin expression. These cells can be isolated and the gene identified by standard techniques.

The selection and screening systems described here have a number of potential applications beyond those stated above. For example, deletion constructs can be generated in order to map the cis-elements of netrin promoter required for regulation by the trk signal transduction pathway.

Compounds that modify the effects of trkB activation on netrin expression may be useful in altering synaptic organization in brain regions that are producing abnormal electrical activity, such as in seizures leading to epilepsy. In addition, abnormal neuronal activity and/or abnormal neural circuitry have been suggested to underlie the pathology of schizophrenia (for review see Barondes S.H. et al., Proc. Natl. Acad. Sci. U.S.A., 94: 1612-1614). Similarly, these compounds may be useful in the treatment of chronic pain by modifying synaptic organization of the DRG afferent fibers in the spinal cord (Woolf C.J. et al., Nature 355:75-78, 1992) . These compounds may also be helpful in the treatment of drug addiction, since some drugs may induce synaptic reorganization; this reorganization is believed to be central to the addictive properties of certain drugs (Nestler E.J. et al., Neuron 11 :995-1006, 1993; Thiele T.E. et al., Nature 396:366-369, 1998). Thus, compounds that inhibit communication between neurons may reverse or prevent this reorganization. In addition, the presence of trk receptors has been linked to the motility and invasiveness of tumor cells (Matsumoto, supra); the effector mechanisms producing this increased motility are not known. Our observation that netrin is a downstream effector of neurotrophin- mediated trk receptor activation is consistent with a role for netrin, produced by tumor cells, promoting the tumor cell's mobility via an autocrine mechanism. We have found that netrin and its receptor, DCC, are both produced by cultured brain derived glial cells. Reagents that promote netrin function would promote glial activation while reagents that interfere with netrin function would decrease glial activation (see Fig. 8). As such, identifying compounds that interfere with trk-mediated upregulation of netrin expression may play a significant role in developing drugs that interfere with tumor cell migration.

Primary screens for compounds that modulate netrin activity, synaptic plasticity, or tumor cell migration

Modulating netrin expression or biological activity regulates neuronal synaptic plasticity and glial tumor cell migration. The finding that netrin expression is regulated by BDNF allows us to provide assays for drugs that modulate synaptic plasticity by monitoring netrin gene or protein expression. Similarly, the present invention provides assays for drugs that inhibit cell migration. Such assays may measure netrin expression using selection-based screens as described above, or by measuring changes in: (a) levels of netrin protein; (b) levels of netrin RNA; (c) levels of netrin-induced synaptic plasticity; (d) levels of trkB phosphorylation; or (e) levels of a reporter gene or protein expressed from a netrin promoter. The latter measurements may be made in vitro or in vivo. These assays allow for the identification of compounds that modulate synaptic plasticity and/or cell migration. Such identified compounds may have therapeutic value in the treatment of memory loss, drug addiction, epilepsy, schizophrenia, brain tumors, multiple sclerosis, and the regulation of pain.

Cells overexpressing netrin can be produced using standard techniques. A neuron, such as a septal neuron, a hippocampal neuron, a spinal cord neuron, or a dorsal root ganglion neuron, overexpressing netrin will likely exhibit a phenotype similar to those exposed to BDNF: increased branching, more neurites, and greater neuritic length. These netrin-overexpressing neurons, exhibiting "hypermorphic" behavior, are suitable for screening for compounds that block BDNF signaling genetically downstream from netrin. Compounds that are identified may bind to netrin or a netrin receptor, such as DCC, and prevent netrin signaling. A strategy similar to the one described above can also be implemented using glial cells or a cell line in which netrin is overexpressed. While screening of compounds can be performed with cultures of primary neurons or glia, cell lines be also be used. Suitable cell lines include any cell lines that exhibit altered netrin expression following exposure to BDNF, NGF, NT-3, or NT-4/5, such as SN 48 cells, described herein, as well as PC 12 (ATCC accession no. CRL-1721), NG108 (HB-12317), 3T3 fibroblasts (CRL- 6361), C6 (CRL-107) and P19 (CRL-1825) cell lines. Cell lines can derived from human glial tumors (e.g., CRL-162) are also suitable for use in compound screening assays.

Cell lines can be modified such that the cells exhibit constitutive activation of the trk receptor signal transduction cascade, including increased expression of netrin. Those skilled in the art will recognize that there are numerous ways to achieve constitutive activation, including, for example, transforming a cell line with a truncated trk receptor that contains the transmembrane and intracellular domains (including the kinase domain). This truncated receptor can dimerize and activate signaling without the presence of ligand (Baxter G.T. et al., J. Neurosci. 17:2683-2690, 1997). Any cell line, such as ones described above, can also be engineered to contain a reporter gene expressed under control of the netrin promoter (described above). A preferred reporter gene encodes for GFP. Typically, the expression of the gene (e.g., the endogenous netrin gene or a recombinant reporter gene expressed under the control of the netrin promoter or fragment thereof) is measured by assaying the RNA or protein levels or both of the expressed gene. For example, the polypeptide expressed by the netrin gene or by the reporter gene produces a detectable signal under conditions such that the compound causes a measurable signal to be produced. Quantitatively determining the amount of signal produced requires comparing the amount of signal produced to the amount of signal detected in the absence of any compound being tested or upon contacting the cell with any other compound, as is described herein. The comparison permits the identification of the compound as one that causes a change in the detectable signal produced by the expressed gene (e.g., at the RNA or protein level) and thus identifies a compound that is capable of inhibiting netrin expression. A decrease in the expression of netrin is likely to be accompanied by an inhibition of synaptic rearrangement in neurons, an inhibition of cell migration, or both.

Compounds identified using the reporter-based screen described above are likely to be genetically upstream of netrin (in contrast to the screens using the netrin-overexpressing cells, also described herein).

Secondary screens for compounds that modulate netrin activity and neuronal cell death

After test compounds that appear to modulate netrin expression, synaptic plasticity, or tumor cell migration are identified, it may be necessary or desirable to subject these compounds to further testing. The invention provides such secondary confirmatory assays. For example, a compound that appears to inhibit netrin activity in early testing will be subject to additional assays to confirm that the compound also inhibits tumor cell migration. Late-stage testing will be performed in vivo to confirm that the compounds initially identified to modulate netrin biological activity in cultured neurons will have the predicted effect on neurons in vivo. In the first round of in vivo testing, synaptic modification is initiated in animals, by well-known methods such as kainic acid treatment (described herein), and the compound is administered by one of the means described in the "Therapy" section, below. Neurons or neural tissue are isolated within hours to days following the insult, and are subjected to assays to assess the level of netrin expression. Such assays are well known to those skilled in the art. Examples of such assays include, but are not limited to, ELISAs, Western blot analysis, RT-PCR, RIA, and Northern blot analysis.

Test Compounds

In general, novel drugs for modulation of synaptic reorganization or tumor cell migration that functions by targeting netrin biological activity are identified from large libraries of both natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively , libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods. In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their therapeutic activities for neurodegenerative disorders should be employed whenever possible.

When a crude extract is found to modulate synaptic reorganization, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having netrin-modulating activities. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using a mammalian epilepsy model, as described herein, or, alternatively, a mammalian chronic pain model, a cell migration assay, or a brain tumor model (described in: Woolf, supra; Albini A. et al, Proc. Natl. Acad. Sci. USA 92:4838-4842, 1995, Koochekpoor S. et al, Cancer Res. 57:5391-5398, 1997; and Tamaki M. et al., J. Neurosurg. 87:602-609, 1997, respectively).

Therapy

Compounds, identified using any of the methods disclosed herein, may be administered to patients or experimental animals with a pharmaceutically- acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients or experimental animals. Although intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, for example, Remington: The Science and Practice of Pharmacy. (19th ed.) ed. A.R. Gennaro AR., 1995, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or Polyoxyethyl ene-polyoxypropyl ene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for antagonists or agonists of the invention include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

As described herein, we have discovered that netrin protein levels increase during synaptic reorganization. It is believed that synaptic reorganization is central to numerous neurologically-based disorders, including drug addiction, epilepsy, schizophrenia, chronic pain, and hyperalgesia. As also described herein, netrin may mediate tumor cell migration. Hence, any compound that prevents the increase in netrin protein levels or blocks the function of netrin, is a candidate compound for therapy in a patient with one of the foregoing disorders. One possible compound is a polypeptide fragment of netrin which maintains its ability to bind to a netrin receptor but has lost its ability to initiate signal transduction. Such a polypeptide will act as an inhibitor of signaling by wild-type netrin protein. Also included in this group of compounds is any compound identified using the screens described herein, as well as RNA that is antisense to netrin mRNA.

Administration of a compound that decreases or prevents netrin expression or function is also appropriate before, during, or after administration of a narcotic such as morphine for the alleviation of pain resulting from invasive surgery, amputation, or the like. Such administration serves to prevent the synaptic reorganization following the surgery itself (due to inflammatory pain or hyperalgesia), as well as any synaptic reorganization due to the administration of an addictive substance.

Other Embodiments

All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the appended claims.

Claims

What we claim is:

1. A method for identifying a candidate compound that decreases synaptic reorganization, said method comprising:

(a) providing a cell expressing recombinant netrin; (b) contacting said cell with a candidate compound; and

(c) detecting or measuring synaptic reorganization following contact of said cell with said candidate compound, wherein a decrease in synaptic reorganization identifies said candidate compound as a compound that decreases synaptic reorganization.

2. A method for identifying a candidate compound that decreases cell migration, said method comprising:

(a) providing a cell expressing recombinant netrin;

(b) contacting said cell with a candidate compound; and

(c) detecting or measuring cell migration following contact of said cell with said candidate compound, wherein a decrease in cell migration identifies said candidate compound as a compound that decreases cell migration.

3. The method of claim 1 or 2, wherein said candidate compound inhibits or decreases said cell's response to BDNF.

4. A method for identifying a gene which modulates netrin expression, said method comprising:

(a) expressing in a cell (i) a first gene operably linked to a netrin gene promoter and (ii) a second candidate gene or a fragment thereof; and (b) monitoring the expression of said first gene, wherein a change in said expression identifies said candidate gene as a gene which modulates netrin expression.

5. The method of claim 1 or 4, wherein said cell is a neuron.

6. The method of claim 5, wherein said neuron is selected from the group consisting of a septal neuron, a hippocampal neuron, a spinal cord neuron, and a dorsal root ganglion neuron.

7. The method of claim 2 or 4, wherein said cell is a glial cell.

8. The method of claim 4, wherein said candidate gene modulates said cell's response to BDNF.

9. A method for determining whether a candidate compound decreases the expression of a gene operably linked to a netrin promoter, said method comprising:

(a) providing a cell expressing a gene operably linked to a netrin promoter;

(b) contacting said cell with a candidate compound;

(c) detecting or measuring expression of said gene following contact of said cell with said candidate compound; and

(d) determining whether said candidate compound decreases the expression of said gene.

10. The method of claim 9, wherein said cell is a neuron and wherein a decrease in the expression of said netrin gene inhibits synaptic reorganization by said neuron.

11. The method of claim 9, wherein said cell is a glial cell wherein a decrease in the expression of said netrin gene inhibits migration of said glial cell.

12. A method for determining whether a candidate compound increases the expression of a gene operably linked to a netrin promoter, said method comprising:

(a) providing a cell capable of expressing a gene operably linked to a netrin promoter;

(b) contacting said cell with a candidate compound;

(d) determining whether said candidate compound increases the expression of said gene.

13. The method of claim 12, wherein said cell is a neuron and wherein an increase in the expression of said netrin gene promotes synaptic reorganization by said neuron.

14. The method of claim 2, 4, 9, or 12, wherein said cell is from a cell line.

15. The method of claim 14, wherein said cell line is selected from the group consisting of SN 48, PC12, NG108, 3T3, and P19.

16. The method of claim 9 or 12, wherein said gene comprises a netrin gene.

17. The method of claim 16, wherein said netrin gene is netrin-1.

18. The method of claim 9 or 12, wherein said gene comprises a reporter gene.

19. The method of claim 18, wherein said reporter gene is gfp.

20. The method of claim 9 or 12, wherein said netrin promoter is the netrin-1 promoter.

21. The method of claim 9 or 12, wherein said netrin promoter is a fragment or deletion of a netrin promoter.

22. The method of claim 9 or 12, wherein said gene expression is measured by assaying the protein level of the expressed gene.

23. The method of claim 9 or 12, wherein said gene expression is measured by assaying the RNA level of the expressed gene.

24. The method of claim 9 or 12, wherein said candidate compound promotes or increases said cell's response to BDNF.

25. A method for inhibiting synaptic reorganization by a neuron, said method comprising the step of contacting said neuron with a compound that decreases netrin expression.

26. A method for inhibiting migration by a tumor cell, said method comprising the step of contacting said tumor cell with a compound that decreases netrin expression.

27. A method of isolating a gene that modulates cell migration or synaptic plasticity, said method comprising:

(a) providing a cell expressing a first gene operably linked to a netrin gene promoter;

(b) mutagenizing said cell;

(c) measuring expression of said first gene, wherein an increase or decrease in the expression of said first gene identifies a mutation in a second gene; and

(d) using said mutation as a marker for isolating said second gene, wherein said second gene modulates cell migration or synaptic plasticity.

28. The method of claim 27, wherein said cell is selected from the group consisting of an SN 48, PC12, NG108, 3T3, and a P19.cell

29. The method of claim 4 or 27, wherein said first gene comprises a netrin gene.

30 . The method of claim 29, wherein said netrin gene is netrin-1.

31. The method of claim 4 or 27, wherein said first gene comprises a reporter gene.

32. The method of claim 31 , wherein said reporter gene is gfp.

33. The method of claim 4 or 27, wherein said netrin gene promoter is the netrin-1 gene promoter.

34. The method of claim 4 or 27, wherein said expression of said first gene is measured by assaying the protein level of the expressed gene.

35. The method of claim 4 or 27, wherein said expression of said first gene is measured by assaying the RNA level of said first gene.

36. The method of claim 1, 2, 4, 9, 12, or 27, wherein said cell is in vitro.

37. The method of claim 1, 2, 4, 9, 12, or 27, wherein said cell is in vivo.

38. A method for decreasing synaptic reorganization in a mammal, comprising administering a composition comprising a compound that decreases netrin signaling.

39. The method of claim 38, wherein said mammal has epilepsy, a drug addiction, schizophrenia, chronic pain, or hyperalgesia.

40. The method of claim 38, wherein said mammal is having a surgical procedure.

41. The method of claim 40, wherein said surgical procedure is an amputation.

42. The method of claim 40, wherein said surgical procedure is accompanied by the administration of an addictive substance.

43. A method for decreasing cell migration in a mammal, comprising administering a composition comprising a compound that decreases netrin signaling.

44. The method of claim 43, wherein said mammal has a tumor.

45. The method of claim 44, wherein said tumor is a glial tumor.