WO2000053735A9 - Methods for identifying modulators of synaptic plasticity and cell motility - Google Patents
Methods for identifying modulators of synaptic plasticity and cell motilityInfo
- Publication number
- WO2000053735A9 WO2000053735A9 PCT/IB2000/000505 IB0000505W WO0053735A9 WO 2000053735 A9 WO2000053735 A9 WO 2000053735A9 IB 0000505 W IB0000505 W IB 0000505W WO 0053735 A9 WO0053735 A9 WO 0053735A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- netrin
- cell
- expression
- candidate compound
- Prior art date
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Definitions
- the invention relates to methods of altering synaptic structure and cellular motility.
- Brain derived neurotrophic factor 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.
- 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 neuroSlastoma 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.
- 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.
- the invention features a method for identifying a compound that modulates synaptic reorganization or cell migration.
- 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.
- 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, PI 9, or a glioma cell line.
- a decrease in the expression of the netrin gene inhibits synaptic reorganization by a neuron or inhibits migration of a glial cell.
- the candidate compound also inhibits or decreases the cell's response to BDNF.
- 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.
- 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, PI 9 or a glioma cell line.
- an increase in the expression of the netrin gene promotes synaptic reorganization by a neuron or promotes migration of a glial cell.
- the candidate compound also promotes or increases the cell's response to BDNF.
- the gene is a netrin gene, such as netrin- 1, or the gene is a reporter gene, such as gfp.
- 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.
- the cell is in a mammal, such as a mouse. In one embodiment, the mammal is a transgenic mammal.
- 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.
- the cell is a neuron, such as a septal neuron, a hippocampal neuron, a spinal cord neuron, or a dorsal root ganglion neuron.
- the candidate compound also inhibits or decreases the cell's response to BDNF.
- 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.
- 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.
- the candidate compound also inhibits or decreases the cell's response to BDNF.
- the invention features a method for inhibiting synaptic reorganization by a neuron by contacting the neuron with a compound that decreases netrin expression.
- the invention features a method for inhibiting migration by a tumor cell by contacting the tumor cell with a compound that decreases netrin expression.
- 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.
- 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.
- the cell is an
- the first gene comprises a netrin gene, (e.g., netrin- J) or a reporter gene (e.g., gfp).
- 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.
- the invention features a method for decreasing synaptic reorganization in a mammal, including administering a composition that decreases netrin signaling.
- the mammal has epilepsy, a drug addiction, schizophrenia, chronic pain, or hyperalgesia.
- the mammal is having a surgical procedure (e.g., an amputation); and the surgical procedure is accompanied by the administration of an addictive substance.
- the composition includes a netrin antisense RNA sequence.
- the composition includes an antibody direct to netrin.
- the invention features a method for decreasing cell migration in a mammal, including administering a composition that decreases netrin signaling.
- the mammal has a tumor (e.g., a glial tumor).
- the composition includes a netrin antisense RNA sequence.
- the composition includes an antibody directed to netrin.
- the invention features a method for increasing cell migration in a mammal, including administering a composition that increases netrin signaling.
- the mammal has multiple sclerosis.
- 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.
- 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.
- 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.
- GAP-43 synapsin, synaptophysin, CDC42, rac, rho, and the actin modifying enzyme NWASP may be increased during synaptic reorganization.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- reporter proteins Many different types 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
- promoter any minimal nucleic acid sequence sufficient to direct transcription of the reporter gene.
- elements e.g., enhancers or suppressors
- elements 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.
- 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.
- the antisense nucleic acid is capable of inhibiting or decreasing synaptic reorganization or cell migration when present in a cell.
- the decrease is at least 10%, relative to a control, more preferably 25%, and most preferably 1-fold or more.
- 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 ⁇ m.
- 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. 4 A) 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.
- 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 ⁇ g/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
- 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.
- BDNF 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.
- Fig. 1 A. 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
- Fig. 2 and Fig. 3 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 ⁇ m sections cut on a cryostat. Antigenicity was enhanced by immersing sections in boiling PBS. Sections were processed with antibodies as follows.
- PBS phosphate buffered saline
- 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. 3 A, 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.
- BDNF 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 10 7 cells/ml in tissue culture dishes.
- the medium contained 500 ⁇ M glutamine, 25 ⁇ M 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.
- 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.
- Tris-buffered saline TBS
- the lysis buffer contained 137 mM NaCl, 20mM Tris, pH 8.0, l%(v/v) NP40, 10% (v/v) glycerol, 1 mM PMSF, 10 ⁇ g aprotinin, and 0.2 ⁇ g leupeptin. After 20 minutes of rocking, the lysate was removed and centrifuged at 15,000 RPM for 15 minutes.
- C l ure ⁇ c t i iiBurQas produced netrin is the presence but not the absence of BDNF (fig. 5A)-
- BDNF induced a significant increase in netrin production above basal levels in primary cultures of hippocampal pagans and in culnires of differentiated SN 48 cells (Fig. 5C), Together, these data demonstrate an increase in netrin expression following BDNF treat ent.
- Kainic acid ireaunent induces netrin expression in vivo
- 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 ⁇ g/ml) were also applied. In the presence of blocking antibodies, the effect of BDNF was strongly attenuated.
- Netrin produced by post-synaptic neurons 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).
- BDNF, trkB activation, and coincident depolarization of the post-synaptic cell together promote the synthesis and release of netrin.
- 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).
- 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).
- Fig. 8 In the control condition, most cells have a flat morphology and express a low level of GFAP (left panel).
- recombinant netrin protein (170 ng/ml) cultured astrocytes exhibit well developed processes characteristic of an activated glial phenotype (center panel).
- BDNF neurotrophic factor
- trkB trkB
- DCC netrin
- 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.
- 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
- netrin- 1 mRNA and netrin protein is present in cells with an oligodendroglial morphology in the adult rat and mouse spinal cord.
- 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.
- 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.
- primary cell cultures, or cell lines, derived from these animals are used in screening assays as described herein.
- genes and compounds can each be screened using the methods described herein.
- 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.
- the netrin promoter can be fused to a gene that confers a growth advantage when gene expression is increased.
- 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.
- gene fusions that link the netrin promoter to a gene product that confers a growth disadvantage can be used to select for genes and compounds that down-regulate netrin expression.
- any gene or compound that decreases and/or eliminates netrin expression will alleviate the growth disadvantage and allow cells to grow.
- 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.
- 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).
- enzymatic or fluorescent reporters such as beta-galactosidase, chloramphenicol acetyltransferase, luciferase, and green fluorescent protein (GFP).
- GFP green fluorescent protein
- 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.
- 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).
- 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) .
- 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.
- 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).
- 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.
- 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.
- 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.
- CRL-162 human glial tumors
- Cell lines can be modified such that the cells exhibit constitutive activation of the trk receptor signal transduction cascade, including increased expression of netrin.
- 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.
- 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
- the expression of the gene is measured by assaying the RNA or protein levels or both of the expressed gene.
- 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.
- 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.
- 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.
- 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.
- 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.
- test extracts or compounds are 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.
- Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI).
- 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).
- natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods.
- any library or compound is readily modified using standard chemical, physical, or biochemical methods.
- dereplication e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof
- elimination of replicates or repeats of materials already known for their therapeutic activities for neurodegenerative disorders should be employed whenever possible.
- 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).
- 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
- 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.
- 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.
- 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 Poly oxy ethyl ene-polyoxypropyl ene copolymers may be used to control the release of the compounds.
- 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.
- 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.
- 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.
- 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.
- a narcotic such as morphine
- 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.
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US5824775A (en) * | 1996-04-19 | 1998-10-20 | The Regents Of The University Of California | Human netrin-1 |
US5939271A (en) * | 1997-02-19 | 1999-08-17 | The Regents Of The University Of California | Netrin receptor |
WO1999061585A1 (en) * | 1998-05-22 | 1999-12-02 | University Technology Corporation | Assays for identifying anti-metastatic and neurite-promoting agents and uses thereof |
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2000
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AU3667600A (en) | 2000-09-28 |
WO2000053735A3 (en) | 2000-12-28 |
WO2000053735A2 (en) | 2000-09-14 |
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