MXPA99004578A - Post-mitotic neurons containing adenovirus vectors that modulate apoptosis and growth - Google Patents

Abstract

A postmitotic neuron containing an adenovirus vector, the neuron having been infected with the adenovirus vector at a multiplicity of infection of approximately 10 to approximately 50, and expressing a gene product encoded by a DNA molecule contained within said vector.

Description

POST-ITOTIC NEURONS CONTAINING ADENOVIRUS VECTORS THAT MODULATE APOPTOSIS AND GROWTH Field of the Invention The field of the invention is neurobiology. BACKGROUND OF THE INVENTION Scheduled cell death (apoptosis) is a continuous process in both the developing and mature nervous systems. In the developing nervous system, neurons suffer apoptosis, unless they receive an adequate supply of neurotrophic substances from the target (eg, the muscle) they innervate. In the mature nervous system, apoptosis occurs in the course of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, which progress slowly over long periods of time, and in acute neurological attacks, such as stroke. Therefore, understanding the manner in which apoptosis is regulated is an important step towards the development of effective treatments for neurodegenerative diseases and stroke. Apoptosis can be induced in a number of different cell types by over-expression of a tumor suppressor gene called p53 (reviewed in Elledge et al., Bioessays 17: 923-930, 1995; White, Genes Dev. 10: 1 -15, 1996). Next to DNA damage, which can cause the cell to proliferate uncontrollably, p53 is expressed, and helps prevent tumor formation by activating the expression of other genes, such as the cyclin kinase inhibitor p21 (also known as WAF-1), which mediates the arrest of the cell cycle and prevents the propagation of damaged DNA (Harper et al., Cell 75: 805-816, 1993; Xiong et al., Nature 366: 701-704, 1996) . The cellular response to p53 overexpression, however, can vary, depending on the type of cell; instead of stopping cell growth, overexpression of p53 can lead to apoptosis (Katayose et al., Int. J. Oncol., 3: 781-788, 1995. Picksley et al. Current Opin. Cell Biol. 6: 853-858 , 1994, White, supra). The precise mechanism by which p53 mediates apoptosis in tumor cells is not well understood, nor is it known whether p53 is directly involved in the apoptosis of post-mitotic (ie non-proliferating) neurons. Recently, a number of studies have shown that adenovirus-based vectors can be used for the transduction of central nervous system neurons that have been cultured (Slack et al., Current Opin. Neuro-biol., 6: 576-583, 1996), but it is not known if these vectors can negatively impact the function of the recipient cell. For adenovirus-derived vectors to be useful for modulating apoptosis in neurons, their influence on the biochemistry and physiology of the neuron must be understood. SUMMARY OF THE INVENTION The invention provides a post-mitotic neuron containing an adenovirus vector that was applied to the neuron, under conditions (such as those described herein) that allow the neuron to be infected, at a multiplicity of infection, preferably, from 1 to 1,000 MOI (multiplicity of infection), more preferably from 1 to 500 MOI, and most preferably from about 10 to about 50 MOI. The neuron can express a genetic product encoded by a DNA molecule contained within the vector. In one embodiment, the post-mitotic neuron is infected while in a tissue culture. In a second embodiment, the post-mitotic neuron is infected in vivo. The invention also provides methods for manufacturing the cell in vi tro and in vivo. The adenovirus vector can be administered according to methods known to those skilled in the art, including intra-cerebrally, intraventricularly, intrathecally, transmucosally, intramuscularly, or subcutaneously. Preferably, the adenovirus vector is applied intramuscularly. The genetic product encoded by the DNA contained within the adenovirus vector can be a structural protein, an enzyme, a transcription factor, or a receptor, such as the low affinity nerve growth factor receptor (NGF) p75, or the high affinity nerve growth factor receptor Trk, or other members of the Trk family, including TrkB, TrkC, NT-3, and NT-4/5. Preferably, the genetic product is a tumor suppressor. More preferably, the genetic product is p53. Alternatively, or in addition, the adenovirus vector may contain DNA that encodes a reporter gene product or marker. Preferably, the reporter gene is alkaline phosphatase, chloramphenicol-acetyltransferase, lacZ, or green fluorescent protein. The invention also provides a method for inducing apoptosis in a post-itotic neuron, by infecting the neuron with an adenoviral vector containing DNA encoding a protein that induces apoptosis, such as p53. In addition, the invention provides a method for inducing apoptosis in a postmitotic neuron, by infecting the neuron with an adenoviral vector containing DNA encoding a protein that inhibits apoptosis, such as Bcl-2, BC1-XL, E1B55K, or Gabl. The invention also provides methods for identifying test compounds that inhibit or induce apoptosis, growth, or proliferation, comprising these methods: (a) culturing a population of post mitotic neurons; (b) infecting the neurons of this population with an adenovirus vector comprising DNA encoding a protein that induces (or inhibits) apoptosis (or growth or proliferation), this vector preferably being applied to neurons at a multiplicity of infection from about 10 to about 50; (c) exposing a subset of the population of neurons infected in step (b) to a test compound, this test compound being a candidate inhibitor (or inducer) of apoptosis, growth, or proliferation; and (d) comparing the approximate number of neurons suffering from apoptosis, growth, or proliferation, as appropriate, in the subset of the population that was infected and exposed to the test compound, with the approximate number of neurons suffering from apoptosis, growth, or proliferation in the population of cells that became infected. The invention can be practiced with DNA molecules that encode full-length proteins or fragments thereof that are biologically active. In a similar manner, the invention can be practiced with DNA molecules that differ from those described herein, by one or more conservative amino acid substitutions. Preferably, the protein contains less than 50 percent substituted amino acid residues, more preferably less than 30 percent substituted amino acid residues, and most preferably less than 10 percent substituted amino acid residues. "Biologically active" means possessing any in vivo or in vi tro activity that is characteristic of the full-length protein. A biologically active fragment generally possesses at least 40 percent, more preferably at least 70 percent, and most preferably at least 90 percent of the activity of the full-length protein. Preferably, the fragment mimics at least one activity of the full-length protein. "Conservative amino acid substitution" means substitution within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. "Apoptosis" means the process of cell death in which a dying cell exhibits a set of well-characterized biochemical signals that include cytolymic membrane blistering, cellular soma shrinkage, chromatin condensation, and DNA scaling. "Stimulus that can induce apoptosis" or "apoptotic stimulus" means any chemical or physical treatment that initiates apoptosis as defined above. For example, withdrawal of nerve growth factor, hypoxia, exposure to staurosporine, and cerebral ischemia, are stimuli capable of inducing apoptosis in neurons. "Neuron" means a cell of ectodermal embryonic origin derived from any part of the nervous system of an animal. Neurons express specific markers of well-characterized neurons that include β-tubulin class III, MAP2, and neurofilament proteins. Neurons include, without limitation, hippocampal, cortex, dopaminergic, midbrain, motor, sensory, and sympathetic neurons.

"Neuronal growth" means an increase in the network density of the process of approximately double or greater, an increase in total neurite length of approximately 1.5 times or more, an increase in cell size (area) of approximately 10 percent or more, preferably 25 percent or more, an increase in a-tubulin Tal mRNA of about five times or more, and / or an increase in tyrosine hydroxylase mRNA of approximately two or more times. "Expose" means allowing contact between an animal, cell, lysate, or extract derived from a cell, or molecule derived from a cell, and a test compound or apoptotic stimulus. "Treat" means subjecting an animal, cells, lysate, or extract derived from a cell, or molecule derived from a cell, to a test compound or apoptotic stimulus. "Test compound" means a chemical, whether occurring naturally or artificially derived, or being studied for its ability to modulate cell death, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof. "Testing" means analyzing the effect of a treatment, be it chemical or physical, administered to intact animals or cells derived from them. The material being analyzed can be an animal, a cell, a lysate, or an extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting altered gene expression, altered stability of RNA, altered protein stability, altered levels of protein, or altered biological activity of the protein. Means to analyze may include, for example, antibody labeling, immunoprecipitation, phosphorylation assays, and methods known to those skilled in the art for detecting nucleic acids. "Modulation" means change, either by decrease or increase. "A decrease" means a reduction in the level of: a) protein, or protein phosphorylation, measured by ELISA; b) reporter gene activity, at least 30 percent, measured by reporter gene assay, for example, lacZ / β-galactosidase, green fluorescent protein, luciferase, etc.; c) mRNA, levels of at least 30 percent, measured by polymerase chain reaction in relation to an internal control, for example a "maintenance" gene product, such as β-actin or glyceraldehyde-3-phosphate dehydroge-nase (GAPDH). In all cases, the reduction is preferably by 30 percent, more preferably by 40 percent, and still more preferably by 70 percent. "An increase" means an elevation in the level of: a) protein, or protein phosphorylation, measured by ELISA; b) activity of the reporter gene, measured by assay of the reporter gene, for example lacZ / β-galactosidase, green fluorescent protein, luciferase, et cetera; c) mRNA, measured by polymerase chain reaction in relation to an inter control (no, for example a "maintenance" gene product, such as β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). , the increase is twice, and more preferably triple. "Alteration in the level of gene expression" means a change in the activity of the gene, in such a way that the amount of a product of the gene is increased, or decreased. , ie mRNA or polypeptide, to increase or decrease the stability of the mRNA or polypeptide. "Reporter gene" means any gene that encodes a product whose expression can be detected and / or quantified by immunological, chemical, biochemical, or biological A reporter gene product, for example, may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ / β-galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin) A), or an ability to specifically bind to a second molecule (e.g., biotin or a detectably labeled antibody). It is understood that any designed variants of reporter genes are also included, which are already available to an expert in this field, without restriction, in the above definition. "Operably linked" means that a gene and a regulatory sequence are connected in such a way as to allow the expression of the genetic product under the control of the regulatory sequence. A "transgene" means a nucleic acid sequence that is artificially inserted into a cell, and becomes a part of that cell's genome and its progeny. This transgene may be partially or completely heterologous to the cell. "Transgenic animal" is an animal that comprises a transgene as described above. "Protein" or "polypeptide" or "polypeptide fragment" means any chain of more than two amino acids, independently of the modification after translation (eg, glycosylation or phosphorylation), which constitutes all or part of a polypeptide or peptide which occurs naturally, or which constitutes a polypeptide or peptide that does not occur naturally. All publications mentioned herein are incorporated by reference. Now the examples of the preferred methods and materials will be described. These examples are illustrative only, and are not intended to be limiting. Those skilled in the art will understand that methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Brief Description of the Drawings Figure 1 is a series of photographs (1A to 1F) illustrating the morphology of sympathetic neurons cultured following viral infection. Figures 1A, 1C, and 1E are photographs of neurons infected with adenovirus at 1, 10, and 100 MOI, respectively. Figures IB, ID, and 1F are photographs of neurons infected with herpes virus at 1, 10, and 100 MOI, respectively. The bar of Figure ID represents 200 microns, and the bar of Figure 1F represents 80 microns. Figures 2 (A-B) are bar graphs comparing the ineffectiveness of the recombinant adenovirus with HSV-1. Figure 2A shows the percentage of neurons infected with recombinant adenovirus in 1-1,000 MOI, and in Figure 2B the percentage of neurons infected with recombinant HSV-1 in 0.1-400 MOI is shown. The data are expressed as the average of two separate experiments; the error bars represent the range. The number of cells per field was 125 + 50 for experiment 1, and 200 + 59 for experiment 2. The data obtained in these two experiments were averaged to give the result shown in Figure 2.

Figure 3 is a bar graph comparing neuronal viability following infection with recombinant adenovirus and HSV-1. The percentage of sympathetic neurons that survived after infection with the recombinant adenovirus is shown by the solid black bars, and the percentage that survived the infection with recombinant HSV-1 is shown with the shaded bars. The bars represent the average of three separate samples + SEM. Figures 4 (A-D) are photographs illustrating changes in the cytoarchitecture of sympathetic neurons following infection with recombinant adenovirus. Figure 4A shows an uninfected sympathetic neuron, which has a normal cytoarchitecture (scale bar = 0.3 microns). Figure 4B shows the same type of neuron after infection with 10 MOI of adenovirus. These cells contain small dense electron inclusions in the nucleus (arrows, scale bar = 0.5 microns), but otherwise they can not be distinguished from uninfected neurons. Figure 4C shows the same type of neuron after infection in 50 MOI with an adenovirus vector. The cytoarchitecture is normal, except for small dense electron inclusions in electrons (scale bar = 0.4 microns). Figure 4D shows the same type of neuron infected at 100 MOI with adenovirus. The cytoarchitecture deteriorates in a significant way: disintegration of chromatin (asteris-eos), dense inclusions in very large electrons (arrow), and accumulations of filaments in the number, are evident (arrowhead) (scale bar = 0.7 microns) ). Figures 5 (AB) are traces representing the potassium currents of the voltage gate on sympathetic neurons expressing ß-galactosidase infected with adenovirus, and uninfected neurons (Figure 5A), and a pair of bar graphs representing the current densities in these cells. The voltage depolarization steps from a Vh of -10 mV activate only a slowly activating non-inactivating current (IK, shown in the uppermost traces of Figure 5A). IA (shown in the lower lines) was isolated by subtracting the currents from Vh of -40 mV (IK + IAs) of the corresponding currents evoked from a Vh of -90 mV (IK + IAs + IA). In Figure 5B, average current densities (pA / pF), which were measured as peak isolated potassium current (pA) divided by membrane capacitance (pF), for β-galactosidase-positive neurons ( n = 6); and uninfected control neurons. Figures 6 (A-B) are photographs showing that overexpression of human p53 is located in the nucleus of sympathetic neurons. Sympathetic neurons infected with a recombinant adenovirus encoding β-galactosidase, AdCA171acZ, are shown in Figure 6A, and comparable cells expressing human wild type p53, Ad tp53, are shown in Figure 6B. The arrows point to the pustotic nuclei that overexpress human p53 (scale bar = 20 microns). Figures 7 (A-C) are photographs showing Western blot analysis of p53 (Figures 7A and 7B) following transduction of sympathetic neurons, and a fragmented DNA agarose gel analysis (Figure 7C). In Figure 7A, Lane 1 contains protein harvested from uninfected neurons, and Lane 2 contains protein harvested from neurons infected with Adwtp53, which express p53. In Figure 7B, Lane 1 contains protein harvested from uninfected neurons, Lane 2 contains protein harvested from neurons infected with 50 MOI of pAdCA17-lacZ, and Lane 3 contains protein harvested from neurons infected with 60 MOI of Ad tp53. Figure 7C is a photograph of a 2 percent agarose gel, over which DNA labeled in end was electrophoresed from neurons infected with 50 MOI of recombinant adenovirus AdCal71acZ (lane 1), or with Ad tp53 (lane 2) . Figures 8 (AB) are line graphs showing the percentage of surviving neurons after infection in different MOIs (Figure 8A), at different times (Figure 8B), with wild-type adenovirus (O), recombinant adenovirus (Ad5CAI7LacZ; LJ), and a recombinant adenovirus carrying the wild-type p53 (Ad tp53; H). Figures 9 (A-F) are photographs of infected neurons following dyeing with TUNNEL. The cells shown in Figures 9A and 9B were photographed at low amplification (scale bar = 400 nanometers), to show a representative view of the cell populations expressing lacZ (9A) and p53 (9B). In a higher amplification, and seen with a phase contrast microscope, the degeneration of the neuritic processes is shown in the cells infected with Ad tp53 (9E) in relation to the cells infected with Ad5CA171acZ (9C). TUNING with corresponding TUNNEL shows pyknotic nuclei, which indicate apoptosis, in cells infected with Adwtp53 (9F). Figure 10 is a graph showing that recombinant adenoviruses expressing the p53 inhibitor, E1B55K, rescue postmitotic sympathetic neurons from death induced by NGF withdrawal. Figure 11 is a graph showing that recombinant adenoviruses expressing p53 and MEKK1, annihilate post mitotic sympathetic neurons in the presence of NGF, while recombinant adenoviruses expressing the anti-apoptotic proteins Bcl-2 and Bcl-xL, rescue neurons from death induced by NGF withdrawal. Figure 12 is a graph showing that a recombinant adenovirus expressing Gabl mediates the survival of post mitotic sympathetic neurons. Figure 13 is a graph showing that the post-mitotic injection of a recombinant adenovirus expressing TrkB mediates the survival of sympathetic neurons.

Figure 14 is a graph showing that induction of neuron survival by TrkB encoded by adenovirus requires both Shc / Ras / PI-3 kinase activation sites and phospholipase C (PLC) activation sites - gamma 1 of TrkB. Figures 15 (A-B) are photographs illustrating infected cells in vivo in the upper cervical ganglia. In Figure 15A, two upper cervical nodes are shown; the ganglion on the left side was harvested following the injection of the adenovirus on that same side (ie, the ipsilateral side), and the ganglion on the right side was harvested from the contralateral 'non-injected side of the same animal. In Figure 15B, the left side ganglion of Figure 15A is shown at a higher amplification. Scale bar of Figure 15A = 300 microns; scale bar in Figure 15B = 240 microns. Figure 16 is a schematic diagram illustrating wild-type p75 (higher illustration), deletion mutations, and substitution mutants. The region of homology with fas and TNFR1 is shown as a dotted bar in the intracellular domain. The potential G protein activation domain is shown as a solid bar. Substitution mutants are indicated by underlining. Figures 17 (A-C) show that cortical progenitor-ras cells and post mitotic cortical neurons efficiently express the proteins encoded by recombinant adenovirus. Cortical progenitor cells (A) and post mitotic neurons (B) were infected with AdlacZ and stained with X-gal. (C) Western blot of cortical progenitor cells infected with a recombinant adenovirus encoding ElA. Figures 18 (A-B) are graphs showing the survival of cortical progenitor cells (A) and post mitotic neurons (B) infected with recombinant adenoviruses encoding lacZ (AdlacZ) and encoding ElA (AdllOl). Survival was measured by MTT assay. Figures 19 (A-L) are photomicrographs showing a comparison of cell viability of cortical progenitor cells and post mitotic neurons infected with recombinant AdlacZ and AdllOl. Figures 20 (A-D) are photomicrographs showing 6-day-old cultures of post-mitotic cortical neurons E18. (A) phase contrast micrograph of (B), and (B) immunoblot anti-BrdU after a 12 hour incubation with BrdU. (C) phase contrast micrograph of (D), and (D) immunostained anti-MAP2. Figures 21 (A-F) are photomicrographs showing that the survival of post mitotic cortical neurons is not affected by functional ablation of members of the pRb family. Figures 22 (A-E) are photomicrographs showing that the growth of sympathetic neurons dependent on NGF respond to NT-3, but not to BDNF. Phase contrast micrographs of cultures of pure sympathetic neurons from 1-day post-natal SCG rat maintained at 10 nanograms / milliliter of NGF for 5 days (A), and then supplemented with 30 nanograms / milliliter of NT-3 ( B) or 30 nanograms / milliliter of BDNF (C). NT-3 improved the number of neurites compared to BDNF, when it was examined two days after the addition. In similar cultures, where NGF was replaced with nanograms / milliliter of BDNF (D), obvious deterioration of the cell body and process was evident. Figures 23 (AD) are graphs showing the density of the process network, the total neurite length, and the cell size of the sympathetic neurons grown in NGF only, or NGF + NT-3, as indicated on the axis X. Detailed Description The compositions and methods described herein provide a means to efficiently transfect postmitotic cells, such as neurons, with adenovirus vectors. The vectors can be used to express useful genes, such as the p53 tumor suppressor gene, and the growth factor receptor genes Trk and p75. The assays described herein may be used to test compounds that decrease cell death and / or stimulate cell growth., and therefore, may have a therapeutic value in the treatment of neurodegenerative disease and neurological trauma. The assays can also be used to screen compounds for the inhibition of neural cell growth and / or for neurotoxicity, these compounds being useful as cancer pesticides or therapeutics, for example. Secondary traces of test compounds that appear to modulate neuronal death After test compounds that appear to have neuronal death and / or growth modulation activity are identified, it may be necessary or advisable to subject these compounds to further testing. The invention provides these secondary confirmation assays. For example, a compound that appears to inhibit neuronal death in the first test will be subject to additional tests to determine whether the compound can stimulate neuronal growth. In later stages, the live test will be performed to confirm that the compounds that were initially identified that affected cell death in cultured neurons have the predicted effect on neurons in vivo. In the first round of live testing, neuronal cell death is initiated in animals, by well-known methods, such as axotomy or cerebral ischemia, and then the compound is administered by one of the means described in the Therapy section immediately below. . Neurons or neuronal tissue are isolated within hours to days following the attack, and are subjected to assays as described in the examples below. Test Compounds In general, novel drugs for the prevention or treatment of neuronal cell death or growth are identified from large libraries of both natural and synthetic (or semi-synthetic) product extracts, or chemical libraries according to the methods known in this field. 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 methods of the invention. In accordance with the foregoing, virtually any number of chemical extracts or compounds can be screened using the example methods described herein. Examples of these extracts or compounds include, but are not limited to, extracts based on plants, fungi, prokaryotes, or animals, fermentation broths, and synthetic compounds, as well as a modification of existing compounds. There are also numerous methods available to generate a random or targeted synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, compounds based on saccharide, lipid, peptide, and nucleic acid. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wl). 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, United Kingdom), Xenova (Slough, UK) , Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, USA (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, for example, by conventional extraction and fractionation methods. In addition, if desired, any library or compound is easily modified using conventional chemical, physical, or biochemical methods. In addition, those skilled in the art of drug discovery and development, readily understand that the methods for re-replication (eg, taxonomic, biological replication, and chemical replenishment, or any combination thereof), or the elimination of replicas or repetitions of materials already known for their therapeutic activities for neurodegenerative or neuroproliferative disorders, should be used whenever possible. When a crude extract is found to prevent or slow down neuronal death or proliferation, an additional fractionation of the positive conductive extract is necessary to isolate the chemical constituents responsible for the observed effect. Therefore, the goal of the extraction, fractionation, and purification process is the characterization and careful identification of a chemical entity within the crude extract that has neuronal apoptosis (or inversely, proliferation) - preventive or palliative activities. The same assays described herein can be used for the detection of activities in mixtures of compounds, for purifying the active component, and for testing their derivatives. The methods of fractionation and purification of these heterogeneous extracts are known in the art. If desired, the compounds shown as useful agents for the treatment are chemically modified according to methods known in the art. Compounds identified as therapeutic value can be subsequently analyzed using a model of apoptosis or mammalian neuronal proliferation, as appropriate. Below are examples of high throughput systems useful for evaluating the efficacy of a molecule or compound in the treatment, prevention, or amelioration of a condition associated with neuronal apoptosis or associated with neuronal proliferation. Therapy The compounds identified using any of the methods disclosed herein, can be administered to patients, or to experimental animals, with a pharmaceutically acceptable diluent, carrier, or excipient, in a unit dosage form. Conventional pharmaceutical practice can be employed to provide formulations or compositions suitable for administering these compositions to patients or experimental animals. Although intravenous administration is preferred, any suitable 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, the 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 the formulations are found, for example, in "Remington's Pharmaceutical Sciences." Formulations for parenteral administration, for example, may contain excipients, sterile water, or serum, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biodegradable, biocompatible lactide polymers, lactide-glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other parenteral delivery systems potentially useful for antagonists or agonists of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

The formulations for inhalation may contain excipients, for example lactose, or they may be aqueous solutions containing, for example, polyoxyethylene 9-lauryl ether, glycocholate, and deoxycholate, or they may be oily solutions to be administered in the form of nasal drops, or like a gel. Use of primary neurons infected with recombinant adenoviral vectors to test the compounds for the purpose of determining their effect on neuronal cell death or growth Primary neurons are cultured, for example, sympathetic neurons of the upper cervical ganglia, or cortical neurons, or cells Neural progenitors, in tissue culture plates of 96 cavities, by conventional methods. Cell death or growth is induced or inhibited by removal or addition of neuronal growth factors, or by infection with recombinant adenoviruses. For example, to induce cell death, neurons can be infected with an adenoviral vector encoding p53, as described in the previous examples. Concomitantly, the compounds to be tested (for example, for the inhibition of p53-mediated cell death) are added to the cells in a range of concentrations. At appropriate time points, for example, between 0 and 36 hours, the treated samples are lysed by standard techniques, and the cellular ones are subjected to the appropriate assay, as described below. ELISA for the detection of compounds that modulate neuronal cell death and growth Enzyme-linked immunosorbent assays (ELISAs) are easily incorporated into high-throughput screening designed to test large numbers of compounds, in order to determine their capacity to modulate the levels of a given protein. When used in the methods of the invention, changes in a given protein level of a sample, relative to a control, reflect changes in the apoptotic or growth state of the cells within the sample. Protocols for ELISA can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1997. Used neuronal cell phones treated with potential cell death or growth modulators are prepared (see, for example, Ausubel et al., Supra), and loaded into microtiter plate cavities coated with antibodies. of "capture", against one of the health markers / neuronal growth described above, for example, α-tubulin Tal, or tyrosine hydroxylase. The unbound antigen is washed off, and an antibody specific to the health / growth marker is added, coupled with an agent to allow detection. Agents that allow detection include alkaline phosphatase (which can be detected following the addition of colorimetric substrates, such as nitrophenol phosphate), horseradish peroxidase (which can be detected by chemiluminescent substrates, such as ECL, commercially available in Amersham), or fluorescent compounds, such as FITC (which can be detected by fluorescence polarization or time-resolved fluorescence). The amount of antibody binding, and consequently, the level of health / growth marker within a sample of lysate, is easily quantified in a microtiter plate reader. As a baseline control for health marker / growth levels in cells that are not dying, a sample that is continuously exposed to NGF is included. As a baseline control for health marker / growth levels in dying cells, a sample is included where the NGF is removed and not replaced. As a baseline control for the levels of health marker / growth in growing cells, a sample that is continuously with NGF is included, and then with a neurotrophin such as NT-3 (see Example X, Assays for Neuronal Growth). MAP kinases, and p85 subunit of PI3 kinase are used as internal standards for absolute protein levels, since their levels do not change during the preferred time course (from 0 to 36 hours after withdrawal of NGF). A positive test result is indicated, for example, the identification of a compound that decreases neuronal apoptosis mediated by p53, by an increase in the levels of the health / growth marker (such as α-tubulin Tal), in relation to the level of health / growth marker observed in the cell that are induced to die without rescue. Reporter gene assays for compounds that modulate neuronal cell death and growth. Assays employing the detection of reporter gene products are extremely sensitive and easily susceptible to automation, making them therefore ideally suited for high throughput screening design. Assays for reporter genes can employ, for example, colorimetric, chemiluminescent, or fluorometric detection of reporter gene products. Many varieties of plasmid and viral vectors containing reporter gene cassettes are easily obtained. These vectors contain cassettes that encode reporter genes such as lacZ / β-galactosidase, green fluorescent protein, and luciferase, among others. The cloned DNA fragments encoding transcriptional control regions of interest are easily inserted, by subcloning the DNA, into these reporter vectors, thereby placing a reporter gene encoded by the vector under the control of transcription of any genetic promoter of interest. . The transcription activity of a promoter operably linked to a reporter gene can then be directly observed and quantified as a function of the reporter gene activity in a reporter gene assay. Reporter gene assay of primary neurons from transgenic mice Primary neurons are cultured from mice containing one or more reporter transgene constructs, death or cell growth is induced or inhibited, and the leaving compounds are added. to test to determine its modulating health / growth activity to neurons. At appropriate time points, the cells are lysed and subjected to appropriate reporter assays, for example, a colorimetric or chemiluminescent enzymatic assay for lacZ / β-galactosidase activity, or fluorescent detection of green fluorescent protein. Changes in the reporter gene activity of the samples treated with the test compounds, in relation to the reporter gene activity of the appropriate control samples, as suggested in the previous section, indicate the presence of a compound that modulates the neuronal cell death. In one embodiment, a transgene could comprise a reporter gene such as lacZ or green fluorescent protein (FPG), operably linked to a promoter from a health / growth marker gene, such as the neurons-specific a-tubulin gene. (see, for example, U.S. Patent Application No. 08 / 215,083). The α-tubulin Tal gene is abundantly expressed in developing neurons during morphological growth, and is also abundantly expressed in mature neurons during the process of objective reinnervation. Accordingly, the amount of activity resulting from a reporter gene that is operably linked to the α-tubulin Tal promoter will indicate the proportion of living (or growing) neurons within a sample, relative to the appropriate controls. There may be transgenes present within the genomic DNA of a neuron to be tested, or they may be transiently introduced into a neuron. A second transgene is included, comprising a second reporter gene operably linked to a second promoter, as an internal control. This could be a reporter gene operably linked, for example, to the neuron-specific T-cell a-tubulin promoter, which is constitutively expressed in the neurons, or alternatively, a promoter from a maintenance gene known to the neurons. experts in this field, for example GAPDH. Reporter Gene Assay in Primary Neurons with Adenovirus Transduction Primary neurons are isolated from transgenic or non-transgenic animals, and are infected with an adenovirus containing a reporter gene construct of interest, such as those described immediately above. The neurons are treated with the test compounds, apoptosis or growth is initiated or inhibited, and the activity of the reporter is measured, and interpreted as provided herein. Alternatively, a gene whose expression modulates neuronal cell death or growth can be introduced by adenovirus-mediated genetic transfer, as described above. For example, an oncogene can be introduced that stimulates neurons to proliferate in an uncontrollable way, in neurons that express α-tubulin Tal: nlacZ, by adenovirus-mediated gene transfer. The expression of the oncogene encoded by the adenoviral vector induces the cells to proliferate, and in this way, test compounds that specifically interfere with neural proliferation can be isolated. For example, a desirable result in a screening of a compound that specifically inhibits neurons that proliferate, but does not induce death in post mitotic neurons, would be a compound that decreases the expression of the reporter gene of health / growth (e.g. -tubulin Tal: nlacZ) in the proliferating neurons that express the oncogene, but which does not alter the expression of the reporter gene in normal postmitotic neurons. Conversely, scans can be performed for compounds that promote neuronal growth, or that inhibit neuronal death, using analogous approaches. Quantxtatxva polymerase chain reaction of health marker / growth mRNA as an assay for compounds that modulate neuronal cell death and growth The polymerase chain reaction (PCR), when coupled with a preceding reverse transcription step (rtPCR ), is a method commonly used to detect the vanishingly small amounts of a target mRNA. When performed within a linear range, with an appropriate internal control objective (using, for example, a maintenance gene, such as actin), this quantitative polymerase chain reaction provides an extremely accurate and sensitive means to detect the slight modulations in mRNA levels. Moreover, this test is easily performed in a 96-cavity format, and therefore, is easily incorporated into a high-throughput screening assay. Neurons are cultured, treated with the test compounds, and growth or death is induced or inhibited as described in the previous examples. Then the neurons are used, the mRNA is reverse transcribed, and the polymerase chain reaction is performed according to the commonly used methods (such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & amp;; Sons, New York, NY, 1997), using oligonucleotide primers that hybridize specifically with the nucleic acid of interest. In one embodiment, the target mRNA could be that of one or more of the health / growth markers, such as a-tubulin T l, or tyrosine hydroxylase. Summary of Conclusions The data presented in the examples found later in this document support several conclusions. The first is that adenovirus-derived vectors can be effectively used as gene transfer agents for post mitotic neurons. When 10 to 50 MOI are used, recombinant adenoviruses can infect more than 75 percent of sympathetic neurons with little disturbance in cell survival, cytoarchitecture, or physiological function for at least 7 days. A second conclusion is that, within the parameters defined with the recombinant adenovirus Ad5CA17LacZ, overexpression of p53 is sufficient to induce apoptosis in post mitotic neurons. Furthermore, p53-mediated cell death can be inhibited by recombinant adenoviruses expressing p53 inhibitors, such as E1B55K, and Bcl-2. In addition, neuronal survival can be mediated with proteins encoding adenoviruses, such as Gab-1, and growth factor receptors, such as TrkB. The first conclusion demonstrates the utility of recombinant adenoviral vectors in mechanical studies of post mitotic neurons, particularly when these studies are performed using the parameters discussed herein. In titers greater than 100 MOI, assays of mitochondria function indicate a decrease in cell survival. Cells that survive excessive infection rates, exhibited remarkable changes in the nuclear ultrastructure, including dense inclusions in electrons, filamentous aggregates, and in severe cases, disintegration of chromatin. The pathological changes resulting from recombinant viral transduction warrant further investigation, particularly with respect to virus-mediated gene therapy. The second conclusion has great implications for the mechanisms of cell death that occur in the nervous system in the case of progressive disease or sudden stroke. Since these data indicate that endogenous p53 is stably expressed in SCG neurons, it appears that the protein must be expressed at a given threshold level before the apoptotic path is triggered. Once this threshold is reached, p53 alone is sufficient to induce neuronal apoptotic cell death. Therefore, the upregulation of p53 following a neuronal lesion may be the key signal that leads to the dismissal of injured neurons. Future studies using adenovirus-derived vectors to introduce p53 suppression mutants will help define the mechanism of p53 action in post mitotic neurons. In addition, proteins encoding recombinant adenoviruses that inhibit p53 activity may be useful as therapeutic inhibitors of apoptosis in post mitotic neurons. Additionally, neurons that express the proteins encoded by adenovirus vectors will be a useful tool to track the therapeutic compounds that regulate neuronal death and proliferation.

EXAMPLE I: GENERAL METHODS Viral Vectors Ad5CA1 7LacZ The adenoviral recombinant was generated carrying an E. coli β-galactosidase expression cassette with a CMV promoter (Ad5CAl7LacZ), by co-transfection * of pCA14 (lacZ) and pJMl7 as it was previously described (Bett et al., Proc. Nati, Acad. Sci. USA 91: 8802-8806, 1994). This virus with deletion of El and E3 contains the reporter gene in the El region. Recombinant adenovirus expressing p53 and expressing the ElA mutant (AdllOl) A recombinant adenovirus carrying the wild-type human p53 was constructed, according to the method from Graham (Bett et al., Supra, see also Katayosc and collaborators, Cell Growth Diff. 6: 1207-1212, 1995). The results obtained with this vector were verified with a second preparation of adenovirus vector that also confers the expression of p53, AdWTp53 (Bacchetti et al., Int. J.

Incol. 3: 781-788, 1993). The adenovirus vector carrying the p300 binding mutant (AdllOl) from ElA was on an ElB lacking 12S (Jelsma et al., Virology 163: 494-502, 1988). The recombinant adenoviruses were amplified in 293 cells, a human embryonic kidney cell line, which expressed the ElA and ElB type 5 adenovirus proteins (Graham et al., In Methods in Molecular Biol. EJ Murray, Ed., The Humana Press, pages 109-128). These vectors were harvested from cellular ones, and were used directly, or were further purified on CsCl gradients according to Graham et al (supra). Infectious titration was determined by plaque assay in 293 cells (also as described by Graham et al., Supra). When comparing the effects of adenovirus or ElA-mediated p53 overexpression (AdllOl) against overexpression of β-galactosidase, the following measures were taken in order to ensure that the observations made could not be attributed to differences in viral preparations: (1) all the viral preparations were purified in an identical manner; (2) several preparations of each virus were examined; (3) the content of particles of each viral preparation, which is a potential source of cytotoxicity, was in a similar range; and (4) the results were reproduced with four different recombinant adenoviruses; two that expressed lacZ, and two that expressed p53. The content of particles was determined by obtaining the proportion of the infectious titration to the titration at an optical density of 260 nanometers, according to conventional procedures (Bett et al., Supra). The ratio of the infectious titre to the particle content is usually about 1: 100. The proportions obtained for the recombinant adenoviruses lacZ and p53 were found in this range, at 1: 110 and 1: 120, respectively. Recently, the emergence of replication-competent virus contamination has been observed in the defective replication adenovirus supplies in 5 of the serial passage (Lochmuller et al., Hum Gene Therapy 5: 1485-1491, 1994). To verify the purity of the virus supplies, both polymerase chain reaction and Southern blot analysis were performed; these procedures are capable of detecting any virus containing the contami- Wf > nante For the polymerase chain reaction analysis, recombinant viral DNA was extracted, and amplified with primers specific for the El region (Lochmuller et al., Supra). The DNA that was purified from the wild-type virus was used as a positive control. When it was detected In the wild-type contamination by polymerase chain reaction analysis, Southern blots of wild-type and recombinant viral DNAs were prepared and probed with radiolabelled DNA fragments that hybridize to the El and E2 region of the viral genome (Lochmuller and collaborators, supra). For In pure preparations, hybridization with a probe for the El region would reveal a positive signal in the wild-type virus only, whereas the probe for E2, a region present in both recombinant and wild-type viruses, should produce a single band that indicates a pure population of viruses in both DNA preparations. If traces of wild-type contamination were detected, the recombinant viruses were further purified in accordance with Graham et al. (Supra). RH105 The HSV vector, RH105, carries the lacZ gene of E. coli inserted into the thymidine kinase (TK) gene, upstream of the first immediate promoter ICP4 (Hoy collaborators, Virol 174: 279-283, 1988). The altered TK gene makes the replication of the virus incompetent in postmitotic cells such as neurons (Boviatsis et al., Human Gene Therapy 5: 183-191, 1994; Don et al., Proc. Nati. Acad. Sci. USA 88 : 1157-1161, 1991; Lipson et al., Proc. Nati, Acad. Sci. USA 86: 6848-6852, Sherley et al., J. Biol. Chem. 263: 8350-8358, 1988). The virus was propagated in Vero cells until a cytopathic effect of 100 percent was observed, after which time, the cells were frozen-thawed and sonified on ice to release the virus particles. The large cell debris was removed by centrifugation at 1,800 X g for 10 minutes. Then the resulting supernatant was layered on a cushion of 25 percent sucrose in phosphate buffered saline (PBS), and centrifuged at 70,000 X g for 18 hours. The granule containing the recombinant herpes virus was resuspended in phosphate-regulated serum, and titrated on Vero cells. The absence of the wild-type virus was confirmed by staining with X-gal plates generated on Vero cells. The multiplicity of infection (MOI) was calculated based on titration on 293 cells for adenovirus-based vectors, and on Vero cells for the HSV RH105 vector, and represents the number of aggregated plaque-forming units per cell. To directly determine whether induction with p53 is sufficient to trigger the establishment of apoptosis in post mitotic neurons, a recombinant adenovirus vector was used to deliver p53 to cultured sympathetic neurons. The efficacy of adenoviral vectors as gene transfer agents for sympathetic neurons was evaluated, and the parameters within which these vectors can be effectively used were well defined. As described below, a recombinant adenovirus carrying the lacZ reporter gene inserted in the deleted region was used (Bett et al., Proc. Nati, Acad. Sci., USA 91: 8802-8806, 1994) to transduce sympathetic neurons. from the superior cervical ganglia in vitro. Examination of ineffectiveness, cytotoxicity, cell physiology, and cytoarchitecture indicated that these recombinant adenoviruses have the potential to serve as highly effective gene transfer agents for sympathetic neurons. Working within the defined parameters, a wild type human p53 expression cassette was introduced into the same base structure of the vector, and used to transduce cultured sympathetic neurons. The overexpression of p53 mediated apoptosis in these neurons. The demonstration that p53 is sufficient to induce apoptosis in post mitotic neurons has important implications for the mechanisms of cell death in the mature traumatized nervous system. Cell Survival Assays To test cell survival, three different assays were used, including in vivo / dead staining, marked with TUNNEL, and a quantitative MTT assay. For Live / Dead dyeing, the Vivo / Dead viability / cytotoxicity kit (Molecular Probes) was used according to the manufacturer's instructions. Said in a brief way, two reagents, calcein-AM and ethidium bromide were added to the cultures in their usual medium. Calcein-AM is converted metabolically by intracellular esterase activity, resulting in the production of a green fluorescent product, calcein, an indicator of cell viability. Ethidium bromide is excluded from living cells, but is easily recovered by dead cells, and stains DNA. The cells are incubated in these reagents for 10 to 15 minutes at 37 ° C, after which time they are examined and photographed immediately, due to the toxicity of these reagents. To detect apoptosis, terminal transferase was used to visualize the fragmented DNA (labeled with TUNNEL). Parallel cultures were infected with AdllOl or AdlacZ at 25 MOI.

After 72 hours, the cells were fixed in acetone / methanol (1: 1) for 10 minutes at -20 ° C. 50 microliters of a cocktail consisting of 1.0 microliters of biotin dUTP, 1.5 was added . -microliters of terminal transferase, 20 microliters of 5X Samples were washed three times in phosphate buffered saline, pH 7.4, and once in TBS, pH 8.0, to stop the reaction. The samples were incubated with a CY3 streptavidin diluted 1: 2000 for 30 minutes. After three 5-minute washes in phosphate-buffered serum, the samples were examined with an inverted fluorescent microscope. For a quantitative measurement of cell survival, the MTT (Cell Titration Assay, Promega, Madison Wl) survival assay was used as described previously (Slack et al., J. Cell Biol. 135: 1085, 1996). This assay measures the mitodrial conversion of the tetrazolium salt to a blue formizano salt, the accumulation of which can be measured colorimetrically. Morological tests for neuronal growth: analysis and quantification of excessive process growth, neurite length, and cell body area Morphological tests to track neuronal growth, for example the tests that measure the total length of the neurite, the size cellular, and the network density of the neuronal process, are well known to those skilled in the art. Protocols for these assays can be found, for example, in Belliveau et al., J. Cell Biol. 136: 375-388, 1997. 5 The analysis of the effects of neurotrophins and other treatments, such as infection with recombinant adenoviruses, on neuronal growth is examined, measuring three parameters: density of the process network, total length of the neurite, and area of the cell body. The analysis was performed Pt) quantitative of the density of the cellular process on high and low density crops, using common statistics applied to random sets of lines, in particular the number of intersection points per unit area. In the microscope, the network of neural processes appears as a random set of lines in one plane. The number of visible cross-links and bifurcations of cellular processes per unit area, therefore, can be considered as a quantitative measure of the density of the cellular process. However, since the number of neurites is a direct function of the number of neurons, only regions of the culture having a similar cell density are comparable. Therefore, in each experiment, 10 to 15 sampling windows (10 square millimeters) were analyzed, each containing seven neuronal cell bodies. All interceptions and bifurcations were counted of the neurites inside these windows, resulting in an estimated value of the neuritic process density. The statistical comparison of the average density values was performed using ANOVA (test F). The total length of the neurite and the area of the cell body were measured in low density cultures within defined controlled areas for the number of cell bodies. The results were analyzed both within the groups of human cultures with different treatments, and grouping the results of similar treatments from different groups of sister crops. Similar results were obtained, and therefore, the pooled data are presented. The t test and ANOVA were used to determine the statistical significance. Gene expression assays for neuronal growth The growth of primary neurons expressing recombinant adenoviruses can be monitored by different gene expression assays that are well known to those skilled in the art. Sympathetic neurons can be cultured under conditions in which their survival is maintained, for example, 10 nanograms / milliliter of NGF, infected with recombinant adenoviruses, and tested to determine the transcription increments in the growth marker genes. Genes that serve as markers for neuronal growth, for example, tyrosine hydroxylase and such a-tubulin, show an increase in transcription activity in healthy versus unhealthy (or dying) neurons. Furthermore, these growth marker genes exhibit even more transcription activity in neurons that grow actively, against passive ones. Therefore, growth marker genes provide a simple and reliable method to monitor neuronal growth. Reporter gene assays can also be used to monitor neuronal growth. For example, we generated transgenic mice containing chimeric transgenes consisting of a lacZ coding region located in the nucleus under the transcriptional regulation of the α-tubulin promoter region Tal. The expression of the α-tubulin T l: nlacZ transgene in sympathetic and cortical neurons cultured from the transgenic α-tubulin mice Tal: nlacZ is proportional to neuronal growth. Therefore, lacZ assays can be used to monitor the health and growth of a-tubulin transgenic neurons Tal: nlacZ infected with recombinant adenoviruses. Alternatively, a recombinant adenoviral vector carrying a reporter gene of a-tubulin Tal: nlacZ, or an analogous reporter gene, can be used to infect the neurons, in order to monitor their health and growth. Gene expression assay for the neuronal phenotype The expression of genes encoded by the recombinant adenovirus can be used to alter the phenotype of a neuron. A genetic expression assay is performed to determine or confirm the phenotype of the resulting transgenic neurons, which are then used as a cell therapy for different neurodegenerative diseases, or alternatively, in assays for the isolation of novel neurotherapeutic compounds. For example, it would be desirable to have a reliable and abundant source of dopaminergic neurons: these neurons would be useful for screening tests, and for implantation in the brains of patients suffering from Parkinson's disease. The dopaminergic neurotransmitter phenotype of cultured neurons expressing a protein encoded by recombinant adenovirus is confirmed by the assay for tyrosine hydroxylase expression, for example, by monitoring mRNA levels by Northern hybridization, or by polymerase chain reaction / reverse transcriptase, by methods known to those skilled in the art. EXAMPLE II: EFFICIENCY OF NEURONAL GENE TRANSFER OF RECOMBINANT ADENOVIRUS AGAINST HERPES SIMPLEX VIRUS (HSV-1) In order to determine the most effective and non-toxic gene transfer vector for post mitotic sympathetic neurons, parallel studies were conducted with the vector adenovirus Ad5CAl7LacZ, and the vector of herpes simplex-1 virus, RH105, where both, as described above, express the lacZ reporter gene of E. coli. Pure cultures of neonatal sympathetic neurons were prepared and infected with defective replication virus of both types as follows: Cell Culture Mass cultures of purified sympathetic neurons were prepared according to the procedure of Ma et al. (J. Cell Biol. 117 : 135-141, 1992). The upper cervical nodes of newborn Sprague-Dawley rat pups (Charles River Laboratories, Charles River Canada, St. Constant, Quebec) were removed and harvested in an L15 medium without sodium bicarbonate. The lymph nodes were washed in phosphate-buffered serum (pH 7.4), and treated with 0.1 percent trypsin (Calbiochem Novabiochem, San Diego, CA) at 37 ° C for 20 minutes., followed by treatment with DNase (10 micrograms / milliliter; Sigma Chemical Co. , St. Louis, MO) for 2 minutes. The ganglia were crushed and passed through a 40 micron mesh (Becton-Dickinson Canada Inc. Mississauga, Ontario) to produce a single cell suspension. Following centrifugation in a clinical centrifuge, the pellet was resuspended in an L15 medium supplemented with sodium bicarbonate (30 mM), vitamin C (1 milligram / milliliter), cytosine-arabinoside (10 μM), rat serum at 3 percent, and 50 nanograms / milliliter of NGF (Cedarlane Laboratories, Hornby, Ontario). The cells were coated at a density of 100,000 cells per milliliter of the medium on tissue culture dishes, which were coated with rat tail collagen. These cells are essentially free of non-neuronal cells (see also Ma et al., Supra). The neurons were cultured for 3 to 5 days before the viral infection, during which time they adhered to the culture dishes, and extended the processes. For viral infection, the medium was removed, and replaced with 25 percent of the usual volume containing the appropriate titration of the virus. The cells were incubated for 1 hour at 37 ° C, and the dishes were oscillated every 15 minutes. The volume of the remaining 75 percent of the medium was then added to each dish. For long-term crops, the medium was changed every 3 days. 48 hours after infection, the infected cultures were stained with X-gal to visualize the expression of β-galactosidase, which is encoded by the transgene. Detection of β-galactosidase-positive cells The dyeing for the expression of β-galactosidase, the product of the lacZ reporter gene, was carried out at different times following the infection, as described by all the examples. Cells were fixed with 0.2 percent glutaraldehyde in phosphate-buffered serum (pH 7.4) for 15 minutes at 4 ° C. Following the washings with phosphate-buffered serum, the cells were incubated for 18 hours in X-gal dye. (2mM MgC12, 1 milligram / milliliter of X-gal, 5mM K3Fe (CN) 6, and K4Fe (CN) 6 in phosphate buffered serum (pH 7.4) To estimate the percentage of cells that were infected, He counted the total number of cells and the number of cells positive for lacZ in five random fields.The data are expressed as the average of two separate experiments, the error bars representing the range.The number of cells per field was 125 + 50 for experiment 1, and 200 + 59 for experiment 2. These experiments demonstrate that both vectors derived from HSV and adenovirus are capable of transducing sympathetic neurons in vitro, however, a closer examination of lacZ dyeing and Cellular morphology indicated clear differences in the efficacy of these two vectors In titers of 1 MOI, infection with adenovirus led to a higher proportion of lacZ-positive neurons; compare Figure 1A, a photograph of sympathetic neurons infected with adenovirus at 1 MOI, with Figure IB, a photograph of the same type of cultured cells infected with herpes virus (HSV-1) at 1 MOI. In MOIs of 1, virtually all neurons infected with adenovirus expressed the transgene (Figure 1C), while many neurons from cultures infected with HSV-1 were negative (Figure ID). In the highest titration examined, 100 MOI, neurons infected with recombinant adenoviruses appeared morphologically normal, exhibited no indication of cytotoxicity, and expressed the transgene at levels that were high enough to produce staining both in cell bodies and in the processes extended (Figure 1E). In contrast, neurons infected with the HSV vector at 100 MOI exhibited signs of severe degeneration, particularly of neuritic processes, within 48 hours (Figure 1F). The quantification of the number of cells positive for lacZ in the sister cultures of sympathetic neurons infected with these two viral vectors, confirmed the qualitative conclusions drawn from observing the cultures, such as those shown in Figure 1. To obtain an estimate of the percentage of cells that were infected with each type of virus, the total number of cells and the number of cells positive for lacZ were counted in five random fields, as described above. Approximately 30 percent of the sympathetic neurons were positive for lacZ following infection with recombinant adenovirus in titers of a plaque-forming unit / cell (Figure 2A), but only 10 percent of the cells that were transduced by the HSV vector were positive in a similar titration (Figure 2B). At 10 plaque / cell forming units, the efficiency of transduction was again higher with adenovirus than with the HSV-1 vector, with 75 percent and 50 percent of lacZ-positive cells in the sibling cultures, respectively (Figures 2A and 2B). Titrations of 100 plaque / cell-forming units or larger, with any vector, resulted in the transduction of more than 95 percent of the cells, but the HSV-1 vector, in these MOIs, appeared to be cytotoxic. Therefore, cell survival was evaluated following infection with these two different vectors. EXAMPLE III: NEURONAL SURVIVAL FOLLOWING INFECTION WITH A RECOMBINANT ADENOVIRAL VECTOR To assess potential cytotoxicity in response to viral infection, sympathetic neurons were infected with the Ad5CA17LacZ adenoviral vector, or the HSV-1 vector, RH105, and tested cell viability, as reflected by mitochondrial function, 2 to 10 days later. Cell survival assay To evaluate cell survival, "neurons were seeded at a density of 5,000 cells per well, in 48-well tissue culture dishes, and were infected with different titers of the adenovirus or HSV-1 vectors., as described herein. Cell viability was measured by the metabolic conversion of a tetrazolium salt to formazan salt according to the CellTiter 96 Assay Kit® (Promega, Madison, Wl). As shown in Figure 3, at 1 MOI, there was no significant difference in the percentage of cells that survived following infection with recombinant adenovirus against infection with recombinant HSV-1. However, as the titrations increased, a large difference in cell survival was evident; at 10 MOI, 90 percent of the neurons in the adenovirus-infected cultures remained alive, while only 45 percent of those in the HSV-infected cultures were alive. When these values were corrected for ineffectiveness, almost all cells transduced with the HSV vector were lost after 10 days in culture (for example, at 10 MOI of HSV, 55% of the cells were infected, and 55 percent of the cells were lost). In contrast, when adenovirus-infected neurons were corrected for infectivity (ie, 75 percent infected / 10 percent lost), only 13 percent of the infected cells were lost 10 days after infection at 10 MOI. A more surprising difference appeared at 50 MOI; at this level of infection, most of the neurons in HSV-infected cultures were lost (90 percent), whereas in cultures infected with adenovirus, only 15 percent of the neurons were lost. Accordingly, HSV-1 has a relatively narrow effective range, and the titers necessary to transduce more than 75 percent of the cell population exhibit severe cytotoxic effects. The results indicate that the adenovirus, in titers of 10 to 50 MOI, can transduce more than 70 percent of the cells with minimal cytotoxicity for at least 10 days. Accordingly, all of the experimentation described in the following examples was performed with the adenovirus-based vectors. EXAMPLE IV: CYTOARCHITECTURE OF TRANSDUCED NEURONS Although the cells appeared to have a normal morphology, and they continued to survive immediately after infection with the adenoviral recombinants, infection was performed with an electron microscope 7 days after infection to determine if the presence of a non-lytic virus carrying the reporter gene of lacZ caused ultrastructural changes in the surviving neurons. Electron Microscope Sympathetic neurons were infected with the recombinant adenovirus Ad5CA17LacZ in titers of 0, 10, 50, 100, and 500 MOI. After 7 days in culture, the cells were fixed with 1.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 hours. In some experiments, the neurons were infected with Adwtp53 at 50 MOI for 2 days, and fixed in the same manner. After initial fixation, the samples were washed three times in 0.1 M sodium cacodylate regulator, for 15 minutes in each wash. The samples were subsequently fixed on ice in 1 percent osmium tetroxide for 90 minutes, dehydrated in ascending concentrations of ethanol and acetone, and embedded in Epon-Araldite. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7100 transmission electron microscope. Three separate grids containing 40 to 60 cells per sample were examined. The cells were first examined immediately after infection in the lower 10 MOI range, which results in the transduction of approximately 70% of the cells. The ultrastructure of these cells was generally indistinguishable from uninfected cells (Figure 4A); the cytoplasms appeared normal, with organelles intact, and the nucleus contained normal chromatin (Figure 4B). In some cells, very small electron-dense inclusions were found in the otherwise healthy cell nuclei (Figure 4B). At 50 MOI, the cellular organelles remained intact, and the core structure appeared normal, although slightly more nuclear inclusions were visible (Figure 4C). At higher adenovirus concentrations, when significant cytotoxicity was revealed by mitochondrial function assays, nuclear abnormalities could easily be noted (Figure 4D). These abnormalities included large dense electron inclusions, and an accumulation of filamentous aggregates that are commonly found in neurons suffering from degeneration. In high titers, some neurons exhibited chromatin disintegration (Figure 4D). Ultrastructural examination did not reveal characteristic features of apoptosis, even in the highest titers examined (500 MOI) in relation to uninfected controls. These results indicate that adenovirus can be used in titers that infect most cells without causing adverse cytological changes. However, virus concentrations should be carefully controlled, since excessive concentrations of infection result in a deterioration of the nuclear structure. EXAMPLE V: ELECTROPHYSIOLOGICAL FUNCTION OF NEURONAS INFECTED WITH RECOMBINANT ADENOVIRUS Although cells infected with 10 to 50 MOI of recombinant adenovirus were normal with respect to mitochondrial function and cytoarchitecture, neuronal function was characterized by examining the electrophysiological properties of the cell. Electrophysiology Upper cervical ganglion (SCG) neurons infected with recombinant and control adenoviruses were subjected to voltage using a cell-wide patch registration technique to measure outward potassium (K +) currents (McFarlane et al. J. Neurosci 13: 2591-2600, 1993). Briefly stated, SCG neurons were harvested on the first postnatal day, cultured for 3 days, infected with Ad5CA17LacZ at 1 MOI of 50 for 24 hours, and then cultured for an additional 7 days before the electrophysiological recordings. Cells were fixed with 2 percent paraformaldehyde-glutaraldehyde 0.2 percent immediately following registration, and stained with X-gal to detect β-galactosidase activity. It was considered that only cells that exhibited β-galactosidase activity had been infected. The total outward current in SCG neurons is formed by three voltage gate currents that differ in their kinetic and voltage-dependent properties: a non-quenching current (IK); a fast transient type A current (AI) that is inactivated with the lapse of time of 10 to 30 milliseconds; and a small slow transient type A current (IAs) that is inactivated with two components, one with a time constant of 100 to 300 milliseconds, the second with a time constant of 1 to 3 milliseconds (McFarlane, J. Neurophys. : 1291-1300, 1992). By holding the membrane at different potentials, it is possible to selectively activate one or two of the currents, and thus characterize the individual currents by subtraction techniques. Briefly, the membrane was held at a potential of -10 or -20 mV, where the depolarizing steps evoked only IK. The IK currents were subtracted from the total currents (IA + IK + IAs) evoked by the steps to the same depolarizing potentials from a more negative potential, -90 mV, to isolate the A currents. For the measurement of the current density ( pA / pF), the current amplitudes IA and IK were determined from the current evoked by a voltage step up to +30 mV after each current was isolated from the other two, and they were divided between the cellular capacitance (pF ). The cellular capacitance was obtained by integrating the capacity current evoked by a hyperpolarizing voltage step of 10 mV, and then dividing this current between the voltage step. The voltage steps were applied by a computer controlled stimulator. The software for the stimulus, data acquisition, and analysis, were written by Mr. A. Sherman (and are publicly available through Alembic Inc., Montreal, Quebec). The membrane streams were filtered with a List EPC-7 amplifier, sampled at 5 kHz, displayed, and stored online. For all experiments, the voltage steps were 125 milliseconds long. All experiments were performed at room temperature (21-24 ° C). The pipettes were filled with intracellular medium (5 mM NaCl, 50 mM potassium acetate, 65 mM KF, 1 mM MgCl 2, 10 mM HEPES (pH 7.4, adjusted with KOH), 10 mM EGTA, 0.5 mM CaCl 2); Pipette current was balanced to 0 with the pipette immersed in the bath solution. The neurons were superfused continuously with extracellular solution (Cl of 140 mM Hill, 2 mM NaCl, 5.4 mM KCl, 0.4 mM CaCl 2, 0.18 mM MgCl 2, 10 mM HEPES (pH 7.4 adjusted with NaOH), glucose 5.6 mM, tetrodoxin 0.5 mM (TTX), and CaCl2 1.5 mM (pH 7.3-7.4)), at a speed of 0.5 milliliters / minute, during the registration session. The extracellular solution included pharmacological agents to block inward sodium and calcium currents, and calcium-dependent currents, as described previously (McFarlane, J. Neurophys, 67: 1291-1300, 1992).

In sympathetic neurons expressing β-galactosidase 7 days following infection, it was found that current densities (pA / pF) for IK and IA are similar to those previously reported for cultured SCG neurons (McFarlane et al. J. Neurosci, 13: 2591-2600, 1993), and they are not significantly different from those of non-infected control SCG neurons (Figures 5A and 5B). Accordingly, neurons infected with up to 50 MOI of recombinant adenovirus appear to function normally for at least 7 days. EXAMPLE VI: OVEREXPRESSION OF P53 INDUCES APOPTOTIC CELLULAR DEATH IN POST-MITICAL NEURONS The discoveries described above indicate that adenovirus vectors can be used to genetically alter primary neurons within controlled parameters. Therefore, this approach was used to determine if overexpression of p53 was sufficient to induce programmed cell death in post mitotic neurons. A recombinant adenovirus, Adwtp53, which carries a wild-type p53 expression cassette on the same base structure of vector pJM17 as the lacZ reporter gene, was used for these studies. Initially, to determine whether adenovirus-mediated delivery of human wild-type p53 could lead to stable overexpression of p53 in cultured sympathetic neurons, cells were infected at 50 MOI with recombinant adenoviruses carrying the human p53 (Adwtp53), or lacZ (AdCA17LacZ), and were immunostained with an antibody specific for human p53. Immuno-luorescence For the detection of immunofluorescence of human p53 supplied by adenovirus vectors, samples were fixed for 5 minutes in methanol: acetone (1: 1), and allowed to air dry for 5 minutes. Following rehydration, the cells were blocked in phosphate-buffered serum containing 3 percent goat serum. A mouse monoclonal antibody that specifically binds an amino-terminal epitope of human p53 (DO-1) (Santa 'Cruz Biotechnology) was used. The primary antibody was diluted in this same blocking solution (1:50), and incubated on slides overnight at 4 ° C. Following three washes in phosphate-buffered serum, a goat anti-mouse secondary antibody conjugated with CY3 (Jackson Laboratories, diluted 1: 2000) was applied and incubated for 1 hour at 25 ° C. After three washes in phosphate-buffered serum, the slides were mounted in glycerol, and examined with a Zeiss Axioskop microscope. Neurons infected with AdCA17LacZ were not immunoreactive for human p53 (Figure 6A), whereas those infected with Adwtp53 exhibited strong nuclear staining in more than 80 percent of the cells (Figure 6B). To determine more precisely the degree of overexpression of p53 in relation to endogenous levels, transduced sympathetic neurons were harvested at 30 and 48 hours following infection with 50 MOI of Ad5CA17LacZ or Adwtp53, and the levels of p53 protein by Western blot analysis, with an antibody that recognized both the p53 of rodent and the human. Western analysis For the detection of p53 protein, cells were harvested in lysis buffer (Slack et al., J. Cell Biol. 129: 779-788, 1995) 48 hours following infection with a titre of 50 MOI. The protein was separated on a 10 percent acrylamide gel, and transferred to a nitrocellulose membrane. After blocking for 2 hours in 5 percent skimmed milk, the membrane was incubated in a solution containing Abl, a mouse monoclonal antibody directed against murine and human p53 (1:10) (Oncogene Science, Cambridge, MA ) overnight at 4 ° C. After five washes in TBST (5 minutes each), the filters were incubated for 1 hour at 25 ° C in goat anti-mouse secondary antibody conjugated with horseradish peroxidase. The filters were again washed five times in TBST for 5 minutes in each wash. The stains Western were revealed by the chemiluminescence system ECL® (Amersham), according to the manufacturer's instructions. These experiments demonstrated that endogenous p53 was expressed stably in sympathetic neurons, and that infection with AdCA17LacZ did not affect the expression of endogenous p53 (Figure 7B). In contrast, at 30 hours following infection with Adwtp53, the p53 protein was detectably overexpressed in the sympathetic neurons (Figure 7A), and at 48 hours, the expression was much higher than the endogenous levels (Figure 7B) . Coinciding with this increased expression of p53 at 48 hours after infection, morphological changes characterized by cellular shrinkage in cells infected with Adwtp53 became apparent, while those carrying AdCA17LacZ appeared normal. Furthermore, a remarkable number of p53-positive and dead neurons were observed (Figure 6B) in relation to the controls, suggesting that overexpression of p53 leads to neuronal death. To quantify the degree of neuronal death following overexpression of p53, cell survival was measured with the metabolic assay described above, where the conversion of the tetrazolium salt to the formazan salt is measured using a Cell Titer 96 Assay Kit® . Three days after the coating, the sympathetic neurons were infected in parallel with Ad5CA17LacZ and Adwtp53 in titers of 5 to 500 MOI. The cell survival assay of lacZ-infected neurons revealed no changes in cell viability 72 hours after infection, even at the highest MOI of 500 (Figure 8A). In contrast, cells infected with Adwtp53, under identical conditions, exhibited a 40% decrease in cell survival at 5 MOI, and a 65 percent decrease at 10 MOI (Figure 8A). Higher levels of virus resulted in a dramatic loss of 75 to 85 percent cell viability at 72 hours. As a cell control experiment, sibling cultures with wild type adenoviruses were infected in similar MOIs. Surprisingly, even the wild-type adenovirus did not affect the survival of sympathetic neurons for up to 72 hours (Figure 8A). These data indicate that overexpression of human p53 leads to the death of sympathetic neurons. To confirm the course of time of neuronal cell death following overexpression of p53, parallel experiments were performed with neurons infected at 50 MOI with Ad5CAl7LacZ and Adwtp53. Survival was assessed at 2, 2.5, 3, 5, 7, and 10 days following infection. First, neuronal cell death could be detected at 48 hours, when a 10 percent decrease in cell survival was evident (Figure 8B), while at 72 hours, a dramatic 60 percent to 70 percent loss in cell viability was detected. Then cell death continued at a very low level for the remainder of the trial. Therefore, cell death begins 48 hours after infection with Adwtp53, with most neurons dying between 2 and 3 days. To determine whether p53-induced cell death was due to apoptosis, three different assays were conducted: (1) nucleosomal DNA isolation to visualize the DNA scales, (2) TUNNEL staining to visualize apoptosis immuno-histochemically , and (3) electron microscope. Isolation of fragmented DNA To examine DNA fragmentation, 106 neurons were seeded on a 60 millimeter tissue culture dish under conventional culture conditions. Cells were infected with recombinant adenoviruses 3 days after coating, and harvested 48 hours following infection. Cells were harvested, washed once with phosphate-regulated serum, and used for DNA isolation as described previously (Slack et al., J. Cell Biol. 129: 779-788, 1995). Lysis buffer in 1.2 milliliters was added to 100 microliters of TE suspended cells (10 mM Tris-HCL, pH 8.0, and 1 mM EDTA). The lysis was allowed to proceed at room temperature for 15 minutes, after which time, the lysate was centrifuged for 15 minutes at 12,000 rpm. The gelatinous granule was removed with a pipette, and the supernatant was digested with 100 micrograms / milliliter of RNAse A at 37 ° C for 30 minutes. Then the DNA was precipitated by adding an equal volume of 100 percent ethanol and NaCl, so that the final concentration was 0.5 M. Following centrifugation, the granule was washed with 70 percent ethanol, and it became to suspend in 50 microliters of TE regulator. The fragmented DNA was marked in extreme with [32-P] -dCTP using Klenow (Promega) for 15 minutes at room temperature. The DNA scales were resolved by passing the labeled DNA in ends on a 2 percent agarose gel, using a scale of 100 base pairs as standard. TUNNEL TUNING To assay for apoptosis immuno-histochemically, terminal transferase was used to visualize the fragmented DNA (stained with TUNNEL). Parallel cultures were infected with Adwtp53 or pCA171acZ at 50 MOI. After 72 hours, the cells were fixed with acetone-methanol (1: 1) for 10 minutes at -20 ° C. 50 microliters of a cocktail consisting of 1.0 microliters of biotin dUTP (Boehringer Mannheim, Indianapolis, IN, Cat. # 109307), 1.5 microliters of terminal transferase (Promega Cat. # M187 / l), 20 microliters of 5X TdT regulator was added. (Promega), and 78 microliters of distilled water, to each slide. Following a 1-hour incubation at 37 ° C, the slides were washed three times in phosphate-buffered serum (pH 7.4), and once in Tris-regulated serum (pH 8.0) to stop the reaction. The slides were incubated with a secondary antibody labeled with streptavidin, CY3 (Jackson Laboratories, West Grove, PA) diluted at 1: 2000 for 30 minutes. After three 5-minute washes in phosphate-buffered serum, the samples were mounted in glycerol, and examined with a Zeiss Axioplan microscope.

The three assays, which were conducted in parallel with cultures infected at 50 MOI with AdCA17LacZ or Adwtp53, indicated that overexpression of p53 leads to neuronal apoptosis. First, neurons infected for 48 hours with Adwtp53 showed significantly more DNA fragmentation than those infected with AdCAl7LacZ (Figure 7C), as demonstrated by gel electrophoresis of DNA. Second, TUNNEL staining revealed significantly higher levels of apoptosis in neurons that overexpressed p53 relative to controls at 72 hours after infection (Figures 9A and 9B). In the cultures infected with Awtp53, there were many picnotic nuclei positive for TUNNEL (Figure 9F), whereas only occasional positive nuclei for TUNNEL were observed in the cultures infected with AdCA17LacZ (Figures 9A and 9D). Coinciding with this increase in TUNNEL labeling, neurons that overexpressed p53 exhibited a dramatic neuritic degeneration (Figure 9E), while those expressing β-galactosidase exhibited a normal morphology (Figure 9C). Finally, the analysis of these cultures by electron microscopy showed a better apoptosis of sympathetic neurons infected with 50 MOI of Adwtp53, as indicated by the collapse and condensation of nuclear chromatin. In contrast, sympathetic neurons infected with AdCA17LacZ did not exhibit a better apoptosis in relation to uninfected controls, even at 500 MOI, at least when measured ultrastructurally. Accordingly, adenovirus-mediated overexpression of p53 is sufficient to cause apoptotic death of post-mitotic sympathetic neurons. EXAMPLE VII: MODULATION OF APOPTOTIC CELLULAR DEATH BY INFECTION IN POSMITTING NEURONS WITH RECOMBINANT ADENOVIRUS Inhibition of p53-mediated cell death by a recombinant adenovirus expressing E1B55K The experiments mentioned above show that post-mitotic neurons are induced to suffer apoptotic cell death on the infection of a retrovirus that encodes p53. Conversely, the survival of post mitotic neurons is improved by infecting them with recombinant retroviruses that encode proteins that inhibit cell death. The graph shown in Figure 10 shows that recombinant adenoviruses expressing the p53 inhibitor, E1B55K, rescue neurons from death induced by NGF withdrawal. Sympathetic neurons were cultured from rat upper cervical lymph nodes (SCG) for 5 days at 50 nanograms / milliliter NGF, after which they were infected with an MOI of 50 or 100 recombinant adenovirus expressing E1B55K, or E1B55K mutant, who can not fix p53. Infections were made in the presence of 50 nanograms / milliliter of NGF. Two days after infection, the cells were washed to free them from NGF, and 2 days later, survival levels were determined using MTT assays.

Cell death induced by p53 and by MEKKl is inhibited by injection of post mitotic neurons with recombinant adenoviruses expressing Bcl-2 and Bcl-xL The experiment shown in Figure 11 demonstrates that recombinant adenoviruses expressing p53 or MEKKl (a member of the mitogen-activated kinase cascade) kill neurons in the presence of NGF, while the recombinant adenovirus expressing the anti-apoptotic proteins Bcl-2 and Bcl-xL, rescue neurons from death induced by NGF withdrawal . Sympathetic neurons were cultured from upper rat cervical ganglia Pl (SCG) for 5 days at 50 nanograms / milliliter NGF, after which they were infected with an MOI of 10 to 100 recombinant adenovirus expressing p53, MEKKl, Bcl-2, and Bcl-xL. Infections were made in the presence of 50 nanograms / milliliter of NGF. Two days after infection, the cells were washed to free them from NGF, and two days later, survival levels were determined using MTT assays. Cell death induced by nerve growth factor (NGF) is inhibited by injection of post mitotic neurons with a recombinant retrovirus that expresses the Gabl coupling protein. The experiment shown in Figure 12 demonstrates that the Gabl adenovirus mediates the survival of neurons. sympathetic Sympathetic neurons (10,000 neurons per test site) were isolated at birth (PO), and cultured for 4 days in 10 nanograms / milliliter of NGF. The neurons were washed to free them from NGF on day 4, and were infected with recombinant adenovirus that encoded Gabl at a MOI of 30 or 100. The experiment shown in Figure 12 shows that the survival percentage after 5 days of the infection, is related to survival in 10 nanograms / milliliter of NGF. Inhibition of neuronal cell death by recombinant adenovirus encoding growth factor receptors The experiment shown in Figure 13 demonstrates that the TrkB adenovirus mediates the survival of sympathetic neurons. Sympathetic neurons expressing TrkA but not TrkB (10,000 neurons per test site) were isolated at birth (P0), and cultured for 4 days at 10 nanograms / milliliter of NGF. The neurons were washed to free from NGF on day 4, and were infected in the presence of BDNF with recombinant adenoviruses encoding wild-type TrkB. The graph shows the survival percentage after 5 days in BDNF (shaded bars), or in the absence of BDNF (black bars) in relation to survival in NGF. The experiment shown in Figure 14 demonstrates that TrkB-mediated sympathetic neuron survival requires both Shc / Ras / PI-3 kinase activation sites and phospholipase C (PLC) -gammal activation sites on TrkB. Sympathetic neurons (10,000 neurons per test site) isolated at birth (PO) were cultured for 4 days at 10 nanograms / milliliter of NGF. The neurons were washed to free from NGF on day 4, and were infected in the presence of BDNF or NGF with recombinant adenoviruses encoding wild-type TrkB, or TrkB containing mutations in the sites we have found that are required for interactions of TrkA with intracellular signaling proteins. The TrkB proteins tested were TrkB wild-type (WT), or the following mutations of TrkB: inactive kinase; mutant Y513F defective in activation of SHC, Ras, and PI-3 kinase; Y814F defective in phospholipase C (PLC) -gamma1 activation; double mutant Y513F / Y814F; or Def, defective in the activation of SHC, Ras, kinase PI-3, phospholipase C (PLC) -gammal, and SN. The survival of sympathetic neurons was evaluated on day 9 by MTT assay. The graph shows the survival percentage after 5 days in BDNF (shaded bars), or in the absence of BDNF (dot bars), in relation to survival in NGF (black bars). EXAMPLE VIII: GENE TRANSFER TO LIVE SYMPATHETIC NEURONS Because the adenovirus appears to be an effective gene transfer vector in vitro, we set out to determine if the recombinant virus could be effectively delivered to SCG neurons in vivo. For live administration, we injected 5xl09 plaque-forming units / milliliter into the ear pavilion of adult mice; The pavilion is one of the targets of the axon terminals that extend from the SCG neurons. Thirty minutes before the injection of the recombinant adenovi-rus, the mice were injected with 0.05 milligrams / kilogram of buprenorphine (Temgesic®, Pickitt and Colman Ltd.), as an analgesic, and then anesthetized by inhalation of Methoxyfluorane (Metofane®, Janssen Pharmaceuti-cals). Fourteen days after administration of the adenovirus, the mice were sacrificed by deep anesthesia consisting of 100 milligrams / kilogram of sodium pentobarbital (Somnitol®, MTC Pharmaceuticals, Cambridge, Ontario). Upper cervical nodes (SCG) were removed, and rinsed in a solution containing 0.1 M NaH2P02 (pH 7.3), 2 mM MgCl2, 0.01 percent sodium deoxycholate, and 0.02 percent NP-40. The β-galactosidase gene product was visualized by incubation of the lymph nodes at 37 ° C in the same rinse solution containing 1 milligram / milliliter of X-gal, 5 mM K3Fe (CN) 6, and K4Fe (CN) 6 5 mM, for 3 hours. Then the lymph nodes were rinsed three times, submerged in fixative (4 percent paraformaldehyde) for 1 hour, and examined microscopically. For histological examination, the lymph nodes were cryoprotected by passage through solutions containing ascending concentrations of sucrose (12 percent, 16 percent, 18 percent) for at least 4 hours each, frozen, and sectioned up 15 mins. The sections were stained with eosin, dehydrated in ascending concentrations of ethanol, followed by xylene, and covered with the coverslip. Numerous LacZ positive cells were seen in the ipsilateral SCGs of the injected animals, indicating that the adenovirus can be delivered to the SCG neurons via retrograde transport. No dyeing was found in 2 of 3 animals in the contralateral ganglia, although one animal exhibited a few positive cells. The general examination of the animals failed to reveal potential side effects, such as inflammation or SCG dysfunction, which would have presented as redness of the ear or ptosis of the eye. No swelling was observed in the ganglia that contained transduced neurons during surgical removal. Those skilled in the art will be able to see that comparable adenovirus vectors can be administered to other animals and to human patients in the same manner, i.e., by targeting the neurons to be transduced. It is well within the ability of the expert to perform this administration. The adenovirus vector can be prepared as described herein, and can be administered intravenously, intraarterially, subcutaneously, intrathecally, intraperitoneally, intramuscularly, intracerebrally, or intraventricularly. The route of administration and effective dosage will depend on other parameters routinely evaluated by practicing clinicians, including the patient's age and general health, and other medications that are being administered concurrently. EXAMPLE IX: INFECTION OF CORTICAL PROGENITOR CELLS AND POSMITTAL CORTICAL NEURONS WITH RECOMBINANT ADENOVIRUS The following experiments demonstrate that recombinant adenoviruses can be used to infect and modulate apoptosis in cortical progenitor cells and in post-mitotic neurons. Cell culture The preparation of cortical progenitors from mouse embryos was based on the method described by Ghosh et al. (1995) for rat cultures, and modified in correspondence with Brewer et al., Na ture 363: 265-266, 1993 Barks of E12-13 mouse embryos were harvested, ground, and coated at a density of 10 5 cells per well of a four-well tissue culture dish. The culture medium consisted of a neurobasal medium (Gibco / BRL), 0.5 mM glutamine, penicillin-streptomycin, 1 percent N2 supplement (Gibco / BRL), or bFGF (40 nanograms / milliliter); Collaborative Research Inc.). After 48 hours, the medium was replaced with the same medium, except that now the 1% N2 supplement was replaced with 2% B27 (Gibco / BRL). The cortical neurons generated from these progenitor cells could be maintained for at least three weeks under these conditions. For the neurite extension tests, after 5 days, the neurons were changed to a medium containing 10 nanograms / milliliter of nerve growth factor (NGF). When the cells were cultured from transgenic mice, the tissue of each embryo was removed, ground, and coated separately before genotyping. Mature post-mitotic neurons were prepared from embryos E17-18, from which the cortices were harvested, they were crushed in a culture medium (Neurobasal with 0.5 mM glutamine, penicillin-streptomycin, 0.5% N2, supplements B27 at 1 percent), and were coated at a density of 0.5x06 cells / milliliter. Infection of cortical neurons with recombinant adenoviruses Cortical progenitor cells were infected at the time of coating, and cortical neurons were infected 8 days after coating, to ensure that the vast majority of neurons in the cultures were post-mitotic. For infection with the virus, the cells were coated in 4-well tissue culture dishes, in 400 microliters of a medium with the addition of another 400 microliters of a medium containing the appropriate titration of the vector. Eighteen hours after the infection, a complete change of the medium was made. The multiplicity of infection (5-100 MOI) indicates the number of plaque-forming units added per cell. Cell survival assays were performed after 72 hours after infection with cortical progenitor cells, and 4 days after infection for cortical neurons. The experiments shown in Figures 17A and 17B demonstrate that infection of cortical progenitor cells and post mitotic cortical neurons with recombinant adenoviruses encoding lacZ (AdlacZ) or mutant ElA (AdllOl), results in efficient expression of the encoded proteins for the adenovirus. At 0 and 8 days in vi tro, respectively, the cortical progenitor cells (Figure 17A) and the neurons (Figure 17B) were infected with recombinant AdlacZ at 25 MOI. After 48 hours, the cells were stained with X-gal to visualize the expression of the β-galactosidase gene. In Figure 17C, the cortical progenitors were infected with AdllOl at 100 MOI on the coating, and the protein was extracted 48 hours later. ElA 1101 was detected by Western blot with M73 antibody. Scale bar = 50 micras. The experiments shown in Figures 18 to 21 demonstrate that cortical progenitor cells, but not postmitotic cortical neurons, are induced to die by ElA encoded by a recombinant adenovirus. Figure 18 shows the quantitative effect of infection with AdlacZ against AdllOl on the survival of cortical progenitor cells and post mitotic neurons, measured using MTT assays. In Figure 18A, the cortical progenitor cells were infected with AdlacZ (black bars) or AdllOl (gray bars) at 0, 10, 25, 50, or 100 MOI, at the time of coating. Cell survival was assayed 3 days later using the MTT assay. A concentration-dependent decrease in cell survival was detected in cells infected with AdllOl in relation to the control cells infected with AdlacZ. In Figure 18B, at 8 days, post mitotic cortical neurons were infected in the same titers as described in Figure 18A, and MTT tests were performed 4 days later. No change in cell survival was detected in any viral titre tested on neuronal cultures. The results represent the average of three different experiments + the standard error of the average. Figures 19A-L show a comparison of cell viability of cortical progenitors and neurons infected with recombinant AdlacZ and AdllOl, using Live / Dead dyeing. Cortical progenitors (the two columns on the left) and cortical neurons (the two columns on the right) were infected on the coating, and 8 days later, respectively, with 25 MOI of AdlacZ (Figure 19E-H) , AdllOl (Figures 19I-L), or were left uninfected (Figures 19A-D). Live cells were measured by enzymatic conversion of perceating calcein-AM to fluorescent calcein (green). The dead cells were detected by the recovery of ethidium bromide into the cell's DNA (red). A dramatic increase in cell death, accompanied by a decrease in cell survival, was detected in progenitor cells infected with AdllOl (Figures 191, J), when compared with uninfected progenitor cells (Figures 19A, B), or with those infected with AdlacZ (Figure 19E, F). In contrast, survival of cortical neurons was not affected by infection with AdlacZ (Figures 19G, H) or AdllOl (Figures 19K, L) relative to uninfected neurons (Figures 19C, D). Size bar for Figures 19A-L = 50 microns. Figures 20A-D show a characterization of the cultures of post-mitotic cortical neurons E18. After 6 days in culture, most of the cells in culture were post mitotic neurons, as indicated by the low level of anti-BrdU immunostaining after a 12 hour incubation with BrdU (Figure 20B) in relation to the total number of cells, as indicated by the phase contrast micrograph of the same field (Figures 20A). The MAP2 neuronal marker was highly expressed at this stage, as indicated by staining with anti-MAP2 (Figure 20D). Figure 20C shows a phase contrast micrograph of the same field. Scale bars: Figures 20A, B) 50 micras; (Figures 20C, D) 50 microns. Figures 21 (A-F) show that the survival of post mitotic cortical neurons is not affected by functional ablation of members of the pRb family. After 8 days, the cortical neurons in vitro were left uninfected (Figures 21A, B), or were infected with 25 MOI of AdlacZ (Figures 21C, D), or AdllOl (Figures 21E, F). Four days later, apoptosis was supervised by TUNNEL marking. No increase was detected in cells positive for TUNNEL, in neurons infected with AdllOl (Figure F) in relation to AdlacZ (Figure 21D), or to uninfected cells (Figure 21B). Figures 21A, C, E are phase contrast micrographs of the fields of Figures 21B, D, F, respectively. The figures are representative of six separate experiments that show similar results. Scale bar for Figures 21A-F = 50 microns. EXAMPLE X: TESTS FOR NEURONAL GROWTH The experiments described below demonstrate methods for measuring the preferential growth response of sympathetic neurons to neuronal growth factor NT-3, in relation to growth in response to neuronal growth factor BDNF. It is understood that analogous assays can be used to analyze the growth response of different types of neurons infected with recombinant adenoviruses, for example, an adenovirus that encodes a neurotrophin receptor, such as a TRK receptor. NT-3 selectively promotes the extension of neurite in NGF-dependent sympathetic eurons. To determine if sympathetic neurons responded to NT-3 or BDNF after becoming dependent on the NGF derived from the target, we selected the NGF-dependent population of neurons neonatal sympathetics, by cultivating in 10 nanograms / milliliter of NGF for 5 days (Figure 22), and we examined the neurotrophin-mediated survival and the neurite extension. To test for survival responses, after selection in NGF, the neurons were changed to 30 nanograms / milliliter of NT-3 or BDNF. BDNF was not sufficient to support the survival of NGF-dependent neurons; at 2 days after the change, all neurons in the cultures were dead, as monitored by counting bright-phase cell bodies. In contrast, 25 to 30 nanograms / milliliter of NT-3 were sufficient to support the survival of a small population of NGF-dependent neurons. To determine whether the addition of NT-3 or BDNF could mediate the extension of the neurite regardless of survival, sympathetic neurons were coated on collagen, and selected at 10 nanograms / milliliter of NGF for 5 days, and then 30 nanograms were added / milliliter of NT-3 or BDNF in the presence of 10 nanograms / milliliter of NGF for 2 additional days. The addition of NT-3 led to a large increase in the density of the neuritic processes (Figure 23A), with an increase of 2 to 2.5 times in the neuritic density in each of three separate experiments. In contrast, the addition of 30 nanograms / milliliter of BDNF did not have a measurable effect (Figures 22C and 23A). To more precisely define the effect of NT-3 on neuritogenesis, low-density sympathetic neurons were coated on poly-D-lysine / laminin, selected for 5 days at 10 nanograms / milliliter of NGF, and then changed to 10 nanograms / milliliter of NGF plus 30 nanograms / milliliter of NT-3, or 30 nanograms / milliliter of NGF. Two days later, the density of the process network, the total length of the neurites, and the size of the cell bodies were measured. As seen in the higher density crops (Figure 23A), the density of the process network was increased from 2 to 2.5 times in the presence of 10 nanograms / milliliter of NGF plus 30 nanograms / milliliter of NT-3 (Figure 23B). A statistically similar increase was observed with 30 nanograms / milliliter of NGF. Similar results were obtained from measurements of the total length of the neurite; both NT-3 and NGF mediated an increase of approximately 1.5 times (Figure 23C). In contrast, NGF and NT-3 differentially regulated the size of the cell body (Figure 23D). Neurons cultured at 10 nanograms / milliliter of NGF plus 30 nanograms / milliliter of NT-3 exhibited a small but significant increase (P = 0.002) of approximately 10 percent, whereas neurons cultured at 30 nanograms / milliliter of NGF were hyperatrophied in approximately 25 to 30 percent, an increase that was significantly greater than that obtained with NGF plus NT-3 (P <0.001). Therefore, although NT-3 was approximately equivalent to NGF in its ability to promote neurite outgrowth, it was significantly less effective in promoting cell body hypertrophy, and was 20 to 40 times less efficient in promoting neuronal survival. . NT-3 selectively induces gene expression associated with growth In neonatal sympathetic neurons, NGF regulates the expression of mRNAs encoding tyrosine hydroxylase, p75 neutrophin receptor, and α-tubulin Tal in a concentration-dependent graduated form . To determine whether NT-3 regulates gene expression as it regulates neurite extension, sympathetic neurons were selected at 10 nanograms / milliliter of NGF for 5 days, followed by 10 or 30 nanograms / milliliter of NT -3 in addition to 10 nanograms / milliliter of NGF. The RNA was isolated at the time points from 6 to 48 hours after the addition. Northern blot analysis revealed that the addition of 30 nanograms / milliliter of NT-3 for 6 hours led to a 5- to 10-fold increase in a-tubulin mRNA Tal, a member of the a-tubulin multigenetic family whose expression is regulated as a function of neuronal growth. This increase was maintained at 24 and 48 hours, consistent with the large increase in the density of the neuritic process induced by the addition of NT-3, and was concentration dependent: 10 nanograms / milliliter of NT-3 did not cause significant increase in a-tubulin T l mRNA. The magnitude of the increase observed with 30 nanograms / milliliter of NT-3 was similar to that observed on the addition of 200 nanograms / milliliter of NGF. The levels of a-tubulin T l mRNA are increased in a concentration dependent manner with increasing levels of NGF, with a plateau at 100-200 nanograms / milliliter. Accordingly, 30 nanograms / milliliter of NT-3 were able to cause such a large increase in a-tubulin Tal mRNA, as the saturation amounts of NGF. In contrast to NT-3, the addition of 30 nanograms / milliliter of BDNF had no effect on the expression of a-tubulin T l mRNA. The addition of 30 nanograms / milliliter of NT-3 also led to a smaller increase, of approximately 2 to 3 times, in the expression of tyrosine hydroxylase mRNA. This increase, which was not caused by 10 nanograms / milliliter of NT-3, was observed first at 6 hours, and subsequently maintained for 48 hours. The addition of BDNF had no effect on the expression of tyrosine hydroxylase mRNA. Other Modalities The following groups of adenovirus constructs can be used in accordance with the methods of the invention, as described herein: (1) p75 wild-type, p75-truncation 1 (no DD), p75-truncation 2 (no ICD - to the Pvull site), p75-truncation 2 (no ICD-to the Narl site), p75 ICD, or p75 mICD; (2) pMAGE or other Mages; (3) Trafl, Traf2, Traf3, Tradd, Fadd / MORT-1, FP, FAP, or FAN; (4) I? B-a, I? B-ß, Bcl-31, I? B-epsilon, or RelA / p65; (5) rhoA, racl, cdc42, PAK1, PAK2, PAK3, G-PAK, or Germinal center kinase (GC); (6) MEKKl, MEKK2, MEKK3, SEK1 / MKK4, or Tpl-2; (7) SEK1, MKK3, MKK6, or MLK (mixed lineage kinases - SPRK, DLK, ZPK, MUK); (8) p54 JNK, p38, or MAPK; (9) jun, atf-2, Elk-1, or Max; (10) Wild type TrkA, TrkA Y490F (defective in Shc and PLC interactions); TrkA Y785F (defective in PLC interactions), TrkA Y490F / Y785F, KFG (dl441-443, defective in SNT interactions), TrkA (kinase-inactive), or truncated TrkA (constitutively active with TM and ICD); (11) TrkA Y490F / Y785F / KFG (TrkAdef); (12) TrkA Y490F / KFG (specific for PLC interactions), or TrkA Y785F / KFG (specific for Shc interactions); (13) TrkAdef + P1-3K, TrkAdef + src, TrkAdef + Grb2, TrkAdef + Syp, TrkAdef + rasGAP, or TrkAdef + STAT1; (14) all retroaggregated mutants in combination with wild-type SNT (intact KFG), PLC sites (Y785), or Shc (Y490); (15) the same series of unpublished mutants and published in the TrkB and TrkC genes; (16) Aktl, Akt2, Pl-3 kinase, or SHP; (17) c-yes, c-src, or c-fyn; (18) SOS, Gabl, Ras, rasGAP, B-raf, Raf-1, KSR, MEK1, MEK2, Rskl, Rsk2, Rsk3, MAPK1, or MAPK2; (19) SH-PTP1, or SH-PTP2; (20) STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or PLC-? L, PKCd, PKCe, or PKC ?; (21) MPK1 or MPK2; (22) p53 or Csk; (23) JAK1, JAK2, JAK3, or GSK3; (24) bcl-2, bcl-x, bcl-xl, bax, or bak; and (25) p75 wild-type or the deletion or substitution mutants shown in Figure 11.

Claims (32)

1. CLAIMS 1. A post-mitotic neuron containing a recombinant adenovirus vector, said neuron (a) having been infected with said recombinant adenovirus vector at a multiplicity of infection of about 10 to about 50, and (b) expressing a product of gene encoded by a DNA molecule contained within said recombinant adenovirus vector.
2. 2. The neuron of claim 1, said neuron being infected - while in tissue culture.
3. 3. The neuron of claim 1, said neuron being infected in vivo.
4. 4. The neuron of claim 1, wherein said adenovirus vector further comprises a reporter gene.
5. The neuron of claim 4, wherein said reporter gene is selected from the group consisting of alkaline phosphatase, chloramphenicol, acetyltransferase, lacZ, and the green fluorescent protein.
6. 6. The neuron of claim 1, wherein said DNA molecule encodes a tumor suppressor gene.
7. 7. The neuron of claim 1, wherein said tumor suppressor gene is p53.
8. 8. The neuron of claim 1, wherein said DNA molecule encodes a growth factor receptor.
9. 9. The neuron of claim 8, wherein said growth factor receptor is a member of the Trk family.
10. 10. The neuron of claim 8, wherein said growth factor receptor is p75.
11. 11. The neuron of claim 1, wherein said neuron is a sympathetic neuron.
12. 12. The neuron of claim 11, wherein said neuron is a dopaminergic neuron.
13. 13. The neuron of claim 1, wherein said neuron is a cortical neuron.
14. 14. The neuron of claim 1, wherein said DNA molecule encodes a protein that inhibits apoptosis.
15. 15. The neuron of claim 14, wherein said protein is Bcl-2.
16. 16. The neuron of claim 14, wherein said protein is Bcl-xL.
17. 17. The neuron of claim 14, wherein said protein is EIB55K.
18. 18. The neuron of claim 14, wherein said protein is Gabl.
19. 19. The neuron of claim 1, wherein said protein encodes a protein that induces apoptosis.
20. 20. A method of inhibiting or inducing apoptosis in a post-mitotic neuron, said method comprising infecting said neuron with a purified adenoviral vector, said vector comprising DNA encoding a protein that inhibits or induces apoptosis.
21. 21. The method of claim 20, wherein said protein induces said apoptosis.
22. 22. The method of claim 21, wherein said protein is p53, or a biologically active fragment thereof.
23. 23. The method of claim 20, wherein said apoptosis is inhibited, and said protein inhibits apoptosis.
24. 24. A method of identifying a substance that inhibits apoptosis, said method comprising (a) culturing a population of post-mitotic neurons; (b) infecting the neurons of said population with an adenovirus vector comprising DNA encoding a protein that induces apoptosis; (c) exposing a subset of the population of infected neurons in step (b) to a substance, said substance being suspected of inhibiting apoptosis; and (d) comparing the approximate number of neurons undergoing apoptosis in the sub-set of the population was infected and exposed to the substance with the approximate number of neurons undergo apoptosis in the population of cells were infected, a relative decrease in the number of apoptotic cells in the subset of the population indicating an effective inhibitor of apoptosis.
25. 25. The method of claim 24, wherein said DNA encodes p53.
26. 26. A method of identifying a substance that induces apoptosis, said method comprising (a) cultivating a population of post-mitotic neurons; (b) infecting the neurons of said population with an adenovirus vector comprising DNA encoding a protein that inhibits apoptosis; fc) (c) exposing a subset of the population of neurons infected in step (b) to a substance, said substance being suspected of inducing apoptosis; and (d) comparing the approximate number of neurons suffering apoptosis in the subset of the population that was infected and exposed to that substance with the approximate number of neurons suffering apoptosis in the population of cells that were infected, an increase Relative in the number of apoptotic cells in the subset of the population indicating an effective inducer of apoptosis.
27. 27. The method of claim 26, wherein said DNA encodes Bcl-2.
28. 28. The method of claim 27, wherein said neuron is induced to re-enter the cell cycle following the expression of said Bcl-2.
29. 29. A method of identifying a substance that inhibits growth or proliferation, said method comprising (a) culturing a population of post-mitotic neurons; (b) infecting the neurons of said population with an adenovirus vector comprising DNA encoding a growth-inducing protein; (c) exposing a subset of the population of neurons infected in step (b) to a substance, said substance being suspected of inhibiting growth or proliferation; and (d) comparing the approximate number of neurons that undergo growth or proliferation in the subset of the population that was infected and exposed to that substance with the approximate number of neurons that suffer growth in the population of cells that were infected, a relative decrease in the expression of T l a-tubulin or a T l a-tubulin transgene indicating an effective inhibitor of growth or proliferation.
30. 30. The method of claim 29, said protein being the TrkB receptor.
31. 31. The method of claim 30, wherein said neuron is induced to re-enter the cell cycle following the expression of said TrkB receptor.
32. 32. The method of claims 20, 24, 26 or 29, wherein said vector is applied to said neurons at a multiplicity of infection of about 10 to about 50.