WO2002015912A1 - Compounds and methods for inhibiting neuronal cell death - Google Patents

Abstract

The invention provides compounds and methods that are useful for the treatment and prevention of a variety of diseases associated with oxidative-stress, DNA damage, or growth factor deprivation. The invention also features methods for detecting the presence of oxidative-stress, DNA damage, or growth factor deprivation in a cell. Additionally, methods are provided for identifying compounds that increase or decrease oxidative-stress, DNA damage, growth factor deprivation, or cell death.

Description

COMPOUNDS AND METHODS FOR TNHTBTTING NFJJRONA CF.T.T.

DEATH

Background of the Invention

Reactive oxygen species (ROS) are generated by the environment (e.g., photo-oxidations and emissions) and normal cellular functions (e.g., mitochondrial metabolism and neutrophil activation) (Gracy et al, Mutat. Res., 428:17-22, 1999). Glutamate, which depletes the glutathione necessary to scavenge ROS, also induces oxidative stress (Kobayashi et al, Free Radio. Res. 32:115-124, 2000). ROS include free radicals {e.g., superoxide and hydroxyl radicals), nonradical oxygen species (e.g., hydrogen peroxide and peroxynitrite), and reactive lipids and carbohydrates (e. g., ketoaldehydes and hydroxynonenal). ROS can oxidize the bases and sugars of DNA and crosslink DNA. Such modifications can lead to DNA mutations, disease, and death. Oxidation of proteins appears to play a causative role in many chronic diseases of aging including cataractogenesis, rheumatoid arthritis, and various neurodegenerative diseases including Alzheimer's Disease (AD) and Parkinson's Disease (Gracy, supra; Arlt et al, Free Radic. Res. 32: 103-114, 2000).

Several lines of evidence suggest that ROS are associated with AD. Lesions that are typically associated with attack by free radicals and metals that have the ability to generate free radicals (e.g., iron, copper, zinc, and aluminum) are found in the brains of AD patients. Beta-amyloid accumulates in the brains ofthese patients and generates additional free radicals once free radicals are present, possibly through stabilization of the more reactive ferrous (Fe²⁺) iron form (Christen, Am. J. Clin. Nutr. 71:621S-629S, 2000; Yang et al, Brain Res. 839:221-226, 1999). Mitochondrial anomalies involving cytochrome-c oxidase that could increase the production of free radicals are associated with AD (Christen, supra). Additionally, oxidated apolipoprotein E and advanced glycation endproducts, which may accumulate due to an accelerated oxidation of glycated proteins, are present in brain plaques of AD patients (Durany et al., Eur. Arch. Psychiatry Clin. Neurosci. 249 Suppl. 3:68-73, 1999; Christen, supra). 3-Nitro-tyrosine is also more abundant in AD patients suggesting that the neuromfammatory process which yields both ROS and reactive nitric oxide species is involved in AD (Floyd, Proc. Soc. Exp. Biol. Med. 22:236-245, 1999).

The antioxidant vitamin E has been shown to inhibit amyloid beta peptide (Abeta)-associated free radical oxidative stress in cortical synaptosomal membranes and hippocampal neuronal cells in culture (Butterfiled et al, Rev. Neurosci. 10:141-149, 1999). Moreover, a clinical trial of vitamin E in AD patients indicated that vitamin E may slow functional deterioration in these patients (Grundman et al.., Am. J. Clin. Nutr. 7 630S- 636S, 2000). Other compounds that have an antioxidant effect—including the antioxidants selegeline, melatonin, Ginkgo biloba extract EGb 761, and alpha-phenyl-tert-butylnitron; the iron chelating agent desferrioxamine; antiflammatroy drugs; and estrogens—have produced promising results in relation to AD (Christen, supra; Lahire et al.., Ann. N.Y. Acad. Sci. 893:325- 330, 1999; Floyd, 2000). Using multiple antioxidants for the prevention of Parkinson's and Alzheimer's disease among high-risk populations and as a treatment ofthese diseases has been suggested (Prasad et al, Curr. Opin. Neruol. 12:761-770, 1999). However, more effective agents are needed to inhibit oxidative-induced damage and death in disease states. In addition, no good markers are available to determine which patients will benefit from antioxidant therapy and which antioxidants reach the sites of active disease in the brain.

Summary of the Invention We have discovered that mithramycin A and chromomycin A₃ inhibit oxidative stress-induced cell death in mammalian cells. Mithramycin A was also shown to inhibit cell death due to DNA damage or growth factor deprivation. Accordingly, the invention features, in one aspect, a method of inhibiting oxidative stress-induced, DNA damage-induced, or growth factor deprivation-induced death in mammalian cells by contacting the cells with a mithramycin, a chromomycin, or a derivative thereof (as defined herein). In one preferred embodiment of this method, the treated cells are neuronal cells, preferably cortical neurons, cerebellar granule cells, or sympathetic neurons. In another preferred embodiment, a mithramycin, a chromomycin, or a derivative thereof is administered to a human patient for the treatment or prophylaxis of a disease or disorder of the nervous system or associated with the aging process. Preferred diseases of the nervous system that may be treated or prevented using this method are neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, Cruetzfeld- Jacob disease, kuru, multiple sclerosis, multiple system atrophy, amyotrophic lateral sclerosis (Lou Gehrig^'s Disease), progressive supranuclear palsy, and spinal cord disease. Other diseases or disorders that may be treated include those associated with apoptosis, oxidative-stress, DNA damage, or growth factor depletion, such as mitochondrial diseases (Adams Victor and Ropper, Principles of Neurology, Sixth Edition, 1997, The

McGraw-Hill Companies, Inc.). Examples of mitochondrial diseases include diseases caused by, or associated with, a genetic mutation in mitochondrial DNA, such as Leber's hereditary optic neuropathy or MELAS (mitochondrial encephalopathy lactic acidosis and stroke). Viral infections such as herpes and HIV infections may also be treated, stabilized, or prevented using this method. Examples of other medical problems that may be treated or prevented include nervous system injuries, such as stroke, spinal cord injury, and brain injury. Examples of stokes that are associated with an increase in blood pressure include focal strokes, such as strokes involving a blot clot or the blockage of a blood vessel. Exemplary stokes that are associated with a decrease in blood pressure include global strokes, such as strokes associated with a heart attack or an insufficient supply of blood. Preferred routes of administration include oral, intramuscular, intravenous, intracranial, intrathecal, or subcutaneous administration. The dosage to be administered and the dosing frequency may be determined during clinical trials using standard procedures. In various embodiments, the amount of a mithramycin or a mithramycin derivative that is administered is less than or equal to 0.1 μg/kg, 0.05 μg/kg, 0.01 μg/kg, 0.005 μg/kg, or 0.001 μg/kg. In still other embodiments, the administered amount is between 0.05 and 0.01 μg/kg, 0.01 and 0.005 μg/kg, or 0.005 and 0.001 μg/kg, inclusive. For example, less than 0.1 μg/kg of a mithramycin, chromomycin, or olivomycin may be administered to treat or prevent Parkinson's disease or multiple sclerosis. In another aspect, the invention features a method of determining whether a compound is capable of inhibiting or inducing oxidative stress, DNA damage, growth factor depletion, or cell death in vitro or in vivo. This method involves contacting the compound with a cell in the presence or absence of an oxidant, DNA-damaging agent, or growth factor depletion agent and determining whether the compound effects a change in the cell in the protein level of an Sp family member or in binding of an Sp family member to DNA. A compound is determined to be an inhibitor of oxidative stress, DNA damage, growth factor depletion, or cell death if it reduces the protein level of an Sp family member or if it decreases the binding of an Sp family member to DNA. In a preferred embodiment, the inhibitor reduces the increase in the protein level of an Sp family member that occurs in the presence of another compound, such as an oxidant, DNA-damaging agent, or growth factor depletion agent. A compound is determined to be an inducer of oxidative stress, DNA damage, growth factor depletion, or cell death if it increases the protein level of an Sp family member or increases the binding of an Sp family member to DNA. Preferably, this change in induction or binding is at least 25%, more preferably at least 50%, and most preferably by at least 90% as determined by standard assays described herein. Preferred cells used in this method include cortical neurons, cerebellar granule cells, and sympathetic neurons. The compound tested may be a member of a library of at least 5, 10, 20, 50, 100, or 500 compounds, all of which are simultaneously contacted with the cell. Additionally the compounds tested may include nucleic acid molecules that have a polynucleotide sequence comprising 20, 50, 100, 250, or more nucleotides that have at least 80, 90, 95, or 100% sequence identity to the corresponding region of a polynucleotide encoding an Sp family member. Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). An antibody specific for a distinct Sp family member may also be tested using this method. In a related aspect, the invention provides a method for preventing or treating a disease or disorder of the nervous system, the aging process, or associated with apoptosis in a mammal (e.g., a human), including administering to the mammal a compound that inhibits the induction of an Sp family member or the binding of an Sp family member to DNA (e.g., a mithramycin, a mithramycin derivative, or a compound identified using a method described herein). Preferably, a cortical neuron, cerebellar granule cell, or sympathetic neuron is contacted with the compound. Preferred routes of administration include oral, intramuscular, intravenous, intracranial, intrathecal, or subcutaneous administration. Preferred diseases of the nervous system that may be treated or prevented using this method are neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, Cruetzfeld- Jacob disease, kuru, multiple sclerosis, multiple system atrophy, amyofrophic lateral sclerosis, progressive supranuclear palsy, and spinal cord disease. Other diseases or disorders that may be treated include those associated with apoptosis, oxidative-stress, DNA damage, or growth factor depletion; such as mitochondrial diseases or viral infections. Examples of other medical problems that may be treated or prevented include nervous system injuries, such as stroke, spinal cord injury, and brain injury. Examples of stokes that are associated with an increase in blood pressure include focal strokes, such as strokes involving a blot clot or the blockage of a blood vessel. Exemplary stokes that are associated with a decrease in blood pressure include global strokes, such as strokes associated with a heart attack or an insufficient supply of blood. The dosage to be administered and the dosing frequency may be determined during clinical trials using standard procedures. In various embodiments, the amount of compound that is administered is less than or equal to 0.1 μg/kg, 0.05 μg/kg, 0.01 μg/kg, 0.005 μg/kg, or 0.001 μg/kg. In still other embodiments, the administered amount is between 0.05 and 0.01 μg/kg, 0.01 and 0.005 μg/kg, or 0.005 and 0.001 μg/kg, inclusive.

In yet another aspect, the invention features a method for detecting oxidative stress, DNA damage, or growth factor depletion in a cell. This method involves analyzing the cell for an increase in the protein level of an Sp family member or an increase in the binding of an Sp family member to DNA. Preferably, this change in induction or binding is at least 25%, more preferably at least 50%, and most preferably at least 90% as determined by standard assays described herein. One preferred embodiment of this method is the comparison of oxidative stress, DNA damage, or growth factor depletion between a transgenic and a wild-type animal. Another embodiment includes analysis of an autopsy tissue to verify or refute the presence of oxidative stress, DNA damage, or growth factor depletion and the effect of treatments, including antioxidants, for a particular disease. Preferred cells that may be analyzed using this method include neuronal cells, such as cortical neurons, cerebellar granule cells, and sympathetic neurons.

In preferred embodiments of various aspects of the invention, the Sp family member is Sp-1 or Sp-3. Preferred mithramycins include mithramycin A, mithramycin B, and mithramycin C. Preferred chromomycins include chromomycin A₃. Because the carbohydrates attached to the aglycon backbone of mithramycin A and chromomycin A₃ differ and because mithramycin A and chromomycin A₃ were both found to inhibit oxidative stress-induced cell death in neurons, derivatives of mithramycins and chromomycins that contain modified carbohydrate residues are also expected to inhibit oxidative stress-induced, DNA damage-induced, and growth factor depletion-induced induction of Sp-1 and Sp-3 protein levels, Sp-1 and Sp-3 DNA binding, and cell death. Standard methods may be used to modify the carbohydrate residues of mithramycins or chromomycins or to replace a carbohydrate in one ofthese molecules with another carbohydrate. For example, chromomycin A₃ contains two ester moieties instead of two hydroxyl groups found in the corresponding position of mithramycin A, and chromomycin A₃ also contains an alkoxy group instead of a hydroxyl group found in the corresponding position of mithramycin A (Figs. 17A and 17B). Thus, the hydroxyl groups on carbohydrates or aglycon rings in mithramycins may be replaced by ester or alkoxy groups (Figs. 18 A), and the resulting mithramycin derivatives may be tested in the assays described herein. Additionally, the ester or alkoxy groups on carbohydrates in chromomycins may be replaced by hydroxyl groups (Fig. 18B). Other preferred mithramycin or chromomycin derivatives include other members of the aureolic acid derivative family, such as daunomycins, WP631, olivomycins, and their derivatives (Figs. 17C, 17D, 18C, and 18D). Other derivatives listed in Figs. 18A-18D are also preferred compounds for use in the methods of the present invention. For these derivatives each R is, independently, a hydroxyl group, an ester group, or an alkoxy group. Preferably, the ester group has the formula -OC(0)R', wherein each R' is, independently, a linear or branched saturated hydrocarbon alkyl group of 1 to 10, 1 to 20, 1 to 50, or 1 to 100 carbon atoms; such as a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, or tetradcyl group; or a cycloalkyl group, such as a cyclopentyl or cyclohexyl group. Other preferred R' groups include aryl groups, such as a monovalent aromatic hydrocarbon radical consisting of one or more fused rings in which at least one ring is aromatic in nature, which may optionally be substituted with one of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino, or diakylamino. Preferred alkoxy groups have the formula -OR', wherein R' is an alkyl or aryl group as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, and isopropoxy groups. For WP631 derivatives (Fig. 18D) R" may be an alkyl or aryl group as defined above. Additionally, R" may contain one or more carbohydrate groups. Examples of carbohydrate groups include those that have been modified (e.g., wherein one or more of the hydroxyl groups are replaced with halogen, alkoxy moieties, aliphatic groups, or are functionalized as ethers, esters, amines, or carboxylic acids). Examples of modified carbohydrates include α- or β-glycosides such as methyl α-D- glucopyranoside or methyl β-D-glucopyranoside; N-glycosylamines; N-glycosides; D-gluconic acid; D-glucosamine; D-galactosamine; and

N-acteyl-D-glucosamine. Preferred carbohydrates include glucose, galactose, fructose, ribose, mannose, arabinose, and xylose. For the mithramycin, chromamycin A₃, and daunomycin derivatives (Figs. 18A, 18B, and 18C), the carbohydrate groups in the derivatives may also be modified or replaced with another carbohydrate group as described above. Other preferred derivatives have a chemical modification that enhances the bioavailability, solubility, stability, or potency of a compound in vivo or in vitro or that reduces the toxicity of a compound in vivo or in vitro. Such modifications are known to those skilled in the field of medicinal chemistry. In other desirable embodiments of any of the above aspects, the inhibition of oxidative stress, DNA damage, growth factor depletion, or cell death by the derivative is at least 10%, 30%, 40%, 50%, 75%, 90%, 95%, or 100% of that by the compound from which the derivative was derived. For example, the derivatives may be tested using the assays described herein to determine whether they modulate oxidative stress-induced, DNA damaged- induced, or growth factor deprivation-induced induction of Sp-1 or Sp-3 protein levels, Sp-1 or Sp-3 DNA binding, or cell death. Compounds that inhibit any ofthese activities may be useful in the treatment or prevention of various diseases, injuries, and infections, such as those described herein. In an embodiment of any one of the aspects of the invention, the compound is in a pharmaceutically acceptable carrier. Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The composition can be adapted for the mode of administration and is typically in the form of a pill, tablet, capsule, spray, powder, or liquid.

Other features and advantages of the invention will be apparent from the following detailed description.

Brief Description of the Drawings Fig. 1A is a picture of a DNA gel showing the increased DNA binding to a ³²P -labeled Sp-1 consensus oligo at three different nuclear extract protein concentrations in the presence of homocysteate (1 mM; HCA), using an electrophoretic mobility shift assay (EMS A). HCA is a glutamate analog which induces cystine deprivation and glutathione depletion in cortical neurons. Fig. IB is picture of a DNA gel showing the identification of Sp-1 in complex "a" and Sp-3 in complexes "b" and "c" from Fig. 1 A. Complex "a" did not contain other Sp family member including Sp-2, Sp-3, and Sp-4. EMS A was performed as in Fig. 1A with 0.5 ug of nuclear extracts from control (lane 1) and HCA (1 mM)-treated cortical neurons (lanes 2-6). Antibodies (denoted "Ab") to Sp-1 (lane 3), Sρ-2 (lane 4), Sp-3 (lane 5), and Sp-4 (lane 6) were added after addition of the radiolabeled oligonucleotide to the nuclear-binding reaction as described herein. Fig. 1C is a picture of a gel showing the time course of induction of Sp-1 and Sp-3 DNA binding activities following HCA (lmM)-freatment. Total glutathione levels (GSSG + GSH) are depleted to 50% of control by two hours after HCA treatment and to 30% of control by five hours after HCA treatment. Fig. ID is a picture of a gel showing the effect of HCA-induced glutathione depletion on Oct-1 DNA binding activity. The EMS A was performed as described in Fig. 1 A with 5 μg of nuclear extracts from control (lane 1) and HCA (1 nιM)-treated cortical neurons (lane 2), using a ³²P labeled Oct-1 -binding site oligonucleotide. EMS As are representative examples of the five experiments that were performed.

Fig. 2A is a picture of a gel demonstrating that the antioxidant iron chelator, DFO, suppresses homocysteate-induced Sp-1 and Sp-3 DNA binding. EMSA was performed with 0.5-1.0 μg of nuclear extracts incubated with buffer (lane 1), HCA (1 mM, lane 2), DFO (100 μM, lane 3), or DFO (100 μM) and HCA (1 mM) (lane 4), using a ³²P-labeled Sp-1 binding site oligonucleotide. Fig. 2B is a picture of a gel demonstrating that the lipid peroxidation inhibitor butylated hydroxanisole (BHA) also suppresses homocysteate-induced Sp-1 and Sp-3 DNA binding. EMSA was performed as described in Fig. 2A with 0.5-1.0 μg of nuclear extracts incubated with buffer (lane 1), HCA (ImM, lane 2), BHA (10 μM, lane 3), or BHA (10 μM) and HCA (1 mM) (lane 4). Fig. 3 A is a picture of a gel showing that the peroxide generating enzyme, D-amino acid oxidase (DAAO) plus its substrate D-Ala induces Sp-1 and Sp-3 DNA binding in a concentration dependent manner. EMSA was performed as described in Fig. 2 A with 0.5 μg of nuclear extract. D-amino acid oxidase from the red yeast, R. gracilicis, was used for these experiments. The amount of D-amino acid oxidase activity was determined with a horseradish peroxidase-coupled, spectrophotometric assay using o-dianisidine as a reducing chromogenic horseradish peroxidase substrate. For lane 1, 20 mM D-alanine (D-ala) was added to the nuclear extract; for lane 2, 1 milliunit (mU) D-amino acid oxidase (DAAO) and 20 mM D-ala were added; for lane 3, 3 mU DAAO and 20 mM D-ala were added; and for lane 4, 5 mU DAAO and 20 mM D-ala were added. Supershift analysis confirmed that the slowest migrating complex contained Sp-1 and the two faster migrating complexes contained Sp-3. Fig. 3B is a picture of a gel showing that addition of the peroxide scavenging enzyme, catalase, abrogates Sp-1 and Sp-3 DNA binding induced by D-ala (20 mM) and DAAO (5 mU). EMSA was performed as described in Fig. 2 A with 0.5 μg of nuclear extracts. For lane 1, 20 mM D-alanine was added to the extract; for lane 2, 5 mU DAAO and 20 mM D-ala were added, for lane 3, 5 mU DAAO, 20 mM D-ala, and 100 units/ml catalase were added; for lane 4, 100 units/ml catalase and 20 mM D-ala were added.

Fig. 4A is a picture of a gel showing the immunoblot analysis of the increase in Sp-1 and Sp-3 levels by incubation with homocysteate for 1, 3, or 5 hours. Whole cell lysates were prepared fiom cortical neurons treated for the designated periods of time with HCA, and subjected to immunoblot analysis using antibodies against Sp-1, Sp-3, and α-tubulin protein. Figs. 4B and 4C are pictures of protein gels showing the increase in induction of Sp-1 and Sp-3 in whole cell lysates prepared from cortical neurons after incubation with 0, 1, or 5 mM homocysteate for five hours. Higher concentrations of HCA induce a more rapid depletion of glutathione levels than lower concentrations. (Zaman et al, J. Neurosci. 19:9821-9830, 1999). Immunoblot analysis using an Sp-1 (Fig. 4B) or Sp-3 (Fig. 4C) antibody was performed as described in Fig. 4A. Immunoblots are representative examples of the five experiments that were performed.

Figs. 5 A-5I are pictures showing immunocytochemical analysis of Sp- 1 (Figs. 5A-5F), Sp-2 (Figs. 5G-5L), and Sp-3 (Figs. 5M-5R) in embryonic cortical neurons after glutathione depletion. The levels of nuclear Sp-1 and Sp-3, but not Sp-2, are increased. These pictures show the immunocytochemical analysis of Sp-1 (Figs. 5A and 5D), Sp-2 (Figs. 5G and 5J), Sp-3 (Figs. 5M and 5P), and the nuclear stain DAPI (Figs. 5B, 5E, 5H, 5K, 5N, and 5Q) in mock (Figs. 5A, 5G, 5M, 5B, 5H, and 5N) or HCA-treated (Figs. 5D, 5J, 5P, 5E, 5K, and 5Q) mixed cortical neuronal cultures. The images in Figs. 5C, 5F, 51, 5L, 50, and 5R are derived from superimposing the Sp-1, Sp-2 or Sp-3 antibody fluorescence (green) on the DAPI fluorescence (blue). The bar in Fig. 5R represents a distance of 10 μm. Fig. 6A is a picture of a gel showing that mithramycin A (MMA) inhibits HCA-induced Sp-1 and Sp-3 DNA binding in embryonic cortical neurons. Cultures were exposed to 1 mM HCA as described herein with varying concentrations of mithramycin A. EMSA was performed with 0.5 μg of nuclear extracts treated with a buffer control (lane 1), lmM HCA (lane 2), 10 nM mithramycin A (lane 3), 10 nM mithramycin A and 1 mM HCA (lane 4), 25 nM mithramycin A (lane 5), or 25 nM mithramycin A and 1 mM HCA (lane 6), using a ³²P-labeled Sp-1 binding site oligonucleotide. Fig. 6B is a picture of a gel showing that mithramycin A abrogates glutathione depletion-induced Sp-1 and Sp-3 DNA binding independent of the route of glutathione depletion in embryonic cortical neurons. EMSA was performed as described in Fig. 6A with 0.5 μg of nuclear extracts treated with a buffer control (lane 1); 1 mM HCA (which depletes glutathione by inhibiting the uptake of its rate- limiting precursor cyst(e)ine, lane 2); 25 nM mithramycin A (lane 3); 200 μM buthionine sulfoximine (BSO which depletes glutathione by inhibiting an enzyme involved in its synthesis, lane 4); 200 μM BSO and 1 mM HCA (lane 5); 25 nM mithramycin A and 1 mM HCA (lane 6); 25 nM mithramycin A and 200 μM BSO (lane 7); or 25 nM mithramycin A, 200 μM BSO, and lmM HCA (lane 8).

Fig. 7 is a picture of a gel showing that mithramycin A fails to non-specifically inhibit the DNA binding of all transcription factors. EMS As were performed using 0.5 μg of extracts treated with a buffer confrol (lane 1), 1 mM HCA (lane 2), 25 nM mithramycin A (lane 3), or 25 nM mithramycin A and 1 mM HCA (lane 4), using a ³²P-labeled CREB-binding site oligonucleotide. Fig. 8A is a graph showing that mithramycin A protects cortical neurons in primary cultures against homocysteate-induced death in a concenfration dependent manner. Fig. 8B is a graph showing that mithramycin A also protects against camptothecin (Campto)-induced death. Fig. 9 is a bar graph showing the effect of mithramycin A on survival of cultured cortical neurons freated with 1 mM or 5 mM homocysteate. Cultures were exposed to HCA as described herein with varying concenfrations of mithramycin A. The cells were harvested after 24 hours and assayed for lactate dehydrogenase activity. Data are means +/- SEM (expressed as a percentage of the non-HCA treated control) from three to five experiments performed in triplicate. Protection by mithramycin A was statistically significant (p < 0.05) at all concenfrations of mithramycin A at or above 5 nM for the 1 mM or 5 mM HCA freatment groups.

Figs. 10A-10C are phase-contrast microscopy pictures of primary cortical neurons cultured for two days. Fig.lOA shows confrol neurons cultured for two days in vitro. Fig. 10B shows cells 24 hours after treatment with 5 mM HCA to induce glutathione depletion. Fig. IOC shows cells exposed to 5 mM HCA and 25 nM mithramycin A. The magnification was 200-fold. Fig. 11 is a bar graph showing that chromomycin A₃, a structural analog of mithramycin A which is also known to displace transcriptional activators from G-C rich DNA binding sites, prevents glutathione depletion-induced death in embryonic cortical neurons. Cell viability was measured in cortical neuronal cultures as described herein. Data are expressed as mean +/- SEM (expressed as a percentage of non-HCA freated confrol) from three experiments performed in duplicate. The differences in the means for the HCA only and the HCA plus chromomycin group was statistically significant (p < 0.05). Fig. 12 is a bar graph showing the effect of mithramycin A on survival of cultured cortical neurons freated with 25 μM or 50 μM camptothecin (CPT), a DNA damaging agent. Cultures were exposed to camptothecin as described herein with varying concenfrations of mithramycin A. The cells were harvested after 24 hours and assayed for MTT activity. Data are means +/- SEM (expressed as a percentage of the non-camptothecin-freated confrol (0.1% DMSO vehicle)) from three to five experiments performed in triplicate. Protection by mithramycin A was statistically significant (p < 0.05) at all concenfrations of mithramycin A at or above 10 nM for the 25 μM or 50 μM CPT treatment groups .

Figs. 13 A to 13H are pictures showing that mithramycin A inhibits CPT-induced apoptotic morphology. Analysis of cellular morphology using the fluorescent nuclear stain, DAPI (Figs. 13A-13D), or Nomarski optics (Figs. 13E-13H) in cells freated with a buffer confrol (Figs. 13A and 13E); 25 μM camptothecin (Figs. 13B and 13F; arrows in Fig. 13B point to cells with compaction and fragmentation of chromatin characteristic of apoptosis); 25 nM mithramycin A and 25 μM camptothecin (Figs. 13C and 13G); or 25 nM mithramycin A (Figs. 13D and 13H).

Fig. 14 is a bar graph showing that the protection by mithramycin A occurs distal to glutathione depletion in the HCA-induced cell death pathway. Total glutathione (nanograms of GSH + GSSG) per microgram protein was measured in cells exposed for six hours to HCA, mithramycin A, HCA and mithramycin A, or BSO. Values represent means +/- SEM based on three to five experiments performed in triplicate. Fig. 15 A is a bar graph showing that protection by mithramycin A is not correlated with inhibition of global protein synthesis. Cultures were exposed to varying concenfrations of mithramycin A for four hours at 37 °C. They were then labeled with [³⁵S]-cysteine/methionine for four hours as described herein. The labeling was stopped by three rapid cold washes. The cells were resuspended in 3% PCA and separated into acid-soluble and acid-precipitable fractions by centrifugation. Each bar represents incorporation of radiolabel into acid-precipitable fractions (protein) expressed as cpm of [³⁵S]cysteine/methionine per milligram protein per four hours of labeling at designated concentrations of mithramycin A. Actinomycin-D, an inhibitor of transcription, inhibits incorporation of radioactive amino acids into protein by 80% and prevents HCA-induced apoptosis. Fig. 15B is a plot of protein synthesis rates at various concenfrations of mithramycin A versus cell viability (in the presence of HCA) at various concenfrations of mithramycin A. This plot indicates that protection by mithramycin A is not correlated with suppression or induction of protein synthesis r = 0.1). These observations are consistent with the notion that mithramycin A acts to inhibit the expression of some genes but does not globally inhibit gene expression. Fig. 16 is a schematic representation of a model for redox regulation of neuronal cell survival and death by Sp-1 and Sp-3. Glutamate or the glutamate analog, homocysteate (HCA), induces glutathione depletion in embryonic cortical neurons by competitively inhibiting the uptake of the amino acid cystine at its plasma membrane transporter. Inhibition of cystine uptake leads to depletion of infracellular cystine. Cystine is normally reduced intracellularly to cysteine. Cysteine is the rate-limiting amino acid precursor for the synthesis of the major cellular antioxidant glutathione. Depletion of infracellular cysteine thus leads to glutathione depletion and an imbalance in cellular oxidants and antioxidants leading to "oxidative sfress." Glutathione depletion-induced oxidative sfress activates the synthesis and increased DNA binding of the zinc finger transcription factors, Sp-1 and Sp-3. Glutathione depletion-induced Sp-1 and Sp-3 activation can be suppressed by antioxidants such as deferoxamine mesylate (DFO), an iron chelator, and butylated hydroxyanisole (BHA), an inhibitor of lipid peroxidation. Increased Sp-1 and Sp-3 DNA binding can also be induced by the addition of hydrogen peroxide. Hydrogen peroxide was generated by addition of D-alanine (D-ala) and D-amino acid oxidase (DAAO) to the bathing medium of cultured embryonic cortical neurons. DAAO catalyzes the oxidative deamination of D-ala to form hydrogen peroxide. Consistent with this scenario, D-ala/DAAO induced Sp-1 and Sp-3 DNA binding can be suppressed by the peroxide scavenging enzyme, catalase. Sp-1 and Sp-3 may regulate the expression of both pro-survival and pro-death proteins, depending on factors such as the death stimulus, cell type, and the level and time of activation relative to the death stimulus.

Figs. 17A-17D are the chemical structures of mithramycin, chromamycin A₃, daunomycin, and the bisintercalating anthracycline WP631, respectively (Gratzer, Methods. Enzymol. 85:475-480, 1985; Majee et a , Eur. J. Biochem. 260: 619-626, 1999).

Figs. 18A-18D are the chemical structures of derivatives of mithramycin, chromamycin A₃, daunomycin, and the bisintercalating anthracycline WP631, respectively, which may be synthesized using standard methods. Fig. 19 is a bar graph demonstrating the ability of mithramycin A to reduce the number of cerebellar granule cells that undergo apoptosis due to a low extracellular concenfration of potassium. Prefreatment with 100 nM of mithramycin A for ten minutes prior to exposure to low potassium levels resulted in a greater decrease in cell death than simultaneous exposure to mithramycin A and low potassium levels ("100-pt" bar graph). Fig. 20A is a table listing the infarct volume (mm³) for mice in a preclinical sfroke model that were prefreated with a vehicle confrol, 150 ug/kg mithramycin A, or 300 ug/kg mithramycin A. Fig. 20B is a bar graph of the mean values for the results in Fig. 20A. Fig. 21 is a graph of a Kaplan-Meier survival curve showing the effect of mithramycin A on the length of survival of Huntington's disease mice.

Fig. 22 is a graph showing the effect of mithramycin A on body weight of Huntington's disease mice.

Fig. 23 is a graph of a Kaplan-Meier survival curve showing the effect of mithramycin A on the length of survival of amyofrophic lateral sclerosis mice.

Fig. 24 is a graph showing the effect of mithramycin A on body weight of amyofrophic lateral sclerosis mice.

Detailed Description Mithramycins A, B, and C are aureolic derivatives that are produced by several Sfreptomyces strains (Steinkamp et al, J. Histochem. Cytochem. 27:273, 1979; Crissman et al, Methods. Cell Biol. 33:97, 1990). Mithramcycins bind GC rich DNA sequences and inhibit RNA synthesis and, to a much lesser extent, inhibit DNA synthesis in mammalian and bacterial cells. Mithramycin A is an antibiotic and an anticancer agent that is nephrotoxic (Fillastre et al, Pathol. Biol. (Paris) 34:1013-1028). Chromomycins are also members of the aureolic acid group of antitumor antibiotics that share a common chromophore, aglycon ring, but differ in the nature of the sugar moieties connected to either side of the aglycon ring via O-glycosidic bonds (Gause, Antibiotics III, Springer- Verlag, Berlin, Germany, pp: 197-202, 1975). Herein, we demonstrate that mithramycin A and its structural analog, chromomycin A₃, are potent inhibitors of neuronal apoptosis induced by glutathione depletion-induced oxidative sfress or the DNA damaging agent, camptothecin. The protective effects of mithramycin are correlated with its ability to inhibit the enhanced DNA binding of the transcription factors Sp-1 and Sp-3 to their cognate "G-C" box that is induced by oxidative sfress or DNA damage. These results suggest that mithramycin and its structural analogs may be propitious agents for the freatment of neurological diseases associated with aberrant activation of apoptosis, and highlight the potential use of sequence-selective DNA binding drugs as neurological therapeutics. With IC₅₀ values in the low nanomolar range, mithramycin and chromomycin are three to four magnitudes more potent as neuroprotectants than other antioxidants, such as vitamin E. Moreover, the ability of mithramycin to prevent oxidative stress-induced death even when added up to eight hours after the death stimulus raises the possibility that this class of neuroprotective compounds may have a wider temporal window of efficacy than currently utilized antioxidants. Suppression of oxidative stress-induced as well as DNA damage-induced apoptosis by mithramycin also suggests the possibility that these agents target a pathway of neuronal injury common to many types of apoptotic insults .

In addition to inhibiting cell death due to oxidative sfress or DNA damage, mithramycin A inhibited death of cerebellar granule cells due to low potassium levels and death of symphatic neurons due to growth factor deprivation. Moreover, mithramycin A was shown to inhibit cell death in vivo. In particular, mithramycin A reduced cell death in a animal sfroke model and prolonged the length of survival in an animal model of Huntington's disease. Thus, the contacting of a cell with a mithramycin, chromomycin, or derivative thereof constitutes a novel method of inhibiting oxidative stress- induced, DNA damage-induced, or growth factor deprivation-induced death in mammalian cells. This method may be used to administer a mithramycin, a chromomycin, or a derivative thereof to a human patient for the treatment or propholyaxis of a disease or disorder of the nervous system or the aging process.

We also discovered that basal Sp-1 and Sp-3 DNA binding activities in cortical neurons are unexpectedly low and are dramatically enhanced by oxidative stress. These changes in Sp-1 and Sp-3 DNA binding occur prior to the onset of apoptotic cell death and can be inhibited by antioxidants that completely suppress cell death. Immunocytochemisfry confirmed that increased levels of Sp-1 and Sp-3 protein are seen in the nuclei of oxidatively-sfressed cortical neurons. Additionally, we demonstrated that increases in Sp-1 and Sp-3 DNA binding are inhibited by deferoxamine mesylate and butylated hydroxyanisole, structurally distinct antioxidants that inhibit glutathione depletion-induced death in cortical neurons. Further, exposure of cortical neurons to hydrogen peroxide induces Sp-1 and Sp-3 DNA binding, and this peroxide-induced activation of Sp-1 and Sp-3 DNA binding is suppressed by catalase or pyruvate. Altogether, these results establish Sp-1 and Sp-3 as oxidative stress-induced transcription factors in cortical neurons that may modulate cell death pathways.

While not meant to limit the invention to any particular theory, it is possible that depletion of the major antioxidant, glutathione or addition of the reactive oxygen species, hydrogen peroxide, to cortical neurons leads to activation of a kinase cascade leading to increased Sp-1 and Sp-3 protein levels and increased DNA binding activity. A likely candidate kinase is Erk2, a Map kinase that has been shown to phosphorylate Sp-1 and increase its DNA binding. Indeed, Erk 2 is activated by glutathione depletion in cortical neurons and Map Kinase inhibitors can prevent glutathione depletion-induced cell death. Additionally, a role for transcriptional activators such as Sp-1 and Sp-3 in regulating the expression of one or more "death protein(s)" would be consistent with observations from several laboratories that RNA and protein synthesis inhibitors abrogate glutathione depletion induced death in cortical neurons. One protein whose levels have be shown to be increased by glutathione depletion and whose activity is required for cell death in cortical neurons is 12-lipoxygenase (12-LOX). 12-LOX catalyzes the formation of 12-hydroxyeicosatefraenoic acid (12-HETE) and also some

15-hydroxyeicosatefranoic acid (15-HETE) from arachidonic acid. Sp-1 and/or Sp-3 may bind to one or more of the two reported Sp-1 recognition motifs in the 12-Lox promoter and activate this promoter, resulting in increased levels of 12-LOX and increased production of arachidonic acid metabolites such as 12-HETE. The catalytic cycles of 12-LOX may lead to yet increased free radical production or alternatively, 12-LOX products such as 12-HETE can increase cytosolic calcium to pathological levels resulting in cell death. Additional Sp-1 and/or Sp-3-regulated genes that are established cell death effectors include Fas, Fas ligand, and the glutamate receptor subunit, NDl.

Sp-1 and/or Sp-3 may also mediate pro-survival responses. Indeed, recent studies suggest that Sp-1 motifs are responsible for the regulation of the inhibitor of apoptosis (IAP) protein survivin. Thus, Sp-1 and Sp-3 may mediate pro-death or pro-survival responses depending on the death stimulus, the cell type, and the level and timing of activation relative to the death stimulus. Using conventional modifications of the methods described herein other compounds, libraries of compounds, antisense constructs to polynucleotides encoding an Sp family member, or specific antibodies to distinct Sp family members may be tested for their ability to enhance or inhibit oxidative stress-induced, DNA damage induced, or growth factor deprivation-induced increases in Sp-1 or Sp-3 protein levels or DNA binding. For example, the gel shift or supershift assays described in Example 1 may be performed in the presence of one or more candidate compounds to determine whether the candidate compounds affect Sp-1 or Sp-3 protein levels or Sp-1 or Sp-3 DNA binding. Inclusion of an oxidant, a DNA-damaging agent, or an agent that reduces the level of a growth factor in these assays may increase the protein levels or DNA binding of Sp-1 or Sp-3 and thus facilitate the detection of a decrease in one ofthese levels that is mediated by candidate compounds. The candidate compounds may also be tested in the presence of an antioxidant or ROS savaging agent to determine whether the agent reduces the effect of the candidate compounds on Sp-1 or Sp-3 protein levels or DNA binding. The ability of such an agent to suppress the effect of the candidate compounds on Sp-1 or Sp-3 protein levels or DNA binding would suggest that the candidate compounds are modulating oxidative stress-induced changes in Sp-1 and Sp-3 levels or activity.

Similar assays may also be performed with recombinant Sp-1 or Sp-3 protein rather than Sp-1 or Sp-3 protein from cell exfracts. For example, in vitro transcribed or translated proteins may be produced using T3 or T7 polymerases (New England Biolabs), pBSK-Sp3/flu or pBSK-Spl/Flu (Kennet et al) as a template, and a coupled reticulocyte lystate system (TNT; Promega, Inc., Madison, WI). In vitro translated proteins may be radiolabeled by using a mixture of radiolabeled amino acids (Express; New England Nuclear, Inc., Boston, MA). Candidate compounds may also be tested as described for mithramycin A or chromomycin A₃ in Example 3 to determine whether they modulate oxidative-stress induced or DNA damage-induced cell death. Additionally, candidate compounds may be analyzed as described in Example 6 to determine whether they increase or decrease cell death due to growth factor depletion or low potassium levels. Compounds may also be tested in animal disease models, such as the stroke, Huntington's disease, and amyofrophic lateral sclerosis models described in Examples 7-9, to determine their ability to prevent, stabilize, or treat a variety of acute and chronic neurological diseases, disorders, and injuries. As described in Examples 4 and 5, candidate compounds may also be tested to determine whether their effect on cell death is independent of modulating glutatione levels or global levels of protein synthesis.

The following examples are provided to illustrate the invention. They are not meant to limit the invention in any way.

Example 1: Induction of Sp-1 and Sp-3 DNA Binding

To determine the effect of oxidative-stress on Sp-1 and Sp-3 DNA binding, elecfrophoretic mobility shift assays (EMSAs) were performed on nuclear exfracts from cortical neuron using a ³²P-labeled oligonucleotide containing a wild-type or mutant Sp-1 binding site (Santa Cruz

Biotechnology, Santa Cruz, California). The sense strand sequences of the double stranded wild-type (wt) and mutant oligonucleotides were 5'-ATTCGATCGGGGCGGGGCGAGC-3' (SEQ ID NO: 1) and 5'-ATTCGATCGGTTCGGGGCGAGC-3' (SEQ ID NO: 2), respectively. Parallel EMSAs were also performed using a radiolabeled Oct-1

(5^,-TGTCGAATGCAAATGACTAGAA-3^,; SEQ ID NO: 3, Santa Cruz Biotechnology) binding site or a radiolabeled CRE

(5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3', SEQ ID NO: 4, Santa Cruz Biotechnology; Example 3) binding site. Embryonic cortical neurons were lightly trypsinized, pelleted, and resuspended in cold PBS. All subsequent steps were performed as previously described at 4 °C (Lin et al, J. Cell Biol. 131:1149-1161, 1995). The cells were suspended in 10 mM Tris (pH 7.5), 1.5 mM MgCl₂, 10 mM KCl, 10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 3 mM PMSF, 3 mM DTT, and 1 mM Na₃V0₄. The cells were lysed by 15 strokes in a Dounce Homogenizer using a type B pestle, and the nuclei were pelleted at 4,500 x g for five minutes, resuspended in three to four packed cell volumes of buffer C (420 mM KCl, 20 mM Tris-HCl, pH 7.8, 1.5 mM MgCl₂,10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 3 mM PMSF, 3 mM DTT and 1 mM Na₃V0₄), and incubated for 30 minutes with gentle agitation. The absence of cytoplasmic contamination of the purified nuclei was verified by the absence of detectable lactate dehydrogenase activity in the nuclear extract. The nuclear extract was centrifuged at 10,000 x g for 30 minutes, and the supernatant was dialyzed twice against 25-50 ml buffer D (20 mM Tris-HCl, pH 7.8, 100 mM KCl, 0.2 mM EDTA, and 20% glycerol). The dialysate was centrifuged at 10,000 x g for 10 minutes at 4 °C, and the supernatants were aliquoted, snap frozen in liquid N₂, and stored at -80 °C. Perchloric acid precipitated protein was measured from a representative aliquot of each sample, and equal amounts were used for binding. Binding reactions were performed at 4 °C for 15 minutes using 0.5- 5 μg of nuclear protein (Sp-1, Oct-1, or CRE) and 0.25 ng (10,000-40,000 cpm) of labeled oligonucleotide in 30 μl of binding buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM KCl, 1 mM MgCl₂, 1 mM EDTA, 5 mM DTT, 5% glycerol, and 0.1 μg sonicated denatured calf thymus DNA. DNA-protein complexes were separated from unbound probe on native 6% polyacrylamide gels at 195 V for two hours. The gels were vacuum dried and exposed to Kodak film for 8-15 hours at -80 °C. Visual inspection of the free probe band at the bottom of the gel confirmed that equivalent amounts of radiolabeled probe were employed for each sample. Five hours of HCA freatment (which leads to a reduction in glutathione levels to 30% of confrol) significantly increased three DNA binding activities in cortical neurons (Fig. 1 A, the three DNA binding activities are designated "a," "b," and "c"). The induction ofthese DNA binding activities appeared to be related to glutathione depletion because it was also observed in the presence of buthionine sulfoximine (100 μM), an agent that depletes glutathione to less than 10% confrol levels by inhibiting one of the enzymes involved in its synthesis rather than inhibiting uptake of its rate-limiting amino acid precursor. All three DNA binding activities were inhibited in a concentration-dependent manner by cold Sp-1 oligonucleotide, and none of the DNA binding activities were inhibited by an oligonucleotide containing mutations in the Sp-1 binding site. Enhanced DNA binding could be observed as early as two hours after HCA freatment (Fig. 1C), permitting sufficient time for genes to be expressed prior to the commitment point of cells to die (approximately 12 hours after HCA treatment). Sp-1 and Sp-3 were identified in this DNA-bound complex using specific antibodies to the distinct Sp family members (Fig. IB). Supershifts were performed with antibodies to Sp-1, Sp-2, Sp-3, or Sp-4 (Santa Cruz Biotechnology, Santa Cruz, California). Each antibody was added to a binding mixture immediately after the addition of the radiolabeled Sp-1 probe. The reaction mixtures were incubated for 20 minutes, and the complexes were resolved by polyacrylamide-electrophoresis as described above. The differences between nuclear Sp-1 and Sp-3 DNA activity in confrol and HCA-treated cortical neurons could not be attributed to global differences in nuclear proteins because levels of Oct-1 DNA binding were similar using 0.5 micrograms ofthese two exfracts (Fig. ID).

If oxidative sfress is responsible for the HCA-induced Sp-1 and Sp-3 DNA binding observed in Figs. 1A-1C, then antioxidants should diminish or suppress the induction. Indeed, addition of the antioxidant iron chelator, deferoxamine (DFO, 100 μM), or the non iron-chelating, lipid peroxidation inhibitor, butylated hydroxyanisole (BHA, 10 μM), suppressed HCA-induced Sp-1 and Sp-3 DNA binding (Figs. 2A and 2B). These results suggests that HCA-induced oxidative sfress mediates enhanced DNA binding of Sp-1 and Sp-3 in cortical neurons. While DFO inhibits oxidative sfress-induced Sp-1 and Sp-3 DNA binding, we previously demonstrated that this agent enhanced the DNA binding of hypoxia-inducible factor- 1 and ATF-1/CREB to the hypoxia response element. These results suggest that the antioxidants that inhibited Sp-1 and Sp-3 activation do not non-specifically inhibit the DNA binding of all transcription factors.

To determine if the addition of the reactive oxygen species, hydrogen peroxide, to cortical neurons is also capable of inducing Sp-1 and Sp-3 DNA binding, varying concentrations of the enzyme D-amino acid oxidase (DAAO; 1-5 milliunits (mU)) and its substrate D-alanine (20 mM) were added to the bathing medium of embryonic rat cortical neurons. DAAO catalyzes the stereoselective oxidative deamination of D-amino acids to form hydrogen peroxide via the following reaction:

D-amino acid + H₂0 + 0₂ =»^■ α-keto acid + NH₃ + H₂0₂.

Hydrogen peroxide generated from DAAO induced three complexes in cortical neurons in a concenfration-dependent manner (Fig. 3A). Supershift analysis confirmed that like glutathione depletion, the slowest migrating complex contained Sp-1 and the two faster migrating complexes contained Sp-3. Additionally, D-alanine/DAAO induced Sp-1 and Sp-3 DNA binding could be completely suppressed by co-application of the peroxide scavenging enzyme catalase (100 units/ml; Fig. 3B), or the non-enzymic peroxide scavenger, pyruvate (2mM).

Example 2: Oxidative Stress-Induced Increase in Nuclear Sp-1 and Sp-3 Protein Levels

Homocysteate at 1 or 5 mM increased the levels of Sp-1 and Sp-3 protein in irnmature embryonic cortical neurons as measured by Western blotting (Figs. 4A-4C). For this assay, cell lysates were obtained by rinsing cortical neurons with cold PBS and adding 100 mM Tris (pH 7.4) buffer containing 1% Triton-X 100, 150 mM NaCl, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 3 mM PMSF, 3 mM DTT, 0.5 μg/ml leupeptin, and 10 μg/ml aprotinin. A 30 μg sample of protein from each cell lysate was boiled in Laemmli buffer, and 5 μg of the protein was electrophoresed under reducing conditions on a 8 % poly acrylamide gel. The protein in the gel was then transferred to a nitrocellulose membrane (Bio Rad). Non-specific binding was inhibited by incubation in Tris buffered saline (TBST: 50 mM Tris HC1, pH 8.0, 0.9% NaCl, and 0.1% Tween 20) containing 5% non-fat dried milk for 0.5 hours. Primary antibodies against Sp-1 (PEP2, Santa Cruz Biotechnology), Sp-2 (K-20, Santa Cruz Biotechnology), Sp-3 (D-20), or Sρ-4 (V-20) were all diluted at 1:1000 in 1% milk TBST and exposed to membranes overnight at 4 °C. Immunoreactive proteins were detected according to the enhanced chemiluminescent protocol (Amersham).

We found that HCA lead to increases in both Sp-1 and Sp-3 protein levels (Fig. 4A). We have previously observed that HCA at a concentration of 5 mM leads to more rapid cystine deprivation and glutathione depletion after 5 hours than 1 mM HCA. As expected from these observations, 5 mM HCA induced greater levels of Sp-1 (Fig. 4B) and Sp-3 (Fig. 4C) protein than did 1 mM HCA. A rabbit polyclonal Sp-1 antibody detected a dominant band at 95 kD and minor band at 105 kD. This slower migrating band at 105 kD likely reflects phosphorylation of the 95 kD Sp-1 polypeptide since the latter form completely disappeared in the presence of the phosphatase inhibitor, calyculin A. A rabbit polyclonal Sp-3 antibody detected a dominant 115 kD band corresponding to Sp-3 previously identified by both immunochemical and molecular genetic techniques. A minor, faster migrating band was observed at 78-80 kD, a species of Sp-3 that has been shown to result from internal translational initiation within Sp-3 mRNA. The increases in Sp-1 and Sp-3 proteins were seen as early as 1 hour after HCA treatment, were maximal at five hours after HCA freatment (Figs. 4B and 4C), and could be observed to be returning to baseline levels by eight hours after HCA freatment

To verify that Sp-1 and Sp-3 protein can be induced in the nuclei of neurons by HCA-induced glutathione depletion, immunocytochemical staining with antibodies to Sp-1 (Figs. 5A and 5D), Sp-2 (Figs. 5G and 5J) and Sp-3 (Figs. 5M and 5P) was performed. For this experiment, dissociated cells from the cerebral cortex (3-5 xlO⁵) were seeded onto poly-D-lysine-coated eight well culture slides (Becton-Dickinson Labware, Bedford, MA) and freated with HCA as described above for four to five hours. The cells were washed with warm PBS and fixed at room temperature for 15 minutes with 4% paraformaldehyde (PFA). After washing with PBS, fixed cells were incubated with blocking solution containing 0.3% Triton-X 100, 5% bovine serum albumin (BSA) and 3% goat serum for 1 hour, followed by incubation with rabbit Sp-1 antibody (1:500 dilution), rabbit anti-Sp-2 polyclonal antibody (1 :500 dilution), or a rabbit polyclonal

Sp-3 antibody (1:500 dilution). After three washes with PBS, the cells were incubated for one hour with FITC-conjugated goat-anti-rabbit IgG antibody (1:200 dilution) which fluoresces green. In parallel, the same cultures were treated with the nuclear stain, DAPI, that fluoresces blue (Molecular Probes, Eugene, Oregon) (Figs. 5B, 5E, 5H, 5K, 5N, and 5Q). All antibodies were diluted in PBS. The slides were washed three times with PBS, mounted with fluorochrome mounting solution (Vector Laboratories). Images were analyzed under a fluorescence microscope (model Olympus AX70TRF). Confrol experiments were performed in the absence of primary antibody. When the Sp-1, Sp-2, and Sp-3 antibody fluorescence images were superimposed on the DAPI fluorescence images, cells stained with an Sp-1 or Sp-3 antibody that were induced to undergo oxidative sfress by treatment with HCA were found to have blue-green nuclei, indicative of enhanced protein level in the nucleus (Figs. 5F and 5R). Moreover, staining with the Sp-2 antibody revealed that levels of this protein were not altered in the cytoplasm or nucleus by HCA-induced glutathione depletion and oxidative sfress (Figs. 5G-5L). Cells that stained positively for Sp-1 and Sp-3 were determined to be neuronal by parallel immunostaining with a neurofilament antibody as previously described (Zaman et α/., J. Neurosci. 19:9821-9830, 1999).

Example 3: Mithramycin A and Chromomycin A₃ Inhibition of Oxidative Sfress in Cortical Neurons

To determine the effects of mithramycin A (also called aureolic acid or plicamycin; Sigma, Saint Louis, Missouri) on oxidative stress-induced Sp-1 and Sp-3 DNA binding in cortical neurons, EMSAs were performed on nuclear exfracts from confrol and HCA-freated cells in the presence of 0, 10, or 25 nM mithramycin A using a radiolabeled oligonucleotide containing a consensus Sp-1 binding site. Addition of mithramycin A to cortical neurons induced to undergo glutathione depletion and oxidative sfress lead to a concentration-dependent inhibition of both Sp-1 and Sp-3 DNA binding activities, with nearly complete inhibition ofthese activities at 25 nM mithramycin A (Fig. 6A, lanes 3-6). To verify that mithramycin can abrogate glutathione depletion-induced Sp-1 and Sp-3 DNA binding, the effects of mithramycin on glutathione depletion induced by buthionine sulfoximine (BSO) rather than by HCA was examined. BSO acts to deplete glutathione by inhibiting one of the enzymes involved in its synthesis rather than by inhibiting uptake of its rate-limiting amino acid precursor. Mithramycin A inhibited Sp-1 and Sp-3 DNA binding induced by BSO or BSO and HCA (Fig. 6B). Together, these results suggest that mithramycin can abrogate glutathione depletion-induced Sp-1 and Sp-3 DNA binding. While mithramycin A inhibited oxidative sfress-induced Sp-1 and Sp-3 DNA binding, it enhanced oxidative sfress-induced CREB DNA binding to a consensus CRE motif (Fig. 7). Since CREB is an established survival factor in neurons, it is possible that the increase in mithramycin-induced CREB DNA binding contributes to the survival promoting effects of this agent. These results also suggest that mithramycin does not non-specifically inhibit the DNA binding of all transcription factors.

HCA results in depletion of the antioxidant glutathione and in oxidative stress-induced apoptosis. Concenfrations of mithramycin A (25 nM) that inhibited HCA-induced Sp-1 and Sp-3 DNA binding completely prevented HCA-induced cell death (Figs. 8 A and 9). Similarly, cliromomycin A₃ (0.5-10 nM) was also found to inhibit HCA-induced cell death (Fig 11). For these experiments, cell cultures were obtained from the cerebral cortex of fetal Sprague Dawley rats (embroyonic day 17) as described previously (Murphy et al, FASEB J. 4:1624-1633, 1990). Experiments were initiated 24-72 hours after plating. Under these conditions, the cells are not susceptible to excitoxicity. For cytotox studies, cells were rinsed with warm PBS and then placed in Minimum Essential Medium (MEM; Life technologies, Gaithersburg, Maryland) with 5.5 gm 1 of glucose, 10 % FCS, 2 mM glutamine, and 100 μM cystine, containing the glutamate analog homocysteate (HCA; 1 mM or 5 mM). Viability was assessed by phase contrast microscopy, lactate dehydrogenase release (Koh and Choi, 1987; Ratan et al, J. Neurosci 14:4385-4392, 1994), or DAPI staining (Molecular Probes, Eugene, OR) under fluorescence microscopy. Mithramycin A (1-100 nM) or chromomycin A₃ (0.5-10 nM) was added at the time cortical neurons were exposed to HCA or up to 10 hrs after HCA freatment. Similar quantitative results were obtained independent of the viability assay used. Cell viability may also be assayed using calcein AM/ethidium homodimer-1 staining (Molecular Probes, Eugen Oregon) under fluorescence microscopy or trypan blue exclusion. The ability of mithramycin to suppress the chromatin condensation and nuclear fragmentation characteristic of apoptosis in HCA-freated cultures was verified by DAPI staining and phase contrast microscopy (Figs. 10A-10C). To determine whether mithramycin is capable of preventing cell death induced by apoptotic stimuli distinct from glutathione depletion, the effects of mithramycin on apoptosis induced by the DNA damaging agent, camptothecin, was also examined (Fig. 8B). Camptothecin is a cytotoxic plant alkaloid that induces cell injury by inhibiting the activity of DNA topoisomerase I, which leads to DNA damage. Previous studies have established that cortical neuronal cell death induced by camptothecin has characteristic morphological features of apoptosis and can be suppressed by global inhibitors of transcription and translation. This assay was performed as described above except that camptothecin (25 μM or 50 μM) was used instead of HCA, and camptothecin was diluted from 1000-fold DMSO solutions. Mithramycin potently inhibited cell death induced by 25 or 50 μM camptothecin (Figs. 8B and 12). The ability of mithramycin to inhibit camptothecin-induced apoptosis was verified by staining with DAPI (Fig. 13A-D) or phase contrast microscopy. As with HCA-induced death, protection by mithramycin could be correlated with suppression of camptothecin-induced Sp-1 and Sp-3 DNA binding.

Example 4: Glutathione Levels in Mithramycin A Treated Cells HCA induces glutathione depletion by competitively inhibiting the uptake of cystine by its plasma membrane transporter. Inhibition of cystine uptake or removal of cystine from the bathing medium leads to depletion of the antioxidant glutathione and death because of oxidative stress. To determine whether mithramycin acts to suppress HCA-induced death by preventing cystine deprivation and glutathione depletion, total glutathione levels [reduced glutathione (GSH) + oxidized glutathione (GSSG)] was measured at several time points after HCA addition (Fig.14). Total glutathione levels were measured by the method of Tietze (Anal. Biochem. 27:502-522, 1969) as described in Ratan et al (J. Neurosci. 14:4385-4392, 1994) with the following modifications. At three and six hours after exposure to potential toxin +/- inhibitors, the cells were washed with PBS, lysed with cold 3% perchloric acid, and centrifuged at 40 °C at 7,400 x g. The supernatants were diluted in nine volumes of 0.1 M Na^PO^ and the remaining steps of the glutathione assay were performed as described (Ratan et al, J. Neurosci. 14:4385-4392, 1994). Glutathione levels were normalized to total protein levels that were determined using the bicinchoninic acid reagent (Pierce, Rockford, IL) method (Smith et al, Anal. Biochem. 150:76- 85, 1985).

Although the rate at which total glutathione levels were depleted by HCA exposure was slowed in the presence of 25 nM mithramycin, total glutathione levels were depleted by greater than 60% in cortical neurons by six hours after freatment with 5 mM HCA or 5 mM HCA and mithramycin (25 nM). In contrast, there was little difference between the total glutathione levels in control and mithramycin-treated cultures (Fig. 14). These results suggest that mithramycin acts to inhibit glutathione depletion-induced apoptosis distal to glutathione depletion. Consistent with this notion, mithramycin was added up to eight hours after HCA addition (two to five hours after glutathione depletion) and still completely prevented cell death.

Example 5: [³⁵S] -Cysteine and Methionine Incorporation Studies in Cells Treated with Mithramycin A

Glutathione depletion-induced death in primary neurons is completely suppressed by global inhibitors of transcription and franslation. To examine the effects of mithramycin on global protein synthesis, incorporation of radioactive cysteine and methionine into perchloric (PCA)-precipitable protein fractions was measured. Radiolabeling experiments were performed using EasyTag express protein labeling mix (New England Nuclear, Boston, MA) as described previously (Ratan et al, J. of Neurosci. 14:4385-4392,

1994) with the following modifications. Embryonic cortical neuronal cultures were plated in 6 well dishes at a density of 2.5 x 10⁶ cells per well. Before labeling, the media was changed and replaced with media containing mithramycin (0-25 nM) for four hours. Then, 2 μCi of [³⁵S] cysteine/methionine was added to each well for four hours.

Treatment of cortical neurons with mithramycin A for four hours did not significantly affect incorporation of methionine/cysteine into protein (Fig. 15 A). Additionally, protein synthesis rates in the presence of increasing concentrations of mithramycin were not correlated with mithramycin' s effects on neuronal survival (Fig. 15B). However, four hours of freatment with the protein synthesis inhibitor, cycloheximide reduced incorporation of methionine/cysteine into protein by greater than 90%, and previous studies have shown a good correlation between the ability of cycloheximide and other protein synthesis inhibitors to prevent oxidative sfress-induced apoptosis and their effects on protein synthesis. These results suggest that mithramycin is acting to selectively inhibit the synthesis of one or more death proteins without affecting global protein synthesis.

Example 6: Mithramycin Inhibits Death of Cerebellar Granule Cells and Symphatic Neurons

During development of the vertebrate nervous system a large proportion of neurons that are generated die by a process referred to as programmed cell death or apoptosis. Although widespread in the nervous system and believed to be critical for normal neuronal development, the molecular mechanisms underlying this process is unknown, and few examples of apoptosis in vitro have been described. Fully differentiated cerebellar granule cells undergo apoptosis when the extracellular level of potassium is lowered. The death process is accompanied by morphological changes characteristic of apoptosis (D'Mello et al, Proc. Natl. Acad. Sci. (USA), 90:10989-10993, 1993 ). Death of granule cells due to low potassium levels can be prevented by IGF-1 but not by several other growth and neurofrophic factors. Thus, culturing granule cells in vitro in the presence of low potassium levels is a useful model for identifying compounds that inhibit apoptosis ofthese cells and of other neuronal cells.

To test the ability of mithramycin to inhibit apoptosis of cerebellar granule cells, cerebellar granule cells were exposed to a medium containing 5 mM potassium in the presence or absence of mithramycin A. In the absence of mithramycin A, a significant number of the cells underwent apoptosis due to the low extracellular level of potassium (Fig. 19). Mithramcyin almost completely prevented apoptosis at concenfrations as low as ~1 micromolar. The level of protection from cell death due to mithramycin A was greater than the level of protection from cell death due to IGF-1. A similar assay was performed in which cerebellar granule cells were prefreated with 100 nM of mithramycin A in a medium containing 25 mM potassium for ten minutes and then shifted to a medium containing 100 nM of mithramycin A and only 5 mM potassium (Fig. 19, "100-pt" bar graph). The level of protection from cell death due to mithramycin A prefreatment was greater than that observed due to simultaneous exposure to mithramycin A and low potassium levels. Additionally, mithramycin A was shown to protect against cell death induced by NGF deprivation of sympathetic neurons. In this assay, sympathetic neurons were originally grown in a medium containing NGF, and then the medium was replaced with a medium that did not contain NGF. An anti-NGF antibody was also added to the later medium to inactivate any residual NGF.(Martin et al, J. Cell. Biol. 106:829-844, 1988). These results indicate that in addition to protecting cortical neurons against DNA damage and oxidative stress, mithramycin A protects cerebellar granule cells and sympathetic neurons from cell death (e.g., cell death due to low potassium levels or growth factor deprivation). Thus, mithramycin A and mithramyin derivatives may be protective in pathological circumstances where insufficient trophic support leads to cell death in vivo. Insufficient levels of growth factors is associated with a variety of diseases and disorders of the nervous system. Thus, these results further support the ability of mithramycin A and mithramyinc derivatives to prevent, stabilize, or treat a variety of neurological diseases and injuries, such as those described herein. Example 7: Mithramycin Inhibits Cells Death in a Preclinical Model of Stroke

To determine whether mithramycin's protective effects extend to in vivo models where apoptosis due to free radicals, DNA damage, and growth factor deprviation is thought to occur, mithramcyin A was tested in a murine focal ischemia model. Briefly, adult male mice (18-22 g; Taconic Farms, Germantown, NY) were housed under diurnal lighting conditions and allowed food and water ad libitum. Animals were anesthetized with 1.5% halothane and maintained in 1.0% halothane in 70% N20 and 30% oxygen by using a Fluotec 3 vaporizer (Colonial Medical, Amherst, NH). Ischemia was induced with a 8.0 nylon monofilament coated with silicon resin/hardner mixture (Xantopren and Elastomer Activator, Bayer Dnetal, Osaka, Japan) as described previously. Animals were injected with 150 ug/kg or 300 ug/kg infraperitoneally at 30 minutes before two hours of focal ischemia, followed by 22 hours of reperfusion.

After 22 hours of postischemic perfusion, brains from the mice were removed and sliced into five coronal sections (2 mm thick). The sections were treated with 2%, 2,3,5-triphenyltefrazolium chloride followed by 10% formalin overnight, as described previously (Bederson et al, Sfroke 17, 1304-1308, 1986). The infarcted areas, outlined in white, were measured on the posterior surface of each section by an image analysis system (MCID version 3 imaging research, St. Catherine's, ON, Canada), and the infarction volume was calculated by summing the infarction volume of sequential 2 mm-thick sections. As illustrated in Figs. 20A and 20B, prefreatment of mice with mithramycin A reduced sfroke volume and thus reduced sfroke related damage (e.g., cell death due to a lack of oxygen and glucose from an insufficient amount of blood). Example 8: Mithramycin Inhibits Cells Death in a Preclinical Model of Huntington's Disease

Huntington's disease is an autosomal dominant, progressive, neurodegenerative disease that starts in midlife and inexorably leads to death. The mean length of survival after the onset of Huntington's disease is 15 to 20 years. At present, there is no effective treatment for Huntington's disease. The mutation that causes the illness is an expanded CAG/polyglutamine repeat stretch that has been postulated to confer toxic effects by several different mechanisms (HDCRG, 1993). The protein product of the Huntington's disease gene, huntington, is expressed ubiquitously in both the nervous system and peripheral tissues.

A breakthrough in Huntington's disease research was the development of transgenic mouse models. Transgenic mice expressing exon 1 of the human Huntington's disease gene with an expanded CAG repeat develop a progressive neurological disorder. These mice (line R6/2) have 141-157 CAG repeats (compared to less than 35 repeats for normal mice) under the confrol of the human Huntington's disease promoter. At approximately six weeks of age, the R6/2 mice show loss of brain and body weight, and at 9-11 weeks they develop an irregular gait, abrupt shuddering, stereotypic movements, resting fremors, and epileptic seizures. The mice also show an early decrease of several neurofransmitter receptors. The brains of the R6/2 mice appear normal in most respects; however, intranuclear inclusions that are immunopositive for huntington and ubiquitin are detected in the striatum at 4.5 weeks. Neuropil, cytoplasmic, and neuronal inclusions are also found in human Huntington's disease.

Transgenic mice from the R6/2 strain and littermate controls were obtained from Jackson Laboratories (Bar Harbor, ME). The male R6/2 mice were bred with females from their background strain (B6 CBAFI/J). The offspring were genotyped by PCR amplification of DNA obtained from tail tissue. Transgenic mice were housed in microisolator cages in a modified barrier facility. A twelve hour light/dark cycle was maintained, and the animals were given ad libitium access to food and water. Groups of transgene negative and positive R6/2 mice from the same "f ' generation were injected with 150 micrograms/kg of mithramcyin A dissolved in PBS intraperitoneally beginning at 21 days of age. Approximately 30 mice were used for the survival studies. The mice were weighed twice a week at the same time of day. For determining the rate of survival, the mice were observed twice daily, in the morning and late afternoon. The criterion used to determine death was the point in time when the mice were unable to initiate movement after being gently prodded for ten minutes. Two independent observers confirmed this criterion, and this point was used as the time of death.

The effect of mithramycin A on survival ofthese Huntington's disease mice is shown in Fig. 21. The mean length of survival increased from 98.6 +/- 2.3 days for control mice freated with buffer to 126.6 +/- 1.0 days (p < .0001) for mice freated with mithramycin A. As illustrated in Fig. 22, mithramycin A also enhanced body weight.

Example 9: Effect of Mithramycin on Amyofrophic Lateral Sclerosis Mouse Model

To study the effect of mithramycin in a mouse model of amyofrophic lateral sclerosis (ALS), hemizygote transgenic mSODl mice, their wild-type littermates, and mice overexpressing wild-type SOD1 (N1029) were used. The mSODl high expressor line (G1H/G93A) carries the point mutation Gly to Ala at codon 93 of the human SOD1 gene and expresses 18-25 copies of the mutated gene (Gurney et al, Science 264:1772-1775 1994). Mice become paralyzed in one or more limbs and die by 4-5 months of age. Paralysis is due to a loss of motor neurons from the spinal cord. The transgenic mSODl and wild-type SOD1 have been found to have equivalent levels of SOD activity relative to non-fransgenic controls (approximately 3.5 fold). Colonies of the G93A transgenic ALS mice line and mice expressing the wild-type SOD (N1029) were established by Dr. Ferrante's group at the Bedford VA Medical Center. These mice were initially obtained from Jackson Laboratories. The offspring were genotyped by standard PCR screening of mouse tails using a pair of primers that is specific for exon-4 of human SOD1 (Kostic et al, Science 277:559-563, 1997). Groups of mice from the same "f generation were injected with 150 micrograms/kg of mithramycin A dissolved in PBS intraperitoneally beginning at 21 days of age. Approximately 17 mice were injected for the survival studies. For determining the rate of survival, the mice were observed twice daily, in the morning and late afternoon. The criterion used to determine death was that point in time when mice were unable to initiate movement after being gently prodded for ten minutes. Two independent observers confirmed this criterion, and this point was used as the time of death.

The effect of mithramycin A at 150 micrograms/kg on the length of survival and body weight ofthese ALS mice is shown in Figs. 23 and 24, respectively. Mithramycin A was shown to enhance body weight of the ALS mice. This mouse model may also be used to test other dosing regimes and other mithramycin derivatives, such as those described herein, for their ability to increase the length of survival or treat the symptoms associated with amyofrophic lateral sclerosis. Additionally, mithramycin and mithramycin derivatives may be tested in other animal models of amyofrophic lateral sclerosis or tested in humans. Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

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

What is claimed is:

Claims

Claims

1. A method of treating or preventing a disease, disorder, injury, or infection in a mammal selected from the group consisting of Alzheimer's disease, Cruetzfeld- Jacob disease, kuru, Huntington's disease, aneurism, sfroke associated with an increase in blood pressure, spinal cord disease, spinal cord injury, brain injury, multiple system afrophy, amyofrophic lateral sclerosis, progressive supranuclear palsy, neurodegeneration associated with the aging process, mitochondrial disease, and viral infection; said method comprising administering a compound to said mammal in an amount sufficient to treat or prevent said disease, disorder, injury, or infection; wherein said compound is a mithramycin, chromomycin, daunomycin, olivomycin, WP631, or derivative thereof.

2. The method of claim 1, wherein said disease is Alzheimer's disease or Huntington's disease.

3. The method of claim 1, wherein said viral infection is an HIV or herpes infection.

4. A method of treating or preventing Parkinson's disease or multiple sclerosis in a mammal, said method comprising administering less than 0.05 μg/kg of a compound to a mammal; wherein said compound is a mithramycin, chromomycin, daunomycin, olivomycin, WP631, or derivative thereof; and wherein said adminisfration is sufficient to treat or prevent Parkinson's disease or multiple sclerosis.

5. The method of claim 1 or 4, wherein said compound is a mithramycin or chromomycin

6. The method of claim 1 or 4, wherein said administration comprises contacting a neuronal cell with said compound.

7. The method of claim 6, wherein said neuronal cell is a cortical neuron, cerebellar granule cell, or sympathetic neuron.

8. The method claim 1 or 4, wherein said adminisfration inhibits cell death.

9. The method claim 8, wherein said adminisfration inhibits cell death associated with oxidative-stress, DNA damage, or growth factor depletion.

10. The method claim 1 or 4, wherein said adminisfration inhibits the induction of an Sp family member or the binding of an Sp family member to DNA.

11. The method of claim 1 or 4, wherein said compound is administered orally, intramuscularly, intravenously, infracranially, intrathecally, or subcutaneously to said mammal.

12. The method claim 1 or 4, wherein said mammal is a human.

13. A method for detecting oxidative sfress, DNA damage, or growth factor depletion in a cell, said method comprising analyzing said cell for an increase in the protein level of an Sp family member or an increase in the binding of an Sp family member to DNA.

14. The method of claim 13, wherein said cell is in a transgenic animal.

15. The method of claim 13, wherein said cell is in an autopsy tissue.

16. The method of claim 13, wherein said cell is a neuronal cell.

17. The method of claim 16, wherein said neuronal cell is a cortical neuron, cerebellar granule cell, or sympathetic neuron.

18. The method of claim 13, wherein said Sp family member is Sp-1 or Sp-3.

19. A method for determining whether a compound is capable of inhibiting or inducing oxidative sfress, DNA damage, growth factor depletion, or cell death in vitro or in vivo, said method comprising steps of:

(a) contacting a cell with a first compound; and

(b) determining whether said first compound effects a change in said cell in the protein level of an Sp family member or in the binding of an Sp family member to DNA.

20. The method of claim 19, wherein step (a) further comprises administering a second compound that increases the protein level of an Sp family member or increases the binding of an Sp family member to DNA

21. The method of claim 20, wherein said second compound is an oxidant, DNA-damaging agent, or growth factor depletion agent.

22. The method of claim 20, wherein said first compound is determined to be an inhibitor of oxidative sfress, DNA damage, growth factor depletion, or cell death if it reduces the increase in the protein level of an Sp family member that occurs in the presence of said second compound, or decreases the binding of an Sp family member to DNA.

23. The method of claim 19, wherein said compound is determined to be an inducer of oxidative sfress, DNA damage, growth factor depletion, or cell death if it increases the protein level of an Sp family member or increases the binding of an Sp family member to DNA.

24. The method of claim 19, wherein said cell is a neuronal cell.

25. The method of claim 24, wherein said neuronal cell is a cortical neuron, cerebellar granule cell, or sympathetic neuron.

26. The method of claim 19, wherein said first compound is a member of a library of at least 100 compounds, all of which are simultaneously contacted with said cell.

27. The method of claim 19, wherein said first compound is a nucleic acid comprising 20 or more nucleotides, and wherein the polynucleotide sequence of said nucleic acid has greater than 80% sequence identity to the corresponding region of a nucleic acid encoding an Sp family member.

28. The method claim 19, wherein said first compound is an antibody specific for a distinct Sp family member.

29. The method of claim 19, wherein said Sp family member is Sp-1 or Sp-3.