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Physiol. Genomics 11: 45-52, 2002. First published September 17, 2002; doi:10.1152/physiolgenomics.00064.2002
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Received 28 May 2002; accepted in final form 27 August 2002.
Physiological Genomics 11:45-52 (2002)
1094-8341/02 $5.00 © 2002 American Physiological Society

Discovery of molecular mechanisms of neuroprotection using cell-based bioassays and oligonucleotide arrays

Satinder S. Sarang, Takumi Yoshida, Rodolphe Cadet, Andrew S. Valeras, Roderick V. Jensen and Steven R. Gullans

Biotechnology Center, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Oxidative injury and the resulting death of neurons is a major pathological factor involved in numerous neurodegenerative diseases. However, the development of drugs that target this mechanism remains limited. The goal of this study was to test a compound library of approved Food and Drug Administration drugs against a hydrogen peroxide-induced oxidant injury model in neuroblastoma cells. We identified 26 neuroprotective compounds, of which megestrol, meclizine, verapamil, methazolamide, sulindac, and retinol were examined in greater detail. Using large-scale oligonucleotide microarray analysis, we identified genes modulated by these drugs that might underlie the cytoprotection. Five key genes were either uniformly upregulated or downregulated by all six drug treatments, namely, tissue inhibitor of matrix metalloproteinase (TIMP1), ret-proto-oncogene, clusterin, galanin, and growth associated protein (GAP43). Exogenous addition of the neuropeptide galanin alone conferred survival to oxidant-stressed cells, comparable to that seen with the drugs. Our approach, which we term "interventional profiling," represents a general and powerful strategy for identifying new bioactive agents for any biological process, as well as identifying key downstream genes and pathways that are involved.

oxidative stress; galanin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
OXIDATIVE INJURY and the resulting apoptotic and necrotic death of neurons is a major pathological factor involved in numerous neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD) (5, 12, 21, 43). In particular, protein oxidation and the aggregation of proteins resulting from oxidative stress play important roles in the pathogenesis of these neurodegenerative diseases (9). These findings have led to the development of numerous in vitro and in vivo models of oxidative stress in neuronal systems which are commonly being used to explore the mechanistic pathways and potential therapies for neurodegeneration (1, 11, 23, 28, 33–35, 40, 50). For example, in familial ALS, mutations in a gene encoding superoxide dismutase (SOD1) have been linked with the rapid progression of the disease (6, 14, 50). In PD, mutations in {alpha}-synuclein and parkin are associated with early onset and rapid progression of PD, and oxidative stress has been implicated (11, 21, 33, 25, 47, 62). In AD, oxidative stress has also been reported to promote the accumulation of ß-amyloid (Aß) through enhancing the amyloidogenic pathway (4, 44). However, drugs that target these neurodegenerative diseases are limited. In the case of ALS there is only one therapy approved to treat ALS, with many shortcomings in toxicity (32, 45, 55). The therapies available for PD are restricted to symptomatic treatment (62). Four compounds are currently approved for the use in treating AD, but these compounds provide only symptomatic benefits rather than modifying the progression of the disease (26).

The drug discovery process is undergoing a revolution with the use of combinatorial chemical libraries, high-throughput drug screening technologies, and DNA microarrays (3, 8, 16, 19, 60). For example, the complementary approaches of high-throughput drug screening and large-scale gene expression profiling were used to screen 60 human cancer cell lines, resulting in the identification of activity profiles of numerous compounds (53, 54). This large-scale human cancer cell line study opened the door to identifying novel "gene-gene, gene-drug, and drug-drug relationships" (54, 60). In addition, numerous bioinformatics methods are currently being used to identify novel gene interactions and genes with related functions (8, 52). Although these studies did not directly assess the influence of drugs on gene expression, they served to show the power of combining large-scale drug screening with gene expression analysis.

In the present study we sought to extend this approach by testing a library of known bioactive compounds, consisting of Food and Drug Administration (FDA)-approved drugs, to identify those that protect human SH-SY5Y neuroblastoma cells from lethal oxidative stress. Once we identified a subset of neuroprotective drugs, we then used oligonucleotide microarrays to measure their influence on large-scale gene expression profiles and thereby elucidate the apparent mechanisms of neuroprotection that involves enhanced expression of the neuropeptide galanin in every case.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
FDA2000 drug library.
Our laboratory developed a drug database that contains information on US FDA-approved drugs including drug indications, contraindications, chemical formulas, and mechanism of action. Using the Physician’s Desk Reference, we identified ~15,000 drugs that are presently marketed in the US. Of those, 1,345 were identified as unique chemical entities. We next created a drug repository of 880 compounds, and the drug collection is known as the FDA2000 drug library. The drugs/compounds were obtained from Sigma-Aldrich Chemicals (St. Louis, MO), the Brigham and Women’s Hospital Pharmacy (Boston, MA), or Athena Rx Home Pharmacy (San Francisco, CA). Each drug was dissolved in water to an approximate concentration of 10 mM. Then, 96-well daughter plates were made, and plates were stored at -20°C.

Cell culture.
Human SH-SY5Y neuroblastoma cells were kindly provided by Dr. E. Feldman (University of Michigan, Ann Arbor, MI). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO BRL; Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) plus 2% penicillin-streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO2. The cells were routinely subcultured using 0.05% trypsin-EDTA solution. The cells were seeded at 103 cells/well in 96-well plates (Corning, Corning, NY) and grown until each well was 75–80% confluent.

Oxidant injury and drug screening.
Our drug screening strategy was multistaged. To take advantage of the potential pleiotropic and latent actions of drugs in cell-based bioassays, we decided to pretreat cells with the drugs for 24 h prior to hydrogen peroxide exposure. Furthermore, as many drugs can have a direct impact on H2O2 itself (e.g., direct antioxidants), we chose to remove the drugs prior to H2O2 treatment. Furthermore, to ensure that the actions of the drugs did not wear off, we opted to perform an initial screen using a high dose of H2O2 for a short exposure time. Then knowing which drugs were active, we performed a secondary screen using a lower dose, longer exposure H2O2 protocol.

Pilot studies were performed to optimize the dose and time for exposure of cells to H2O2. SH-SY5Y cells were incubated with various doses of H2O2, ranging from 1 to 10 mM, and for different times, ranging from 4 to 24 h (data not shown). An optimal dose was determined to be that which resulted in ~70% loss of cell viability, as this provided maximal signal-noise in identifying drugs that rescued cells from death. For the initial high-dose screen, cells were exposed to 6 mM H2O2 for 4 h. In the secondary low-dose H2O2 screen, to confirm drug efficacy and optimize drug doses, cells were exposed to 100 µM H2O2 for 24 h. In each 96-well plate, 8 wells were used as controls in which no H2O2 was added.

Drugs were prepared by prediluting them to a concentration of 10–100 µM in DMEM containing 10% fetal bovine serum. Cell culture medium was removed from the cells in the 96-well plates and replaced with the fresh medium containing the drug (10–100 µM). There was only one drug per well. Cells were incubated with the drug for 24 h at 37°C. Then cell culture medium and drugs were removed and the cells washed once with Dulbecco’s PBS (D-PBS). Those drugs found to be protective in this first screen were then investigated in the secondary low-dose H2O2 screen. Drug dose-response experiments were performed using one protective drug from six different therapeutic classes to identify a peak effective dose. The SH-SY5Y cells were incubated with each drug (10–100 µM) for 24 h, drugs were removed, and cells were exposed to 100 µM H2O2 for 24 h at 37°C.

To measure cell viability, the cell culture medium containing H2O2 was removed and replaced with D-PBS containing 10 µM of the acetomethoxy ester of calcein (calcein-AM; Molecular Probes, Eugene OR), and cells were then incubated at 25°C for 30 min. Fluorescence was measured using a Victor2 Multilabel fluorescence plate reader (PerkinElmer Life Sciences, Boston, MA).

RNA isolation and oligonucleotide arrays.
Cells were incubated for 24 h with drugs at peak effective concentrations as follows: 30 µM megestrol, 60 µM meclizine, 30 µM verapamil, 100 µM methazolamide, 10 µM sulindac, and 10 µM retinol; there was no exposure to H2O2. Total RNA was isolated using Trizol (GIBCO BRL; Life Technologies), and RNA integrity was tested by visualization of 18S and 28S bands. Total RNA (5–7.9 µg) was used for in vitro transcription and labeled with biotin following procedures described previously (31, 39, 61). Following verification of cRNA quality on Test2 GeneChips, Affymetrix HG-U95A GeneChip probe arrays were used to determine mRNA expression levels.

Data analysis and informatics.
Drug screening data were analyzed using Microsoft Excel to assess standard statistical parameters. The oligonucleotide arrays were analyzed using MicroArray Suite 4.0 software from Affymetrix, with a "target intensity" (mean expression level) of 100. Significant changes in gene expression were identified by an average twofold or greater change across different protective drug treatments with P < 0.003 calculated using a single sample, two-tailed t-test applied to the logarithms of the ratio of the drug-treated gene expression levels to the control levels. This analysis was repeated for two sets of microarrays for the control and drug treatments (with n = 5 and n = 6). A small set of genes was identified that satisfied these requirements in both of the replicate experiments. These stringent criteria were designed to minimize the number of false positives and to generate a short list of informative genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
For our primary drug screen, SH-SY5Y cells were incubated with each of the 880 drugs in the library at concentrations of 10–100 µM for 24 h. The drugs were then removed, a lethal dose of H2O2 was added for 4 h, and cell viability was assayed. As shown in Table 1, we identified 26 compounds that provided 50–100% cytoprotection of SH-SY5Y cells, compared with control cell cultures in which no drugs were added. Interestingly, these compounds fell into multiple therapeutic classes, suggesting multiple mechanisms may be involved. The drugs were grouped into therapeutic classes, and one drug was chosen from the different classes for further testing, namely, megestrol, meclizine, verapamil, methazolamide, sulindac, and retinol. To determine the optimal protective doses, the six protective drugs were added at concentrations ranging from 1 to 300 µM for 24 h and assayed for their ability to abrogate high-dose H2O2 (6 mM)-mediated cell death (Table 2). Each drug showed a classic dose-response. Increasing doses provided greater cytoprotection until an optimal protective dose was attained, after which additional drug caused toxicity.


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  		Table 1. Cytoprotective drugs identified in the initial screen

 

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  		Table 2. Cytoprotective drugs with high-dose H2O2

 
Having identified a subset of six neuroprotective compounds, we next evaluated their efficacy in a low-dose H2O2 toxicity assay. The optimal doses of each compound were added to SH-SY5Y cells for 24 h, drugs were removed, and cell survival was assessed following a 100 µM H2O2 exposure for 24 h in the absence of the drug. As shown in Fig. 1, this prolonged application of H2O2 resulted in 66 ± 9% cell death in the absence of any drug. All six drugs were cytoprotective, with all compounds except sulindac promoting survival that was no different from control cells. Sulindac showed partial protection with 59 ± 9% viability observed.



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  		Fig. 1. Drug inhibition of low-dose H2O2-induced oxidant injury. SH-SY5Y cells were incubated with 30 µM megestrol, 60 µM meclizine, 30 µM verapamil, 100 µM methazolamide, 10 µM sulindac, or 10 µM retinol for 24 h. The drugs were removed and the cells were incubated with 100 µM H2O2 for 24 h, and cell viability measured. Bars represent percent protection compared with the controls in which no H2O2 was added (±SD, n = 4–8).

 
To check that the measured levels of neuroprotection were not the result of enhanced proliferation induced by the drugs, we used Hoechst 33258 pentahydrate nucleic acid stain and calcein-AM. After 24 h of exposure to the drugs, the normalized cell numbers averaged 109 ± 8% for Hoechst stain and 85 ± 9% for calcein-AM compared with untreated controls (100 ± 11% Hoechst stain and 100 ± 16% calcein-AM).

Having identified a highly diverse group of neuroprotective pharmaceutical compounds whose known mechanisms of action are highly unrelated to each other or to cell survival, we sought to discover a biological process that would interconnect them. Using high-density oligonucleotide microarrays, RNA expression levels of ~12,000 genes were measured in SH-SY5Y cells incubated for 24 h with optimal doses of megestrol, meclizine, verapamil, methazolamide, sulindac, and retinol. RNA was also isolated from vehicle-treated control cells. Both the drug treatments and the control were repeated on two separate occasions. These replicate experiments provided two sets of microarray measurements of the gene expression changes due to the protective drugs (however, one of the microarrays for megestrol was discarded from the data set due to poor chip quality). These two sets of measurements of gene expression changes induced by the drugs were then analyzed using single-sample, two-tailed Student’s t-tests to determine a small subset of genes with the most significant changes in expression. As shown in Fig. 2, data analysis identified a small subset of genes that were differentially expressed in response to all six drugs. These included increased expression of tissue inhibitor of matrix metalloproteinase (TIMP1, D11139), clusterin (M25915), galanin (M77140), and growth associated protein (GAP43, M25667) and decreased expression of ret-proto-oncogene (HG4677-HT5102). Quantitative RT-PCR was used to confirm the increased expression for two of these genes, clusterin and galanin, for every drug treatment. Although the five genes do not appear to be involved in any common pathways, they all play critical roles in the normal physiological functions of neural cells.



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  		Fig. 2. mRNAs differentially expressed in response to the drugs. Data are expressed as gene expression ratios of log2(drug/control) across the drug treatments. The points on the graph represent the log2 (drug/control) ratio across the drug treatments (n = 2; n = 1 for 30 µM megestrol).

 
Galanin is a known to be a secreted neuropeptide that is involved in the neurotransmission of neural cells (49). Being a secreted peptide, we reasoned that galanin may also act as an autocrine cytoprotective agent. Thus, we incubated SH-SY5Y cells with exogenous galanin for 24 h to assess its ability to confer cell survival with subsequent exposure to 100 µM H2O2 for 24 h. As shown in Fig. 3, galanin alone blocked oxidant-induced cell death of the SH-SY5Y cells. We verified that the galanin alone did not enhance cell proliferation as determined using Hoechst stain (110 ± 25 vs. 100 ± 18% control). Collectively, these findings suggest that the drugs that provide protection to SH-SY5Y cells were influencing the transcription of a small set of genes, and in particular that galanin plays a key role in the neurodegenerative pathways of oxidant-induced neural injury.



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  		Fig. 3. Inhibition of oxidant-induced injury by the neuropeptide galanin. SH-SY5Y cells were incubated with galanin for 24 h, followed by 100 µM H2O2 for 24 h, and then cell viability was measured. Bars represent percent protection compared with the controls in which no H2O2 was added. Data are means ± SD (n = 4–6).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study we showed that with a cell-based bioassay it is possible to identify existing pharmaceuticals that are neuroprotective in a model of oxidant injury. Furthermore, by directly evaluating the effects of several of these neuroprotective agents on mRNA expression, we identified five differentially expressed genes, including galanin, which appear to be associated with cytoprotection. Exogenous addition of galanin alone recapitulated the drug-mediated neuroprotection. This approach of using drug screening followed by global analysis of gene expression represents a powerful paradigm for biological and drug discovery that could be used in many systems.

Our cell bioassay involved the use of human SH-SY5Y neuroblastoma cells which are derived from the sympathetic nervous system and possess many properties of mature sympathetic neurons. Using the well-established H2O2 oxidant injury model, we implemented a two-step drug screening process: first using a high-dose, acute cellular H2O2 exposure to identify candidate neuroprotective compounds and then confirming the efficacy of these agents using a lower dose and more chronic H2O2 exposure protocol. The drug library comprised 880 known pharmaceuticals. For both the acute and chronic protocols, cells were exposed to the drugs for 24 h prior to H2O2 exposure, enabling the compounds to directly or indirectly modulate many processes within the cells. Moreover, this is more reflective of an in vivo setting where drugs are taken chronically, giving cells an opportunity to manifest both primary and secondary responses.

Overall, we identified 26 drugs that provide neuroprotection, representing more than 6 different therapeutic classes. This relatively high "hit rate" of 3% was attained without optimizing doses or formulations in the initial screen, underscoring the multipotent activities of existing pharmaceuticals. Additional neuroprotective compounds may have been missed in our primary screen since they were not all tested under optimal conditions.

Our FDA2000 drug library is composed of a nonredundant set of FDA approved drugs that are presently marketed in the US. Most of these compounds have never been implicated in neuroprotection, for either their prescribed indication or known mechanisms of action. However, due to the cross-reactivity of many of these drugs with other possible targets or their actions on non-target neuronal cells, a number of compounds emerged as having unanticipated actions to prevent oxidative damage in neuronal cells. This observation suggests that through the interconnected network of biological pathways and processes, drugs of highly unrelated structures and actions can have similar effects.

The six drugs examined in detail, namely sulindac, retinol, verapamil, megestrol, meclizine, and methazolamide, are highly unrelated with regard to their known targets and primary actions. Of these, prior studies suggested sulindac, retinol, and verapamil could be cytoprotective during oxidant injury. Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) used for the treatment of inflammatory diseases and rheumatoid arthritis. It inhibits prostaglandin synthesis by decreasing the activity of cyclooxygenase. Sulindac has been shown to scavenge oxidant products of prostaglandin cyclooxygenase/peroxides (51). Moreover, epidemiological studies have revealed a reduction in the prevalence of AD among people taking NSAID compounds (58), although sulindac has not yet been specifically implicated.

Retinol (vitamin A or all-trans-retinoic acid) is used a topical treatment of acne. Retinoic acids act upon various biological process including cell proliferation, differentiation, and cellular morphogenesis (36). Human trials of retinol revealed a reduction in reactive oxygen metabolites such as those produced by endogenous cellular H2O2 (13). Pretreatment of mesangial cells with retinol blocked morphological and biochemical markers of apoptosis typically induced by H2O2 (46). The anti-apoptotic effect of retinol against H2O2 was also observed in fibroblasts (46). This anti-apoptotic pathway was shown to act through the dual suppression of the cell death pathway mediated by c-Jun N-terminal kinase (c-Jun) and activator protein 1 (AP-1). The trans-retinoic acid anti-apoptotic pathway acts on both nuclear receptor-dependent and -independent mechanisms (36).

Verapamil belongs to the antianginal and antihypertensive therapeutic class of compounds and is specifically an antiarrhythmic class IV drug. Verapamil inhibits calcium ions from entering the slow channels and select voltage-sensitive areas of the vascular smooth muscle. Verapamil has possible neuroprotective effects on normal neurons exposed to high concentrations of ethanol, and it has been suggested that verapamil should be evaluated as a drug for treatment of alcohol-induced brain damage and neurodegenerative disorders (37).

Megestrol, meclizine, and methazolamide, as well as many compounds identified in the primary screen (Table 1), are not known to be neuroprotective or cytoprotective during oxidant stress. Megestrol is an antineoplastic agent that is a synthetic analog of progesterone (10). The chemical structure of megestrol is similar to that of norethindrone and flunisolide, which were also found to be protective to a lesser extent in the primary H2O2 screen. Meclizine is an antiemetic and an antihistamine H1 blocker used for the prevention and treatment of motion sickness and the management of vertigo with diseases affecting the vestibular system. Meclizine has central anticholinergic actions by blocking the chemoreceptor trigger zones (48). Methazolamide is a diuretic drug and acts as a noncompetitive inhibitor of carbonic anhydrase (38).

To investigate potential biological actions of the protective drugs, we evaluated their effects on mRNA expression 24 h after drug exposure using large-scale oligonucleotide microarray analysis (Fig. 2). A set of five genes, TIMP1, ret-proto-oncogene, clusterin, galanin, and GAP43, were all identified as differentially expressed in response to all six drugs (Fig. 2). Their encoded proteins all play critical roles in the normal physiological functions of neural cells, and several have been implicated in cell survival.

TIMP1 was upregulated and is a member of a family of TIMP genes involved in cell proliferation and cell survival. TIMP1 may specifically inhibit apoptosis (18, 27) and can confer resistance to oxidative stress (22). Interestingly, the inhibition of matrix metalloproteinase showed a significant decrease in liver ischemia/reperfusion injury as assessed by histological and serum hepatic levels and has been proposed to have clinical relevance in liver-associated ischemic disease (15). H2O2 was reported to be an important intermediate in the downstream signaling pathway leading to the induction of an increased steady state of matrix metalloproteinase-1 mRNA levels (7). These findings provide supporting evidence for our experiments in which TIMP1 was upregulated by the protective compounds and prevented oxidant-induced injury.

Clusterin (apolipoprotein J) was significantly upregulated in all the drug treatments compared with the control cells. Clusterin is an 80-kDa glycoprotein that has been implicated in cytoprotection of fibroblasts and is induced by numerous cellular stresses. Overexpression of clusterin may be associated with cell survival after oxidative injury (2, 17, 57). Clusterin has been suggested to protect cells against apoptotic cell death and neurodegeneration (59). Interestingly, in AD, lower cellular expression levels of the clusterin protein was suggested to be associated with neuronal degeneration and death (20). Using an antisense approach, researchers have found that suppression of clusterin mRNA and protein expression made cells more sensitive to apoptotic cell death induced by heat shock or H2O2-induced oxidant stress (57). These studies indicate that clusterin confers cellular protection against heat shock and oxidative stress (57).

RET was decreased more than twofold in all the drug treatments. The ret-proto-oncogene encodes a cell membrane tyrosine kinase receptor protein whose ligands belong to the glial cell line-derived neurotrophic factor family (56). Its role in cytoprotection during oxidant stress still needs to be elucidated.

Galanin expression was enhanced by all six drugs. This 29-amino acid secreted neuropeptide colocalizes with choline acetyltransferase (49) and has been implicated in cell injury recovery processes in neurons. Rat dorsal root ganglion (DRG) were incubated with various forms of ß-amyloid and a decrease in galanin immunoreactive neurons was identified (41). Cytokines and galanin have been suggested to function in a molecular cascade mediating injury-induced regeneration (29). Peripheral nerve damage upregulates cytokine interleukin-6 (IL-6) in DRG neurons, and these changes increase the levels of galanin in the DRG neurons. The increased levels of galanin in sensory neurons contributes to the initiation and maintenance of axonal regeneration in injured neurons (29).

GAP43, which was induced by the drugs, encodes a protein that localizes at the growth cone of neurite outgrowths. In astrocytoma tumors it is involved in attachment, spreading, and motility (24). Cellular changes in GAP43 and galanin protein immunoreactivity have been studied in an axonal injury model (30). The axonal injury model revealed that neurons in the middle and caudal part of the ganglia survived the injury and showed an increase in GAP43 and galanin immunoreactivity, indicating a sign of regeneration/neuronal plasticity (30). In AD patients, GAP43 levels were decreased, suggesting in part that synaptic injury in the frontal cortex is an early event in AD (42).

Since galanin is a secreted neuropeptide, we hypothesize that it could act as autocrine survival factor. In fact, we found that exogenous galanin alone blocked the H2O2-induced cell death of the SH-SY5Y cells (Fig. 3). Thus, galanin can act as a neuroprotective factor and should be considered for its therapeutic potential in treating neurodegeneration. Moreover, the galanin receptor could represent a therapeutic target for small drug discovery. These results have important ramifications for understanding and developing therapeutics for neuronal repair and preventing further oxidant-induced neural damage.

Collectively, our studies have identified a set of highly protective drugs that blocked oxidant-induced injury in SH-SY5Y cells, a small set of genes that were highly upregulated and downregulated by the protective compounds, and the specific involvement of the neuropeptide galanin in blocking oxidant injury to SH-SY5Y cells.


   ACKNOWLEDGMENTS
 
We thank Dr. L.-L. Hsiao, Dr. J. Randall, K. Clark, and F. Beato for conducting the oligonucleotide microarray experiments. We thank Peter Clark of the Hope for ALS Foundation. We also thank the Ride for ALS, ALS Treatment Development Foundation, and Project ALS for continued support.

S. S. Sarang was supported by a fellowship from the Hope for ALS Foundation, and S. R. Gullans was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-36031 and DK-58849.

Editor M. A. Marra served as the review editor for this manuscript submitted by Editor S. R. Gullans.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S. R. Gullans, Center for Neurologic Diseases, Brigham and Women’s Hospital, 65 Landsdowne St., Rm. 301, Cambridge, MA 02139 (E-mail: ssarang{at}rics.bwh.harvard.edu).

10.1152/physiolgenomics.00064.2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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