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 Womens Hospital, Harvard Medical School, Cambridge, Massachusetts 02139
 |
ABSTRACT
|
---|
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
|
---|
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), Parkinsons disease (PD), and Alzheimers 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, 3335, 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
-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
|
---|
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 Physicians 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 Womens 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 Dulbeccos modified Eagles 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 7580% 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 10100 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 (10100 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 Dulbeccos 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 (10100 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 (57.9 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
|
---|
For our primary drug screen, SH-SY5Y cells were incubated with each of the 880 drugs in the library at concentrations of 10100 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 50100% 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.
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.

View larger version (15K):
[in this window]
[in a new window]
|
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 = 48).
|
|
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 Students 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.

View larger version (15K):
[in this window]
[in a new window]
|
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.

View larger version (8K):
[in this window]
[in a new window]
|
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 = 46).
|
|
 |
DISCUSSION
|
---|
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 Womens Hospital, 65 Landsdowne St., Rm. 301, Cambridge, MA 02139 (E-mail: ssarang{at}rics.bwh.harvard.edu).
10.1152/physiolgenomics.00064.2002.
 |
References
|
---|
- Abe K, Morita S, Kikuchi T, and Itoyama Y. Protective effect of a novel free radical scavenger, OPC-14117, on wobbler mouse motor neuron disease. J Neurosci Res 48: 6370, 1997.[ISI][Medline]
- Bach UC, Baiersdorfer M, Klock G, Cattaruzza M, Post A, and Koch-Brandt C. Apoptotic cell debris and phosphatidylserine-containing lipid vesicles induce apolipoprotein J (clusterin) gene expression in vital fibroblasts. Exp Cell Res 265: 1120, 2001.[ISI][Medline]
- Barry CE III, Slayden RA, Sampson AE, and Lee RE. Use of genomics and combinatorial chemistry in the development of new antimycobacterial drugs. Biochem Pharmacol 59: 221231, 2000.[ISI][Medline]
- Blass JP. Brain metabolism and brain disease: is metabolic deficiency the proximate cause of Alzheimer dementia? J Neurosci Res 66: 851856, 2001.[ISI][Medline]
- Bogdanov MB, Ramos LE, Xu Z, and Beal MF. Elevated "hydroxyl radical" generation in vivo in an animal model of amyotrophic lateral sclerosis. J Neurochem 71: 13211324, 1998.[ISI][Medline]
- Bowling AC, Barkowski EE, McKenna-Yasek D, Sapp P, Horvitz HR, Beal MF, and Brown RH Jr. Superoxide dismutase concentration and activity in familial amyotrophic lateral sclerosis. J Neurochem 64: 23662369, 1995.[ISI][Medline]
- Brenneisen P, Briviba K, Wlaschek M, Wenk J, and Scharffetter-Kochanek K. Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts. Free Radic Biol Med 22: 515524, 1997.[ISI][Medline]
- Butte AJ, Ye J, Haring HU, Stumvoll M, White MF, and Kohane IS. Determining significant fold differences in gene expression analysis. Pac Symp Biocomput : 617, 2001.
- Butterfield DA, Howard BJ, and LaFontaine MA. Brain oxidative stress in animal models of accelerated aging and the age-related neurodegenerative disorders, Alzheimers disease and Huntingtons disease. Curr Med Chem 8: 815828, 2001.[ISI][Medline]
- Chang AY. Megestrol acetate as a biomodulator. Semin Oncol 25: 5861, 1998.[ISI][Medline]
- Choi P, Golts N, Snyder H, Chong M, Petrucelli L, Hardy J, Sparkman D, Cochran E, Lee JM, and Wolozin B. Co-association of parkin and alpha-synuclein. Neuroreport 12: 28392843, 2001.[ISI][Medline]
- Cookson MR and Shaw PJ. Oxidative stress and motor neurone disease. Brain Pathol 9: 165186, 1999.[ISI][Medline]
- Cornelli U, Terranova R, Luca S, Cornelli M, and Alberti A. Bioavailability and antioxidant activity of some food supplements in men and women using the D-Roms test as a marker of oxidative stress. J Nutr 131: 32083211, 2001.[Abstract/Free Full Text]
- Cudkowicz ME, McKenna-Yasek D, Sapp PE, Chin W, Geller B, Hayden DL, Schoenfeld DA, Hosler BA, Horvitz HR, and Brown RH. Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann Neurol 41: 210221, 1997.[ISI][Medline]
- Cursio R, Mari B, Louis K, Rostagno P, Saint-Paul MC, Giudicelli J, Bottero V, Anglard P, Yiotakis A, Dive V, Gugenheim J, and Auberger P. Rat liver injury after normothermic ischemia is prevented by a phosphinic matrix metalloproteinase inhibitor. FASEB J 16: 9395, 2002.[Abstract/Free Full Text]
- Debouck C and Goodfellow PN. DNA microarrays in drug discovery and development. Nat Genet 21: 4850, 1999.[ISI][Medline]
- Dumont P, Burton M, Chen QM, Gonos ES, Frippiat C, Mazarati JB, Eliaers F, Remacle J, and Toussaint O. Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast. Free Radic Biol Med 28: 361373, 2000.[ISI][Medline]
- Fassina G, Ferrari N, Brigati C, Benelli R, Santi L, Noonan DM, and Albini A. Tissue inhibitors of metalloproteases: regulation and biological activities. Clin Exp Metastasis 18: 111120, 2000.[ISI][Medline]
- Gerhold D, Lu M, Xu J, Austin C, Caskey CT, and Rushmore T. Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays. Physiol Genomics 5: 161170, 2001.[Abstract/Free Full Text]
- Giannakopoulos P, Kovari E, French LE, Viard I, Hof PR, and Bouras C. Possible neuroprotective role of clusterin in Alzheimers disease: a quantitative immunocytochemical study. Acta Neuropathol (Berl) 95: 387394, 1998.[ISI][Medline]
- Giasson BI, Duda JE, Murray IV, Chen Q, Souza JM, Hurtig HI, Ischiropoulos H, Trojanowski JQ, and Lee VM. Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions. Science 290: 985989, 2000.[Abstract/Free Full Text]
- Gonzalez W, Fontaine V, Pueyo ME, Laquay N, Messika-Zeitoun D, Philippe M, Arnal JF, Jacob MP, and Michel JB. Molecular plasticity of vascular wall during NG-nitro-L-arginine methyl ester-induced hypertension: modulation of proinflammatory signals. Hypertension 36: 103109, 2000.[Abstract/Free Full Text]
- Gurney ME, Cutting FB, Zhai P, Doble A, Taylor CP, Andrus PK, and Hall ED. Benefit of vitamin E, riluzole, and gabapentin in a transgenic model of familial amyotrophic lateral sclerosis. Ann Neurol 39: 147157, 1996.[ISI][Medline]
- Gutmann DH, Wu YL, Hedrick NM, Zhu Y, Guha A, and Parada LF. Heterozygosity for the neurofibromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes. Hum Mol Genet 10: 30093016, 2001.[Abstract/Free Full Text]
- Gwinn-Hardy K and Farrer M. Parkinsons genetics: an embarrassment of riches. Ann Neurol 51: 78, 2002.[ISI][Medline]
- Hake AM and Farlow MR. New concepts in the drug therapy of Alzheimers disease. Expert Opin Pharmacother 2: 19751983, 2001.[Medline]
- Han X, Sun Y, Scott S, and Bleich D. Tissue inhibitor of metalloproteinase-1 prevents cytokine-mediated dysfunction and cytotoxicity in pancreatic islets and beta-cells. Diabetes 50: 10471055, 2001.[Abstract/Free Full Text]
- Hayward LJ, Rodriguez JA, Kim JW, Tiwari A, Goto JJ, Cabelli DE, Valentine JS, and Brown RH Jr. Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial ALS. J Biol Chem, 2002.
- Holmes FE, Mahoney S, King VR, Bacon A, Kerr NC, Pachnis V, Curtis R, Priestley JV, and Wynick D. Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity. Proc Natl Acad Sci USA 97: 1156311568, 2000.[Abstract/Free Full Text]
- Hou XE, Lundmark K, and Dahlstrom AB. Cellular reactions to axotomy in rat superior cervical ganglia includes apoptotic cell death. J Neurocytol 27: 441451, 1998.[ISI][Medline]
- Hsiao LL, Jensen RV, Yoshida T, Clark KE, Blumenstock JE, and Gullans SR. Correcting for signal saturation errors in the analysis of microarray data. Biotechniques 32: 330332, 334, 336, 2002.[ISI][Medline]
- Hurko O and Walsh FS. Novel drug development for amyotrophic lateral sclerosis. J Neurol Sci 180: 2128, 2000.[ISI][Medline]
- Junn E and Mouradian MM. Human
-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine. Neurosci Lett 320: 146150, 2002.[ISI][Medline]
- Kaal EC, Vlug AS, Versleijen MW, Kuilman M, Joosten EA, and Bar PR. Chronic mitochondrial inhibition induces selective motoneuron death in vitro: a new model for amyotrophic lateral sclerosis. J Neurochem 74: 11581165, 2000.[ISI][Medline]
- Klaidman LK, Mukherjee SK, and Adams JD Jr. Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide. Biochim Biophys Acta 1525: 136148, 2001.[ISI][Medline]
- Konta T, Xu Q, Furusu A, Nakayama K, and Kitamura M. Selective roles of retinoic acid receptor and retinoid x receptor in the suppression of apoptosis by all-trans-retinoic acid. J Biol Chem 276: 1269712701, 2001.[Abstract/Free Full Text]
- Liesi P, Wright JM, and Krauthamer V. BAPTA-AM and ethanol protect cerebellar granule neurons from the destructive effect of the weaver gene. J Neurosci Res 48: 571579, 1997.[ISI][Medline]
- Lindskog S. Structure and mechanism of carbonic anhydrase. Pharmacol Ther 74: 120, 1997.[ISI][Medline]
- Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, and Brown EL. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol 14: 16751680, 1996.[ISI][Medline]
- Ludolph AC. Animal models for motor neuron diseases: research directions. Neurology 47: S228232, 1996.[Abstract]
- Ma W, Zheng WH, Belanger S, Kar S, and Quirion R. Effects of amyloid peptides on cell viability and expression of neuropeptides in cultured rat dorsal root ganglion neurons: a role for free radicals and protein kinase C. Eur J Neurosci 13: 11251135, 2001.[ISI][Medline]
- Masliah E, Mallory M, Alford M, DeTeresa R, Hansen LA, McKeel DW Jr, and Morris JC. Altered expression of synaptic proteins occurs early during progression of Alzheimers disease. Neurology 56: 127129, 2001.[Abstract/Free Full Text]
- McCord JM. The evolution of free radicals and oxidative stress. Am J Med 108: 652659, 2000.[ISI][Medline]
- Misonou H, Morishima-Kawashima M, and Ihara Y. Oxidative stress induces intracellular accumulation of amyloid ß-protein (Aß) in human neuroblastoma cells. Biochemistry 39: 69516959, 2000.[ISI][Medline]
- Mitsumoto H. Clinical trials: present and future. Amyotroph Lateral Scler Other Motor Neuron Disord 2, Suppl 1: S10S14, 2001.
- Moreno-Manzano V, Ishikawa Y, Lucio-Cazana J, and Kitamura M. Suppression of apoptosis by all-trans-retinoic acid. Dual intervention in the c-Jun n-terminal kinase-AP-1 pathway. J Biol Chem 274: 2025120258, 1999.[Abstract/Free Full Text]
- Mouradian MM. Recent advances in the genetics and pathogenesis of Parkinson disease. Neurology 58: 179185, 2002.[Abstract/Free Full Text]
- Murray JB. Psychophysiological aspects of motion sickness. Percept Mot Skills 85: 11631167, 1997.[ISI][Medline]
- OMeara G, Coumis U, Ma SY, Kehr J, Mahoney S, Bacon A, Allen SJ, Holmes F, Kahl U, Wang FH, Kearns IR, Ove-Ogren S, Dawbarn D, Mufson EJ, Davies C, Dawson G, and Wynick D. Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons. Proc Natl Acad Sci USA 97: 1156911574, 2000.[Abstract/Free Full Text]
- Pasinelli P, Borchelt DR, Houseweart MK, Cleveland DW, and Brown RH Jr. Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase. Proc Natl Acad Sci USA 95: 1576315768, 1998.[Abstract/Free Full Text]
- Pekoe G, Van Dyke K, Peden D, Mengoli H, and English D. Antioxidation theory of non-steroidal anti-inflammatory drugs based upon the inhibition of luminol-enhanced chemiluminescence from the myeloperoxidase reaction. Agents Actions 12: 371376, 1982.[ISI][Medline]
- Reis BY, Butte AS, and Kohane IS. Extracting knowledge from dynamics in gene expression. J Biomed Inform 34: 1527, 2001.[ISI][Medline]
- Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, Spellman P, Iyer V, Jeffrey SS, Van de Rijn M, Waltham M, Pergamenschikov A, Lee JC, Lashkari D, Shalon D, Myers TG, Weinstein JN, Botstein D, and Brown PO. Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet 24: 227235, 2000.[ISI][Medline]
- Scherf U, Ross DT, Waltham M, Smith LH, Lee JK, Tanabe L, Kohn KW, Reinhold WC, Myers TG, Andrews DT, Scudiero DA, Eisen MB, Sausville EA, Pommier Y, Botstein D, Brown PO, and Weinstein JN. A gene expression database for the molecular pharmacology of cancer. Nat Genet 24: 236244, 2000.[ISI][Medline]
- Silani V, Braga M, Cardin V, and Scarlato G. The pathogenesis of ALS: implications for treatment strategies. Neurol Neurochir Pol 35: 2539, 2001.
- Tallini G and Asa SL. RET oncogene activation in papillary thyroid carcinoma. Adv Anat Pathol 8: 345354, 2001.[ISI][Medline]
- Viard I, Wehrli P, Jornot L, Bullani R, Vechietti JL, Schifferli JA, Tschopp J, and French LE. Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress. J Invest Dermatol 112: 290296, 1999.[Abstract/Free Full Text]
- Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, Findlay KA, Smith TE, Murphy MP, Bulter T, Kang DE, Marquez-Sterling N, Golde TE, and Koo EH. A subset of NSAIDs lower amyloidogenic Aß42 independently of cyclooxygenase activity. Nature 414: 212216, 2001.[ISI][Medline]
- Wehrli P, Charnay Y, Vallet P, Zhu G, Harmony J, Aronow B, Tschopp J, Bouras C, Viard-Leveugle I, French LE, and Giannakopoulos P. Inhibition of post-ischemic brain injury by clusterin overexpression. Nat Med 7: 977979, 2001.[ISI][Medline]
- Weinstein JN, Myers TG, OConnor PM, Friend SH, Fornace AJ Jr, Kohn KW, Fojo T, Bates SE, Rubinstein LV, Anderson NL, Buolamwini JK, van Osdol WW, Monks AP, Scudiero DA, Sausville EA, Zaharevitz DW, Bunow B, Viswanadhan VN, Johnson GS, Wittes RE, and Paull KD. An information-intensive approach to the molecular pharmacology of cancer. Science 275: 343349, 1997.[Abstract/Free Full Text]
- Wodicka L, Dong H, Mittmann M, Ho MH, and Lockhart DJ. Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat Biotechnol 15: 13591367, 1997.[ISI][Medline]
- Zhang Y, Dawson VL, and Dawson TM. Oxidative stress and genetics in the pathogenesis of Parkinsons disease. Neurobiol Dis 7: 240250, 2000.[ISI][Medline]