Novel function of the C-terminal region of the Hsp110 family member Osp94 in unfolded protein refolding.

Osp94 (also known as HSPA4L or HSPH3), a member of the Hsp110/Sse1 family of heat-shock proteins, has a longer C-terminus than found in Hsc70/Hsp70 family proteins, composed of the loop region with a partial substrate-binding domain (SBD) ß (L), and the SBDα and the C-terminal extension (H), but the functions of these domains are poorly understood. Here, we found that Osp94 suppressed heat-induced aggregation of luciferase (Luc). Osp94-bound heat-inactivated Luc was reactivated in the presence of rabbit reticulocyte lysate (RRL) and/or a combination of Hsc70 and Hsp40 (also known as HSPA8 and DNAJB1, respectively). Targeted deletion mutagenesis revealed that the SBDß and H domains of Osp94 are critical for protein disaggregation and RRL-mediated refolding. Reactivation of Hsp90-bound heat-inactivated Luc was abolished in the absence of RRL but compensated for by PA28α (also known as PSME1), a proteasome activator. Interestingly, the LH domain also reactivated heat-inactivated Luc, independently of PA28α. Biotin-tag cross-linking experiments indicated that the LH domain and PA28α interact with Luc bound by Hsp90 during refolding. A chimeric protein in which the H domain was exchanged for PA28α also mediated disaggregation and reactivation of heat-inactivated Luc. These results indicate that Osp94 acts as a holdase, and that the C-terminal region plays a PA28α-like role in the refolding of unfolded proteins.

LNA-mediated microRNA silencing in non-human primates.

microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg(-1) LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg(-1) LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

Better therapeutics through microarrays.

DNA microarrays are an integral part of the process for therapeutic discovery, optimization and clinical validation. At an early stage, investigators use arrays to prioritize a few genes as potential therapeutic targets on the basis of various criteria. Subsequently, gene expression analysis assists in drug discovery and toxicology by eliminating poor compounds and optimizing the selection of promising leads. Integral to this process is the use of sophisticated statistics, mathematics and bioinformatics to define statistically valid observations and to deduce complex patterns of phenotypes and biological pathways. In short, microarrays are redefining the drug discovery process by providing greater knowledge at each step and by illuminating the complex workings of biological systems.

Transcriptome sequencing of malignant pleural mesothelioma tumors.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

ATF3 protects against renal ischemia-reperfusion injury.

Oxidative stress-induced cell death plays a major role in the progression of ischemic acute renal failure. Using microarrays, we sought to identify a stress-induced gene that may be a therapeutic candidate. Human proximal tubule (HK2) cells were treated with hydrogen peroxide (H2O2) and RNA was applied to an Affymetrix gene chip. Five genes were markedly induced in a parallel time-dependent manner by cluster analysis, including activating transcription factor 3 (ATF3), p21(WAF1/CiP1) (p21), CHOP/GADD153, dual-specificity protein phosphatase, and heme oxygenase-1. H2O2 rapidly induced ATF3 approximately 12-fold in HK2 cells and approximately 6.5-fold in a mouse model of renal ischemia-reperfusion injury. Adenovirus-mediated expression of ATF3 protected HK2 cells against H2O2-induced cell death, and this was associated with a decrease of p53 mRNA and an increase of p21 mRNA. Moreover, when ATF3 was overexpressed in mice via adenovirus-mediated gene transfer, ischemia-reperfusion injury was reduced. In conclusion, ATF3 plays a protective role in renal ischemia-reperfusion injury and the mechanism of the protection may involve suppression of p53 and induction of p21.

Identification, coassembly, and activity of gamma-aminobutyric acid receptor subunits in renal proximal tubular cells.

Although the properties and functions of GABA(A) receptors in the mammalian central nervous system have been well studied, the presence and significance of GABA(A) receptors in non-neural tissues are less clear. The goal of this study was to examine the expression of GABA(A) receptor alpha(1), alpha(2), alpha(4), alpha(5), beta(1), gamma(1), gamma(2), and delta subunits in the kidney and to determine whether these subunits coassemble to form an active renal epithelial cell GABA(A) receptor. Using reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and brain or cerebellum through polymerase chain reaction (PCR) and sequencing of the PCR products, we revealed that rat kidney cortex contained the alpha(1), alpha(5), beta(1), gamma(1), and gamma(2) subunits and that they were similar to the neuronal subunits. Sequencing of the PCR products revealed that the rabbit kidney cortex contained the alpha(1) and gamma(2) subunits and that they were similar to their neuronal counterparts. Immunoprecipitation and immunoblot studies using GABA(A) receptor subunit-specific antibodies and detergent-solubilized rat kidney cortex membranes identified a GABA(A) receptor complex containing alpha(5), beta(1), and gamma(1). Isolated rat renal proximal tubular cells exhibited GABA-mediated, picrotoxin-sensitive (36)Cl(-) uptake. These studies demonstrate the presence of numerous GABA(A) receptor subunits in the kidneys of two species, the assembly of the subunits into at least one novel receptor complex, and an active GABA(A) receptor in renal proximal tubular cells.

Transcriptional activation of placental growth factor by the forkhead/winged helix transcription factor FoxD1.

Stromal-epithelial interactions play an important role in renal organogenesis. Expression of the forkhead/winged helix transcription factor FoxD1 (BF-2) is restricted to stromal cells in the embryonic renal cortex, but it mediates its effects on the adjacent ureteric bud and metanephric mesenchyme, which fail to grow and differentiate in BF-2 null mice. BF-2 is therefore likely to regulate transcription of factors secreted by stromal cells that modulate the differentiation of neighboring epithelial cells. Here, we used cells with inducible expression of BF-2, combined with microarray analysis, to identify Placental Growth Factor (PlGF), a Vascular Endothelial Growth Factor (VEGF) family member previously implicated in angiogenesis, as a downstream target of BF-2. BF-2 binds to a conserved HNF3beta site in the PlGF promoter and activates transcription. PlGF is precisely coexpressed with BF-2, both temporally and spatially, within the developing renal stroma, and it is completely absent in BF-2 null kidney stroma. Addition of PlGF to in vitro kidney organ cultures stimulates branching of the ureteric bud. Our observations indicate that PlGF is a direct and physiologically relevant transcriptional target of BF-2. The contribution of PlGF toward stromal signals that regulate epithelial differentiation suggests novel functions for a growth factor previously implicated in reactive angiogenesis.

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA.

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of +/-0.7 microm or +/-2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension.

Single DNA molecule stretching in sudden mixed shear and elongational microflows.

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.

Endogenous ouabain: upregulation of steroidogenic genes in hypertensive hypothalamus but not adrenal.

BACKGROUND: Mammalian tissues contain a presumed endogenous Na+, K(+)-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides. METHODS AND RESULTS: Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and Delta5-3beta-hydroxysteroid dehydrogenase/Delta5-Delta4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue. CONCLUSIONS: Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.

12-lipoxygenase in opioid-induced delayed cardioprotection: gene array, mass spectrometric, and pharmacological analyses.

12-lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a delta-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic-mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (P<0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50+/-4% and 42+/-3% versus 29+/-2%, P<0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.

HugeIndex: a database with visualization tools for high-density oligonucleotide array data from normal human tissues.

High-density oligonucleotide arrays are a powerful tool for uncovering changes in global gene expression in various disease states. To this end, it is essential to first characterize the variations of gene expression in normal physiological processes. We established the Human Gene Expression (HuGE) Index database (www.HugeIndex.org) to serve as a public repository for gene expression data on normal human tissues using high-density oligonucleotide arrays. This resource currently contains the results of 59 gene expression experiments on 19 human tissues. We provide interactive tools for researchers to query and visualize our data over the Internet. To facilitate data analysis, we cross-reference each gene on the array with its annotation in the LocusLink database at NCBI.

Using gene expression ratios to predict outcome among patients with mesothelioma.

BACKGROUND: We have recently demonstrated that simple ratios of the expression levels of selected genes in tumor samples can be used to distinguish among types of thoracic malignancies. We examined whether this technique could predict treatment-related outcome for patients with mesothelioma. METHODS: We used gene expression profiling data previously collected from 17 mesothelioma patients with different overall survival times to define two outcome-related groups of patients and to train an expression ratio-based outcome predictor model. A Student's t test was used to identify genes among the two outcome groups that had statistically significant, inversely correlated expression levels; those genes were used to form prognostic expression ratios. We used a combination of several highly accurate expression ratios and cross-validation techniques to assess the internal consistency of this predictor model, quantitative reverse transcription-polymerase chain reaction of tumor RNA to confirm the microarray data, and Kaplan-Meier survival analysis to validate the model among an independent set of 29 mesothelioma tumors. All statistical tests were two-sided. RESULTS: We developed an expression ratio-based test capable of identifying 100% (17/17) of the samples used to train the model. This test remained highly accurate (88%, 15/17) after cross-validation. A four-gene expression ratio test statistically significantly (P =.0035) predicted treatment-related patient outcome in mesothelioma independent of the histologic subtype of the tumor. CONCLUSIONS: Gene expression ratio-based analysis accurately predicts treatment-related outcome in mesothelioma samples. This technique could impact the clinical treatment of mesothelioma by allowing the preoperative identification of patients with widely divergent prognoses.

Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma.

The pathological distinction between malignant pleural mesothelioma (MPM)and adenocarcinoma (ADCA) of the lung can be cumbersome using established methods. We propose that a simple technique, based on the expression levels of a small number of genes, can be useful in the early and accurate diagnosis of MPM and lung cancer. This method is designed to accurately distinguish between genetically disparate tissues using gene expression ratios and rationally chosen thresholds. Here we have tested the fidelity of ratio-based diagnosis in differentiating between MPM and lung cancer in 181 tissue samples (31 MPM and 150 ADCA). A training set of 32 samples (16 MPM and 16 ADCA) was used to identify pairs of genes with highly significant, inversely correlated expression levels to form a total of 15 diagnostic ratios using expression profiling data. Any single ratio of the 15 examined was at least 90% accurate in predicting diagnosis for the remaining 149 samples (e.g., test set). We then examined (in the test set) the accuracy of multiple ratios combined to form a simple diagnostic tool. Using two and three expression ratios, we found that the differential diagnoses of MPM and lung ADCA were 95% and 99% accurate, respectively. We propose that using gene expression ratios is an accurate and inexpensive technique with direct clinical applicability for distinguishing between MPM and lung cancer. Furthermore, we provide evidence suggesting that this technique can be equally accurate in other clinical scenarios.

Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.

BACKGROUND: Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions, ES cells can differentiate into specialized cells, including cardiac myocytes, but the efficiency is typically low and the process is incompletely understood. METHODS AND RESULTS: We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific alpha-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP), 880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid, also known as vitamin C, markedly increased the number of EGFP-positive cells, which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and alpha-actinin. Furthermore, ascorbic acid induced the expression of cardiac genes, including GATA4, alpha-MHC, and beta-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine, Tiron, or vitamin E. CONCLUSIONS: Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells.

Expression profiling of substantia nigra in Parkinson disease, progressive supranuclear palsy, and frontotemporal dementia with parkinsonism.

BACKGROUND: Parkinson disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra. Genes contributing to rare mendelian forms of PD have been identified, but the genes involved in the more common idiopathic PD are not well understood. OBJECTIVES: To identify genes important to PD pathogenesis using microarrays and to investigate their potential to aid in diagnosing parkinsonism. DESIGN: Microarray expression analysis of postmortem substantia nigra tissue. PATIENTS: Substantia nigra samples from 14 unrelated individuals were analyzed, including 6 with PD, 2 with progressive supranuclear palsy, 1 with frontotemporal dementia with parkinsonism, and 5 control subjects. MAIN OUTCOME MEASURES: Identification of genes significantly differentially expressed (P<.05) using Affymetrix U133A microarrays. RESULTS: There were 142 genes that were significantly differentially expressed between PD cases and controls and 96 genes that were significantly differentially expressed between the combined progressive supranuclear palsy and frontotemporal dementia with parkinsonism cases and controls. The 12 genes common to all 3 disorders may be related to secondary effects. Hierarchical cluster analysis after exclusion of these 12 genes differentiated 4 of the 6 PD cases from progressive supranuclear palsy and frontotemporal dementia with parkinsonism. CONCLUSIONS: Four main molecular pathways are altered in PD substantia nigra: chaperones, ubiquitination, vesicle trafficking, and nuclear-encoded mitochondrial genes. These results correlate well with expression analyses performed in several PD animal models. Expression analyses have promising potential to aid in postmortem diagnostic evaluation of parkinsonism.

Regulation of expression of the stress response gene, Osp94: identification of the tonicity response element and intracellular signalling pathways.

Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5'-flanking region of the gene. Sequence analysis of the proximal 4 kb 5'-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus TonE/ORE (tonicity-response element/osmotic response element) as well as the consensus HSE (heat-shock element). Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE; 5'-TGGAAAGGACCAG-3') and HSE enhanced reporter gene expression under hypertonic stress and heat-shock stress respectively. Electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the Osp94-TonE probe, probably representing binding of TonEBP (TonE binding protein) to this enhancer element. Furthermore, treatment of mIMCD3 cells with MAPK (mitogen-activated protein kinase) inhibitors (SB203580, PD98059, U0126 and SP600125) and a proteasome inhibitor (MG132) suppressed the increase in Osp94 gene expression caused by hypertonic NaCl. These results indicate that the 5'-flanking region of Osp94 gene contains a hypertonicity sensitive cis -acting element, Osp94-TonE, which is distinct from a functional HSE. Furthermore, the MAPK and proteasome systems appear to be, at least in part, involved in hypertonic-stressmediated regulation of Osp94 through Osp94-TonE.

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

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.

Molecular signature of late-stage human ALS revealed by expression profiling of postmortem spinal cord gray matter.

Little is known about global gene expression patterns in the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). To address this, we used high-density oligonucleotide microarray technology to compare expression levels of approximately 6,800 genes in postmortem spinal cord gray matter obtained from individuals with ALS as well as normal individuals. Using Fisher discriminant analysis (FDA) and leave-one-out cross-validation (LOOCV), we discerned an ALS-specific signature. Moreover, it was possible to distinguish familial ALS (FALS) from sporadic ALS (SALS) gene expression profiles. Characterization of the specific genes significantly altered in ALS uncovered a pro-inflammatory terminal state. Moreover, we found alterations in genes involved in mitochondrial function, oxidative stress, excitotoxicity, apoptosis, cytoskeletal architecture, RNA transcription and translation, proteasomal function, and growth and signaling. It is apparent from this study that DNA microarray analysis and appropriate bioinformatics can reveal distinct phenotypic changes that underlie the terminal stages of neurodegeneration in ALS.