ACTH (Acthar Gel) Reduces Toxic SOD1 Protein Linked to Amyotrophic Lateral Sclerosis in Transgenic Mice: A Novel Observation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a complex etiology and pathology that makes the development of new therapies difficult. ACTH has neurotrophic and myotrophic effects, but has not been tested in an ALS mouse model. The G93A-SOD1 mouse model of ALS was used to test the ability of this drug to delay ALS-like symptoms. We showed that within a specific dose range, ACTH significantly postponed the disease onset and paralysis in the mouse model. To our surprise and of greater significance is that ACTH significantly reduced the levels of soluble SOD1 in the spinal cord and CNS tissues of G93A-SOD1 treated mice as well as cultured fibroblasts.

Increased mitochondrial fission and volume density by blocking glutamate excitotoxicity protect glaucomatous optic nerve head astrocytes.

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation. GLIA 2015;63:736-753.

Pharmacodynamics of potassium channel openers in cultured neuronal networks.

A novel class of drugs - potassium (K(+)) channel openers or activators - has recently been shown to cause anticonvulsive and neuroprotective effects by activating hyperpolarizing K(+) currents, and therefore, may show efficacy for treating tinnitus. This study presents measurements of the modulatory effects of four K(+) channel openers on the spontaneous activity and action potential waveforms of neuronal networks. The networks were derived from mouse embryonic auditory cortices and grown on microelectrode arrays. Pentylenetetrazol was used to create hyperactivity states in the neuronal networks as a first approximation for mimicking tinnitus or tinnitus-like activity. We then compared the pharmacodynamics of the four channel activators, retigabine and flupirtine (voltage-gated K(+) channel KV7 activators), NS1619 and isopimaric acid ("big potassium" BK channel activators). The EC50 of retigabine, flupirtine, NS1619, and isopimaric acid were 8.0, 4.0, 5.8, and 7.8µM, respectively. The reduction of hyperactivity compared to the reference activity was significant. The present results highlight the notion of re-purposing the K(+) channel activators for reducing hyperactivity of spontaneously active auditory networks, serving as a platform for these drugs to show efficacy toward target identification, prevention, as well as treatment of tinnitus.

Discovery of 1,3,4-oxidiazole scaffold compounds as inhibitors of superoxide dismutase expression.

The treatment of neurodegenerative diseases is difficult because of multiple etiologies and the interplay of genetics and environment as precipitating factors. In the case of amyotrophic lateral sclerosis (ALS), we have knowledge of a handful of genes that cause disease when mutated. However, drugs to counteract the effect of genetic mutations have not yet been found. One of the causative genes, Cu, Zn-superoxide dismutase (SOD1) is responsible for about 10-15% of the genetically linked autosomal dominant disease. Our rationale was that compounds that reduce expression of the mutant protein would be beneficial to slow onset and/or disease progression. We screened candidate compounds using a cell-based in vitro assay for those that reduce mutant SOD1 (G93A) protein expression. This led to the discovery of 2-[3-iodophenyl)methylsulfanyl]-5pyridin-4-yl-1,3,4-oxadiazole, a known protein kinase inhibitor that decreases G93A-SOD1 expression in vitro and in the brain and spinal cord in vivo. However, this compound has a biphasic dose response curve and a likely toxophore which limit its therapeutic window for chronic disease such as ALS. Therefore, we designed and tested a focused library of analogs for their ability to decrease SOD1 expression in vitro. This exercise resulted in the identification of a lead compound with improved drug-like characteristics and activity. Development of small molecules that reduce the expression of etiologically relevant toxic proteins is a strategy that may also be extended to familial ALS linked to gain of function mutations in other genes.

Kcnq1-5 (Kv7.1-5) potassium channel expression in the adult zebrafish.

BACKGROUND: KCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ1), epilepsy in adults (KCNQ2/3), benign familial neonatal convulsions in children (KCNQ3), and hearing loss or tinnitus in humans (KCNQ4, but not KCNQ5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues. RESULTS: Overall expression of the kcnqx channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues. CONCLUSIONS: We conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus.

Tumor-suppressive maspin functions as a reactive oxygen species scavenger: importance of cysteine residues.

Maspin is a member of the serine protease inhibitor (serpin) superfamily and displays tumor-suppressing activity by controlling cell migration, proliferation, apoptosis, and adhesion. Here, we provide evidence that maspin acts as a reactive oxygen species (ROS) scavenger through oxidation of three structurally exposed cysteine thiols to sulfenic acid. Ablation of these cysteine residues in maspin resulted in a significant increase in total ROS production in mouse mammary TM40D cells. Also, cells containing a triple-cysteine mutant of maspin showed elevated ERK1/2 activity, a downstream target of ROS, and enhanced proliferation and colony formation. These findings establish a novel mechanism by which maspin utilizes its cysteine thiols to inhibit oxidative stress and cell growth.

Isolation and culture of astrocytes from the retina and optic nerve.

Detailed methods for the preparation of optic nerve head (ONH) astrocytes from human donor eyes and retinal astrocytes from rat eyes are described. Included is the immunopanning method used for ONH astrocyte isolation as well as cell characterization. The isolation of purified retinal astrocytes is outlined as a method applicable to rodent and other mammalian retinas.

An in vitro model for testing drugs to treat tinnitus.

Tinnitus affects approximately 50 million people in the USA alone, with 10 million being highly debilitated. Pharmacotherapy for tinnitus is still in emerging stages due to time consuming clinical trials and/or animal experiments. We tested a new cellular model where induced rapid neuronal firing or spiking was used as a mimic for the type of aberrant activity that may occur in tinnitus. Spontaneously active auditory cortical networks growing on microelectrode arrays were exposed to pentylenetetrazol (PTZ), a proconvulsant and an antagonist of GABA(A) receptor, which is implicated in tinnitus. Auditory cortical networks were then exposed to experimental tinnitus drugs linopirdine (Dup966, a potassium channel blocker), L-carnitine (an antioxidant), or selective Ca(2+) channel antagonists pregabalin (Lyrica), or gabapentin (Neurontin) at various concentrations. PTZ increased spike rate by 139.6±27% and burst rate by 129.7±28% in auditory cortical networks with a phenotypic high firing of excitable neurons. Reductions of increased activity were observed to varying degrees using the experimental tinnitus drugs. The potency of the drugs was linopirdine (EC(50): 176±7.0 µM)>L-carnitine (EC(50): 1569±41 µM)>pregabalin (EC(50): 8360±340 µM), >gabapentin, with 34.2±7.5% efficacy (EC(50): 2092±980 µM). These studies provide proof of principle for the use of auditory cortical networks on microelectrode array as a feasible platform for semi-high throughput application for screening of drugs that might be used for the treatment of tinnitus.

Modulation of factors affecting optic nerve head astrocyte migration.

PURPOSE: The authors investigated the role of myosin light chain kinase (MYLK) and transforming growth factor beta (TGFbeta) receptor pathways in optic nerve head (ONH) astrocyte migration. They further investigated how the expression of these genes is altered by elevated hydrostatic pressure (HP). METHODS: PCR was used to determine the isoforms of MYLK expressed in ONH astrocytes. siRNAs against MYLK (all isoforms) and TGFbeta receptor 2 (TGFBR2) were prepared and tested for effects on the migration of cultured ONH astrocytes. Finally, the effects of elevated HP (24-96 hours) on the expression of MYLK isoforms and selected TGFbeta pathway components were measured. RESULTS: Multiple isoforms of MYLK are present in ONH astrocytes from Caucasian (CA) and African American (AA) donors. Both populations express the short form (MYLK-130) and the long form (MYLK-210) of MYLK and a splicing variant within MYLK-210. MYLK-directed siRNA decreased MYLK expression and cell migration compared with control siRNA. siRNA directed against TGFbeta receptor 2 also decreased cell migration compared with control and decreased extracellular matrix genes regulated by TGFbeta signaling. Elevated HP increased the expression of MYLK-130 and MYLK-210 in both populations of astrocytes. However, TGFbeta2 was uniquely upregulated by exposure to elevated HP in CA compared with AA astrocytes. CONCLUSIONS: Differential expression of TGFbeta pathway genes and MYLK isoforms observed in populations of glaucomatous astrocytes applies to the elevated HP model system. MYLK may be a new target for intervention in glaucoma to alter reactive astrocyte migration in the ONH.

Age-related increase in mitochondrial DNA damage and loss of DNA repair capacity in the neural retina.

With age, there is increased mitochondrial DNA (mtDNA) damage in the central nervous system (CNS) that may underlie, contribute or increase the susceptibility to certain neurodegenerative diseases. We examined retinas from the eyes of young and old rodents for mtDNA damage and for changes in selected DNA repair enzymes. We found increased levels of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) by immunohistochemical labeling for the oxidative DNA damage marker in aged rodent retinas, which was confirmed by quantitative ELISA. 8-OHdG co-localized with the mitochondrial enzyme superoxide dismutase (MnSOD), suggesting damage to mtDNA. Most of the damaged mtDNA was in the photoreceptors and retinal ganglion cells. Measurements of nuclear DNA (nDNA) and mtDNA lesions indicated that DNA damage was primarily in mtDNA in aged retinas. The increased damage to mtDNA may be due to decreased levels of DNA repair enzymes in the aged retina. Using qPCR, Western blots and immunohistochemistry, we determined the levels of DNA repair enzymes for oxidative damage. In retinas from old eyes compared to retinas from young eyes, we found decreased levels of poly (ADP-ribose) polymerase 1 (PARP1), mutY homolog (MYH) and endonuclease III homologue 1 (NTH1). Our results suggest that normal, age-related, increased mtDNA damage, likely due to decreased repair capacity in aged retinas, may be a susceptibility factor that underlies age-related retinal diseases.

Using LC3 to monitor autophagy flux in the retinal pigment epithelium.

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation. Autophagy begins with the formation of membranes, arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. However, few studies of autophagy in retinal disease have been reported.

Hydrostatic pressure-dependent changes in cyclic AMP signaling in optic nerve head astrocytes from Caucasian and African American donors.

PURPOSE: Investigate the effect of hydrostatic pressure (HP) on 3', 5'-cyclic adenosine monophosphate (cAMP) levels and downstream signaling in cultures of normal optic nerve head (ONH) astrocytes from Caucasian American (CA) and African American (AA) donors. METHODS: Intracellular cAMP levels were assayed after exposing ONH astrocytes to HP for varying times. Quantitative RT-PCR was used to determine the expression levels of selected cAMP pathway genes in human ONH astrocytes after HP treatment. Western blots were used to measure changes in the phosphorylation state of cAMP response element binding protein (CREB) in astrocytes subjected to HP, ATP, and phosphodiesterase or kinase inhibitors. RESULTS: The basal intracellular cAMP level is similar among AA and CA astrocytes. After exposure to HP for 15 min and 30 min in the presence of a phosphodiesterase inhibitor a further increase of intracellular cAMP was observed in AA astrocytes, but not in CA astrocytes. Consistent with activation of the cAMP-dependent protein kinase pathway, CREB phosphorylation (Ser-133) was increased to a greater extent in AA than in CA astrocytes after 3 h of HP. Exposure to elevated HP for 3-6 h differentially altered the expression levels of selected cAMP pathway genes (ADCY3, ADCY9, PTHLH, PDE7B) in AA compared to CA astrocytes. Treatment with ATP increased more CREB phosphorylation in CA than in AA astrocytes, suggesting differential Ca(2+) signaling in these populations. CONCLUSIONS: Activation of the cAMP-dependent signaling pathway by pressure may be an important contributor to increased susceptibility to elevated intraocular pressure and glaucoma in AA, a population at higher risk for the disease.

Changes in retinal pigment epithelium related to cigarette smoke: possible relevance to smoking as a risk factor for age-related macular degeneration.

Age-related Macular Degeneration (AMD) is a major cause of central vision loss in the elderly and smoking is a primary risk factor associated with the prevalence and incidence of AMD. To better understand the cellular and molecular bases for the association between smoking and AMD, we determined the effects of Benzo(a)Pyrene (B(a)P), a toxic element in cigarette smoke, on cultured retinal pigment epithelia (RPE) and we examined the RPE/choroid from mice exposed to chronic cigarette smoke. We measured: mitochondrial DNA (mtDNA) damage, phagocytic activity, lysosomal enzymes, exosome markers and selected complement pathway components. In the presence of a non-cytotoxic dose of B(a)P, there was extensive mtDNA damage but no nuclear DNA damage. RPE phagocytic activity was not altered but there were increased lysosomal activity, exocytotic activity and complement pathway components. Retinas from mice exposed to cigarette smoke contained markers for mtDNA damage, exosomes and complement pathway components surrounding Bruch's membrane. Markers for these processes are found in drusen from AMD patients. Thus, smoking may cause damage to mtDNA and increased degradative processes in the RPE. These altered cell biological processes in the RPE may contribute to the formation of drusen in individuals who are cigarette smokers and underlie susceptibility to genetic mutations associated with AMD.

Early cellular signaling responses to axonal injury.

BACKGROUND: We have used optic nerve injury as a model to study early signaling events in neuronal tissue following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury. RESULTS: We found evidence of cell to cell signaling within 30 min of axonal injury. We detected differences in phosphoproteins and gene expression within the 6 hrs time period. Activation of TNFalpha and glutamate receptors, two pathways that can initiate cell death, begins in RGCs within 6 hrs following axonal injury. Differential gene expression at 6 hrs post injury included genes involved in cytokine, neurotrophic factor signaling (Socs3) and apoptosis (Bax). CONCLUSION: We interpret our studies to indicate that both neurons and glia in the retina have been signaled within 30 min after optic nerve injury. The signals are probably initiated by the RGC soma. In addition, signals activating cellular death pathways occur within 6 hrs of injury, which likely lead to RGC degeneration.

Autophagy, exosomes and drusen formation in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch's membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch's membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.

Selectivity in the post-translational, transglutaminase-dependent acylation of lysine residues.

Transglutaminases (TGs) are known to exhibit remarkable specificities not only for the Q (or Gln) sites but also for the K (or Lys) sites of proteins with which they react. To gain further insight into K-site specificity, we examined the reactions of dansyl-epsilon-aminocaproyl-GlnGlnIleVal with three chemically and structurally well-characterized proteins (bovine pancreatic ribonuclease A, bovine pancreatic trypsin inhibitor, and chicken egg white lysozyme), as catalyzed by TG2, a biologically important post-translational enzyme. The substrates represent a total of 20 potential surface sites for acylation by the fluorescent Gln probe, yet only two of the lysine side chains reacted with TG2. While the K1 site of ribonuclease and the K15 site of the trypsin inhibitor could be readily acylated by the enzyme, none of the lysines in lysozyme were modified. The findings lead us to suggest that the selection of lysine residues by TG2 is not encoded in the primary amino acid sequence surrounding the target side chain but depends primarily on its being positioned in an accessible segment of the protein structure.

Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch's membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch's membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.

Dysfunction of the retinal pigment epithelium with age: increased iron decreases phagocytosis and lysosomal activity.

PURPOSE: Iron accumulation with age in the retinal pigment epithelium (RPE) may be one important source of oxidative stress that contributes to age-related macular degeneration (AMD). Young and old rodent RPE/choroid were compared to assess iron homeostasis during normal aging and the effects of increased iron on the functions of retinal pigment epithelial cells. METHODS: The iron level, mRNA expression, and protein level of iron-regulatory molecules in RPE/choroid were quantitatively compared between young and old animals. To test the effects of increased intracellular iron on the functions of retinal pigment epithelial cells, in vitro ARPE-19 cells were treated with high levels of iron and assessed for phagocytosis activity and lysosomal activity. RESULTS: Iron level was significantly increased in the aged RPE/choroid. Ferritin and ceruloplasmin mRNAs were significantly increased in the aged RPE/choroid, whereas transferrin, transferrin receptor, and ferroportin mRNAs did not change with age. At the protein level, decreased transferrin and transferrin receptor, increased ferritin and ceruloplasmin, and unchanged ferroportin were observed in the aged RPE/choroid. Exposure of ARPE-19 cells to increased iron markedly decreased phagocytosis activity, interrupted cathepsin D processing, and reduced cathepsin D activity in retinal pigment epithelial cells. CONCLUSIONS: The RPE/choroid of aged animals demonstrates iron accumulation and associated alterations in iron homeostasis. Iron accumulation with age may impair the phagocytosis and lysosomal functions of retinal pigment epithelial cells in the aged RPE/choroid. Therefore, age-related changes of iron homeostasis in the RPE could increase the susceptibility of the tissue to genetic mutations associated with AMD.

Changes in iron-regulatory proteins in the aged rodent neural retina.

Iron accumulation is associated with age-related neurodegenerations and may contribute to age-related increased susceptibility of neurons to damage. We compared young and old rodent retinas to assess iron homeostasis during normal aging and the effects of increased iron on the susceptibility of retinal neurons to degeneration. Retinal iron was significantly increased with age. Quantitative RT-PCR showed that transferrin and ferritin genes were upregulated in the aged retina. At the protein level, we found decreased transferrin, and increased transferrin receptor, ferritin, ferroportin, and ceruloplasmin in the aged retina. These results support an increased steady state of iron with age in the retina. We tested susceptibility of retinal neurons with increased intracellular iron to damage in vitro. Exposure of RGC-5 cells to increased iron potentiated the neurotoxicity induced by paraquat, glutamate, and TNFalpha. Our results demonstrate that iron homeostasis in the retina is altered with age and suggest that iron accumulation, due to altered levels of iron-regulatory proteins in the aged retina, could be a susceptibility factor in age-related retinal diseases.

Gene expression and functional studies of the optic nerve head astrocyte transcriptome from normal African Americans and Caucasian Americans donors.

PURPOSE: To determine whether optic nerve head (ONH) astrocytes, a key cellular component of glaucomatous neuropathy, exhibit differential gene expression in primary cultures of astrocytes from normal African American (AA) donors compared to astrocytes from normal Caucasian American (CA) donors. METHODS: We used oligonucleotide Affymetrix microarray (HG U133A & HG U133A 2.0 chips) to compare gene expression levels in cultured ONH astrocytes from twelve CA and twelve AA normal age matched donor eyes. Chips were normalized with Robust Microarray Analysis (RMA) in R using Bioconductor. Significant differential gene expression levels were detected using mixed effects modeling and Statistical Analysis of Microarray (SAM). Functional analysis and Gene Ontology were used to classify differentially expressed genes. Differential gene expression was validated by quantitative real time RT-PCR. Protein levels were detected by Western blots and ELISA. Cell adhesion and migration assays tested physiological responses. Glutathione (GSH) assay detected levels of intracellular GSH. RESULTS: Multiple analyses selected 87 genes differentially expressed between normal AA and CA (P<0.01). The most relevant genes expressed in AA were categorized by function, including: signal transduction, response to stress, ECM genes, migration and cell adhesion. CONCLUSIONS: These data show that normal astrocytes from AA and CA normal donors display distinct expression profiles that impact astrocyte functions in the ONH. Our data suggests that differences in gene expression in ONH astrocytes may be specific to the development and/or progression of glaucoma in AA.