Purification of Retinal Ganglion Cells from Differentiation Through Adult via Immunopanning and Low-Pressure Flow Cytometry.

The isolation and culturing of rodent retinal ganglion cells (RGC) is a key step in studying the function and cellular response of this crucial cell type. Typical methods used for isolation of RGCs include immunopanning or magnetic bead separation with antibodies targeting RGC specific protein markers. However, in developmental research, many of the most common markers, such as Thy-1, are not expressed in early stages of development. To help study these crucial early stage RGCs, we have developed a novel method that utilizes a transgenic mouse with a GFP tag on the protein BRN3 and a low-pressure fluorescence-activated cell sorter (FACS) system.

N-acylethanolamide metabolizing enzymes are upregulated in human neural progenitor-derived neurons exposed to sub-lethal oxidative stress.

N-acyl amides (NAAs) are a class of lipids that consist of an acyl group N-linked to an amino acid, neurotransmitter, taurine or ethanolamide group (N-acylethanolamines or NAEs) and include some endocannabinoids (eCB) such as anandamide. These lipids are synthesized in a wide variety of organisms and in multiple cell types, including neurons. NAEs are involved in numerous cellular and physiological processes and their concentrations are elevated in response to ischemia and physical trauma to play a role in neuroprotection. The neuroprotective properties of eCB NAEs make the protein targets of these compounds attractive targets for clinical intervention for a variety of conditions. The most promising of these targets include cannabinoid receptor type 1 (CB1), cannabinoid receptor type 2 (CB2), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA), and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD). Further characterization of these targets in a more contemporary model system of neurodegeneration and neuroprotection will allow us to fully describe their role and mechanism of action in neuroprotection against oxidative stress leading to better utilization in the clinical setting. Human stem cell-derived or human neural progenitor cell-derived cells, such as ReN cells, have become more utilized for the study of human neuronal development and neurodegenerative diseases. ReN cells can be easily differentiated thereby circumventing the need for using transformed cell lines and primary neurons as cell model systems. In this study, we determined whether ReN cells, a superior cell model system for studying neurodevelopment, differentiation, and neuroprotection, express proteins involved in canonical eCB NAE signaling and whether oxidative stress can induce their expression. We determined that sublethal oxidative stress upregulates the expression of all eCB proteins tested. In addition, we determined that oxidative stress increases the nuclear localization of FAAH, and to a lesser extent, NAAA and NAPE-PLD. This study is a first step toward determining how oxidative stress affects CB1, CB2, FAAH, NAAA, and NAPE-PLD expression and their potential defense against oxidative stress. As such, our data is important for further determining the role of eCB metabolizing proteins and eCB receptors against oxidative stress.

Analysis of Glaucoma Associated Genes in Response to Inflammation, an Examination of a Public Data Set Derived from Peripheral Blood from Patients with Hepatitis C.

Introduction: Glaucoma is the second leading cause of blindness worldwide and despite its prevalence, there are still many unanswered questions related to its pathogenesis. There is evidence that oxidative stress and inflammation play a major role in disease progression. Glaucoma patients from several studies showed altered gene expression in leukocytes, revealing the possibility of using peripheral biomarkers to diagnose or stage glaucoma. The fact that glaucoma is associated with gene expression changes in tissues distant from the retina underscores the possible involvement of systemic oxidative stress and inflammation as potential contributing or compounding factors in glaucoma. Methods: We assembled a list of oxidative stress and inflammatory markers related to glaucoma based on a review of the literature. In addition, we utilized publicly available data sets of gene expression values collected from peripheral blood mononuclear cells and macrophages from two patient groups: those chronically infected by the hepatitis C virus and those who have cleared it. Activation of the innate immune response can render cells or tissues more responsive to a second delayed proinflammatory stimulus. Additional gene expression data from these cells after subsequent polyinosinic:polycytidylic acid treatment, used to elicit an acute inflammatory response, allowed for the investigation of the acute inflammatory response in these groups. We used fold-change comparison values between the two patient groups to identify genes of interest. Results: A comparison analysis identified 17 glaucoma biomarkers that were differentially expressed in response to HCV-mediated inflammation. Of these 17, six had significant p-values in the baseline vs treated values. Expression data of these genes were compared between patients who had cleared the Hepatitis C virus versus those who had not and identified three genes of interest for further study. Discussion: These results support our hypothesis that inflammation secondary to Hepatitis C virus infection affects the expression of glaucoma biomarker genes related to the antioxidant response and inflammation. In addition, they provide several potential targets for further research into understanding the relationship between innate responses to viral infection and inflammatory aspects of glaucoma and for potential use as a predictive biomarker or pharmacological intervention in glaucoma.

The efficacy and tolerability of auto-stimulation-VNS in children with Lennox-Gastaut syndrome.

OBJECTIVE: Lennox-Gastaut syndrome (LGS) is a severe drug-resistant epilepsy (DRE) of childhood. The Vagus Nerve Stimulator (VNS) is established as a safe and effective treatment for DRE. This study assesses efficacy and tolerability of the auto-stimulation VNS models in pediatric patients with LGS. METHODS: This is a retrospective chart review of a cohort of pediatric patients (Age 1-18 years old) with LGS implanted with an auto-stimulation VNS model at a single level four pediatric epilepsy center. Patient responder's rate was measured as seizure reduction over baseline and improvements in five quality-of-life measures as reported by the patients and families. Efficacy and tolerability were assessed at 1, 3, 6, 12, 18 and 24 months compared to baseline. RESULTS: This cohort includes 71 consecutive children with Lennox-Gastaut syndrome who underwent implantation with one of two models of the auto-stimulation VNS. The average age of the children at implantation was 20.82 months. Of those patients, 55 % of patients achieved greater than 50 % seizure reduction at six months, 67.7 % at 12 months, and 65 % at 24 months. At 12 months 11 % of the patients were completely seizure free and at 24 months 17 % were seizure free. By 24 months post implantation most of the patient families reported at least a 50 % improvement rate in one or more of the quality-of-life measures. The most commonly reported adverse events were dysphonia, paresthesia, and shortness of breath, all of which were tolerated and subsided by 24 months. SIGNIFICANCE: This study provides evidence that VNS models with the auto-stimulation paradigm based on detection of tachycardia are well tolerated and effective in a pediatric population with LGS. Furthermore, this study shows that for this population, the auto-stimulation models of the VNS may provide additional benefits over the earlier VNS versions.

The Contribution of Anterior Segment Abnormalities to Changes in Intraocular Pressure in the DBA/2J Mouse Model of Glaucoma: DBA/2J-Gpnmb +/SjJ Mice as Critical Controls.

The contributions of anterior segment abnormalities to the development of ocular hypertension was determined in the DBA/2J mouse model of glaucoma. Intraocular pressure (IOP) was measured non-invasively. Iris pigment dispersion (IPD) and corneal calcification were measured weekly starting at 20 weeks of age in DBA/2J and DBA/2J-Gpnmb +/SjJ mice. Thickness, surface area, auto-fluorescence intensity, and perimeter length of calcified regions were measured in postmortem corneas using confocal microscopy. DBA/2J mice developed elevated IOP between 9 and 12 months of age, but DBA/2J-Gpnmb +/SjJ mice did not. Corneal calcification was found at all ages observed and at similar frequencies in both strains with 83.3% of DBA/2J eyes and 60.0% of DBA/2J-Gpnmb +/SjJ eyes affected at 12 months (P = 0.11). Calcification increased with age in both DBA/2J (P = 0.049) and DBA/2J-Gpnmb +/SjJ mice (P = 0.04) when assessed qualitatively and based on mixed-effects analysis. No differences in the four objective measures of calcification were observed between strains or ages. At 12 months of age, DBA/2J mice with corneal calcification had greater mean IOP than DBA/2J mice without corneal calcification. IOP was not correlated with the qualitatively assessed measures of calcification. For the subset of eyes with ocular hypertension, which were only found in DBA/2J mice, IOP was negatively correlated with the qualitative degree of calcification, but was not correlated with the four quantitative measures of calcification. Differences in IOP were not observed between DBA/2J-Gpnmb +/SjJ mice with and without calcification at any age. IPD increased with age and demonstrated a moderate correlation with IOP in DBA/2J mice, but was not observed in DBA/2J-Gpnmb +/SjJ mice. In the DBA/2J mouse model of glaucoma, increased IPD is positively correlated with an increase in IOP and corneal calcification is present in the majority of eyes at and after age 9 months. However, while IPD causes ocular hypertension, corneal calcification does not appear to contribute to the elevation of IOP, as the control strain DBA/2J-Gpnmb +/SjJ exhibits corneal calcification similar to DBA/2J mice, but does not develop ocular hypertension. Corneal calcification, therefore, does not appear to be a contributing factor to the development of elevated IOP in DBA/2J mice.

RNA Sequencing of Human Peripheral Nerve in Response to Injury: Distinctive Analysis of the Nerve Repair Pathways.

The development of regenerative therapies for central nervous system diseases can likely benefit from an understanding of the peripheral nervous system repair process, particularly in identifying potential gene pathways involved in human nerve repair. This study employed RNA sequencing (RNA-seq) technology to analyze the whole transcriptome profile of the human peripheral nerve in response to an injury. The distal sural nerve was exposed, completely transected, and a 1 to 2 cm section of nerve fascicles was collected for RNA-seq from six participants with Parkinson's disease, ranging in age between 53 and 70 yr. Two weeks after the initial injury, another section of the nerve fascicles of the distal and pre-degenerated stump of the nerve was dissected and processed for RNA-seq studies. An initial analysis between the pre-lesion status and the postinjury gene expression revealed 3,641 genes that were significantly differentially expressed. In addition, the results support a clear transdifferentiation process that occurred by the end of the 2-wk postinjury. Gene ontology (GO) and hierarchical clustering were used to identify the major signaling pathways affected by the injury. In contrast to previous nonclinical studies, important changes were observed in molecular pathways related to antiapoptotic signaling, neurotrophic factor processes, cell motility, and immune cell chemotactic signaling. The results of our current study provide new insights regarding the essential interactions of different molecular pathways that drive neuronal repair and axonal regeneration in humans.

Review of bilirubin neurotoxicity I: molecular biology and neuropathology of disease.

Despite the availability of successful prevention strategies to prevent excessive hyperbilirubinemia, the neurological sequelae of bilirubin neurotoxicity (BNTx) still occur throughout the world. Kernicterus, encephalopathy due to BNTx, is now understood to be a spectrum of severity and phenotypes known as kernicterus spectrum disorder (KSD). A better understanding of the selective neuropathology and molecular biology of BNTx and using consistent clinical definitions of KSDs as outcome measure can lead to more accurately predicting the risk and causes of BNTx and KSDs. In Part I of our two-part review, we will summarize current and recent advances in the understanding of the selective neuropathology and molecular biology of the disease. Herein we emphasize the role of unbound, free unconjugated bilirubin as well as genetic contributions to the susceptibility BNTx and the development of KSDs. In Part II, we focus on current and possible novel methods to prevent BNTx and ABE and treat ABE and KSDs.

Review of bilirubin neurotoxicity II: preventing and treating acute bilirubin encephalopathy and kernicterus spectrum disorders.

Previously in Part I of this two-part review, we discussed the current and recent advances in the understanding of the molecular biology and neuropathology of bilirubin neurotoxicity (BNTx). Here in Part II, we summarize current treatment options available to treat the severely jaundiced infants to prevent significant brain damage and improve clinical outcomes. In addition, we review potential novel therapies that are in various stages of research and development. We will emphasize treatments for both prevention and treatment of both acute bilirubin encephalopathy (ABE) and kernicterus spectrum disorders (KSDs), highlighting the treatment of the most disabling neurological sequelae of children with mild-to-severe KSDs whose "rare disease" status often means they are overlooked by the clinical research community at large. As with other secondary dystonias, treatment of the dystonic motor symptoms in kernicterus is the greatest clinical challenge.

Novel Protective Role of Nicotinamide Phosphoribosyltransferase in Acetaminophen-Induced Acute Liver Injury in Mice.

Acetaminophen overdose is the most common cause of acute liver injury (ALI) or acute liver failure in the United States. Its pathogenetic mechanisms are incompletely understood. Additional studies are warranted to identify new genetic risk factors for more mechanistic insights and new therapeutic target discoveries. The objective of this study was to explore the role and mechanisms of nicotinamide phosphoribosyltransferase (NAMPT) in acetaminophen-induced ALI. C57BL/6 Nampt gene wild-type (Nampt+/+), heterozygous knockout (Nampt+/-), and overexpression (NamptOE) mice were treated with overdose of acetaminophen, followed by histologic, biochemical, and transcriptomic evaluation of liver injury. The mechanism of Nampt in acetaminophen-induced hepatocytic toxicity was also explored in cultured primary hepatocytes. Three lines of evidence have convergently demonstrated that acetaminophen overdose triggers the most severe oxidative stress and necrosis, and the highest expression of key necrosis driving genes in Nampt+/- mice, whereas the effects in NamptOE mice were least severe relative to Nampt+/+ mice. Treatment of P7C3-A20, a small chemical molecule up-regulator of Nampt, ameliorated acetaminophen-induced mouse ALI over the reagent control. These findings support the fact that NAMPT protects against acetaminophen-induced ALI.

The Neurological Sequelae of Neonatal Hyperbilirubinemia: Definitions, Diagnosis and Treatment of the Kernicterus Spectrum Disorders (KSDs).

BACKGROUND: Despite its lengthy history, the study of jaundice, hyperbilirubinemia and kernicterus suffers from a lack of clarity and consistency in the key terms used to describe both the clinical and pathophysiological nature of these conditions. For example, the term Bilirubin-induced Neurological Dysfunction (BIND) has been used to refer to all neurological sequelae caused by exposure to high levels of bilirubin, to only mild neurological sequelae, or to scoring systems that quantitate the progressive stages of Acute Bilirubin Encephalopathy (ABE). OBJECTIVE: We seek to clarify and simplify terminology by introducing, defining, and proposing new terms and diagnostic criteria for kernicterus. METHODS: We propose a systematic nomenclature based on pathophysiological and clinical criteria, presenting a logical argument for each term. Acknowledging observations that kernicterus is symptomatically broad and diverse, we propose the use of the overarching term Kernicterus Spectrum Disorders (KSDs) to encompass all the neurological sequelae of bilirubin neurotoxicity including Acute Bilirubin Neurotoxicity (ABE). We further suggest subclassification of KSDs based on the principal disabling features of kernicterus (motor, auditory). Finally, we suggest the term subtle KSD to designate a child with a history of significant bilirubin neurotoxicity with mild or subtle developmental delays. RESULTS AND CONCLUSION: We conclude with a brief description of the limited treatments currently available for KSD, thereby underscoring the importance of further research. We believe that adopting a systematic nomenclature for the spectrum of clinical consequences of hyperbilirubinemia will help unify the field and promote more effective research in both prevention and treatment of KSDs.

Fate of Neural Progenitor Cells Transplanted Into Jaundiced and Nonjaundiced Rat Brains.

High levels of bilirubin in infants can cause kernicterus, which includes basal ganglia damage and dystonia. Stem cell transplantation may be an effective treatment for this disease. In this study, we transplanted human neural progenitor cells differentiated toward propriospinal interneurons into the striatum of 20-day-old spontaneously jaundiced (jj) Gunn rats and nonjaundiced (Nj) littermates. Using immunohistochemical methods, we found that grafted cells survived and grew fibers in jj and Nj brains 3 weeks after transplantation. Grafted cells had a higher survival rate in jj than in Nj brains, suggesting that slightly elevated bilirubin may protect graft survival due to its antioxidative and immunosuppressive effects. Despite their survival, only a small portion of grafted neurons expressed GAD-6 or ChAT, which mark GABAergic and cholinergic neurons, respectively, and are the cells that we are attempting to replace in kernicterus. Thus, NPCs containing large populations of GABAergic and cholinergic neurons should be used for further study in this field.

A Hypothesis for Using Pathway Genetic Load Analysis for Understanding Complex Outcomes in Bilirubin Encephalopathy.

Genetic-based susceptibility to bilirubin neurotoxicity and chronic bilirubin encephalopathy (kernicterus) is still poorly understood. Neonatal jaundice affects 60-80% of newborns, and considerable effort goes into preventing this relatively benign condition from escalating into the development of kernicterus making the incidence of this potentially devastating condition very rare in more developed countries. The current understanding of the genetic background of kernicterus is largely comprised of mutations related to alterations of bilirubin production, elimination, or both. Less is known about mutations that may predispose or protect against CNS bilirubin neurotoxicity. The lack of a monogenetic source for this risk of bilirubin neurotoxicity suggests that disease progression is dependent upon an overall decrease in the functionality of one or more essential genetically controlled metabolic pathways. In other words, a "load" is placed on key pathways in the form of multiple genetic variants that combine to create a vulnerable phenotype. The idea of epistatic interactions creating a pathway genetic load (PGL) that affects the response to a specific insult has been previously reported as a PGL score. We hypothesize that the PGL score can be used to investigate whether increased susceptibility to bilirubin-induced CNS damage in neonates is due to a mutational load being placed on key genetic pathways important to the central nervous system's response to bilirubin neurotoxicity. We propose a modification of the PGL score method that replaces the use of a canonical pathway with custom gene lists organized into three tiers with descending levels of evidence combined with the utilization of single nucleotide polymorphism (SNP) causality prediction methods. The PGL score has the potential to explain the genetic background of complex bilirubin induced neurological disorders (BIND) such as kernicterus and could be the key to understanding ranges of outcome severity in complex diseases. We anticipate that this method could be useful for improving the care of jaundiced newborns through its use as an at-risk screen. Importantly, this method would also be useful in uncovering basic knowledge about this and other polygenetic diseases whose genetic source is difficult to discern through traditional means such as a genome-wide association study.

Application of CRISPR/Cas9 for biomedical discoveries.

The Clustered Regions of Interspersed Palindromic Repeats-Cas9 (CRISPR/Cas9), a viral defense system found in bacteria and archaea, has emerged as a tour de force genome editing tool. The CRISPR/Cas9 system is much easier to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA rather than an engineered protein as in the systems of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and meganucleases. Although it still suffers from some off-target effects, the CRISPR/Cas9 system has been broadly and successfully applied for biomedical discoveries in a number of areas. In this review, we present a brief history and development of the CRISPR system and focus on the application of this genome editing technology for biomedical discoveries. We then present concise concluding remarks and future directions for this fast moving field.

A critical role of nicotinamide phosphoribosyltransferase in human telomerase reverse transcriptase induction by resveratrol in aortic smooth muscle cells.

Aging is the predominant risk factor for cardiovascular diseases and contributes to a considerably more severe outcome in patients with acute myocardial infarction. Resveratrol, a polyphenol found in red wine, is a caloric restriction mimetic with potential anti-aging properties which has emerged as a beneficial nutraceutical for patients with cardiovascular disease. Although resveratrol is widely consumed as a nutritional supplement, its mechanism of action remains to be elucidated fully. Here, we report that resveratrol activates human nicotinamide phosphoribosyltransferase (NAMPT), SIRT4 and telomerase reverse transcriptase (hTERT) in human aortic smooth muscle cells. Similar observations were obtained in resveratrol treated C57BL/6J mouse heart and liver tissues. Resverotrol can also augment telomerase activity in both human pulmonary microvascular endothelial cells and A549 cells. Blocking NAMPT and SIRT4 expression prevents induction of hTERT in human aortic smooth muscle cells while overexpression of NAMPT elevates the telomerase activity induced by resveratrol in A549 cells. Together, these results identify a NAMPT-SIRT4-hTERT axis as a novel mechanism by which resveratrol may affect the anti-aging process in human aortic smooth muscle cells, mouse hearts and other cells. These findings enrich our understanding of the positive effects of resveratrol in human cardiovascular diseases.

ß-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)-deficient mice exhibit a close homolog of L1 (CHL1) loss-of-function phenotype involving axon guidance defects.

BACE1 is the ß-secretase enzyme that initiates production of the ß-amyloid peptide involved in Alzheimer disease. However, little is known about the functions of BACE1. BACE1-deficient mice exhibit mild but complex neurological phenotypes suggesting therapeutic BACE1 inhibition may not be completely free of mechanism-based side effects. Recently, we have reported that BACE1 null mice have axon guidance defects in olfactory sensory neuron projections to glomeruli in the olfactory bulb. Here, we show that BACE1 deficiency also causes an axon guidance defect in the hippocampus, a shortened and disorganized infrapyramidal bundle of the mossy fiber projection from the dentate gyrus to CA3. Although we observed that a classical axon guidance molecule, EphA4, was cleaved by BACE1 when co-expressed with BACE1 in HEK293 cells, we could find no evidence of BACE1 processing of EphA4 in the brain. Remarkably, we discovered that the axon guidance defects of BACE1(-/-) mice were strikingly similar to those of mice deficient in a recently identified BACE1 substrate, the neural cell adhesion molecule close homolog of L1 (CHL1) that is involved in neurite outgrowth. CHL1 undergoes BACE1-dependent processing in BACE1(+/+), but not BACE1(-/-), hippocampus, and olfactory bulb, indicating that CHL1 is a BACE1 substrate in vivo. Finally, BACE1 and CHL1 co-localize in the terminals of hippocampal mossy fibers, olfactory sensory neuron axons, and growth cones of primary hippocampal neurons. We conclude that BACE1(-/-) axon guidance defects are likely the result of abrogated BACE1 processing of CHL1 and that BACE1 deficiency produces a CHL1 loss-of-function phenotype. Our results imply the possibility that axon mis-targeting may occur in adult neurogenic and/or regenerating neurons as a result of chronic BACE1 inhibition and add a note of caution to BACE1 inhibitor development.

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.

Susceptibility to glaucoma: differential comparison of the astrocyte transcriptome from glaucomatous African American and Caucasian American donors.

BACKGROUND: Epidemiological and genetic studies indicate that ethnic/genetic background plays an important role in susceptibility to primary open angle glaucoma (POAG). POAG is more prevalent among the African-descent population compared to the Caucasian population. Damage in POAG occurs at the level of the optic nerve head (ONH) and is mediated by astrocytes. Here we investigated differences in gene expression in primary cultures of ONH astrocytes obtained from age-matched normal and glaucomatous donors of Caucasian American (CA) and African American (AA) populations using oligonucleotide microarrays. RESULTS: Gene expression data were obtained from cultured astrocytes representing 12 normal CA and 12 normal AA eyes, 6 AA eyes with POAG and 8 CA eyes with POAG. Data were normalized and significant differential gene expression levels detected by using empirical Bayesian shrinkage moderated t-statistics. Gene Ontology analysis and networks of interacting proteins were constructed using the BioGRID database. Network maps included regulation of myosin, actin, and protein trafficking. Real-time RT-PCR, western blots, ELISA, and functional assays validated genes in the networks. CONCLUSION: Cultured AA and CA glaucomatous astrocytes retain differential expression of genes that promote cell motility and migration, regulate cell adhesion, and are associated with structural tissue changes that collectively contribute to neural degeneration. Key upregulated genes include those encoding myosin light chain kinase (MYLK), transforming growth factor-beta receptor 2 (TGFBR2), rho-family GTPase-2 (RAC2), and versican (VCAN). These genes along with other differentially expressed components of integrated networks may reflect functional susceptibility to chronic elevated intraocular pressure that is enhanced in the optic nerve head of African Americans.