Publications by authors named "Robert W Nickells"

56 Publications

BclX (Bcl2l1) gene therapy lessens retinal ganglion cell soma loss but not axonal degeneration after acute axonal injury.

Cell Death Discov 2022 Jul 22;8(1):331. Epub 2022 Jul 22.

Department of Ophthalmology, Flaum Eye Institute, University of Rochester Medical Center, Rochester, NY, USA.

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http://dx.doi.org/10.1038/s41420-022-01111-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9307748PMC
July 2022

Increased Susceptibility and Intrinsic Apoptotic Signaling in Neurons by Induced HDAC3 Expression.

Invest Ophthalmol Vis Sci 2021 08;62(10):14

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison WI, United States.

Purpose: Inhibition or targeted deletion of histone deacetylase 3 (HDAC3) is neuroprotective in a variety neurodegenerative conditions, including retinal ganglion cells (RGCs) after acute optic nerve damage. Consistent with this, induced HDAC3 expression in cultured cells shows selective toxicity to neurons. Despite an established role for HDAC3 in neuronal pathology, little is known regarding the mechanism of this pathology.

Methods: Induced expression of an HDAC3-mCherry fusion protein in mouse RGCs was accomplished by transduction with AAV2/2-Pgk-HDAC3-mCherry. Increased susceptibility to optic nerve damage in HDAC3-mCherry expressing RGCs was evaluated in transduced mice that received acute optic nerve crush surgery. Expression of HDAC3-FLAG or HDAC3-mCherry was induced by nucleofection or transfection of plasmids into differentiated or undifferentiated 661W tissue culture cells. Immunostaining for cleaved caspase 3, localization of a GFP-BAX fusion protein, and quantitative RT-PCR was used to evaluate HDAC3-induced damage.

Results: Induced expression of exogenous HDAC3 in RGCs by viral-mediated gene transfer resulted in modest levels of cell death but significantly increased the sensitivity of these neurons to axonal damage. Undifferentiated 661W retinal precursor cells were resilient to induced HDAC3 expression, but after differentiation, HDAC3 induced GFP-BAX recruitment to the mitochondria and BAX/BAK dependent activation of caspase 3. This was accompanied by an increase in accumulation of transcripts for the JNK2/3 kinases and the p53-regulated BH3-only gene Bbc3/Puma. Cell cycle arrest of undifferentiated 661W cells did not increase their sensitivity to HDAC3 expression.

Conclusions: Collectively, these results indicate that HDAC3-induced toxicity to neurons is mediated by the intrinsic apoptotic pathway.
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http://dx.doi.org/10.1167/iovs.62.10.14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8375002PMC
August 2021

BCLX gene therapy moderates neuropathology in the DBA/2J mouse model of inherited glaucoma.

Cell Death Dis 2021 08 10;12(8):781. Epub 2021 Aug 10.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA.

Axonal degeneration of retinal ganglion cells (RGCs) causes blindness in glaucoma. Currently, there are no therapies that target axons to prevent them from degenerating. Activation of the BAX protein has been shown to be the determining step in the intrinsic apoptotic pathway that causes RGCs to die in glaucoma. A putative role for BAX in axonal degeneration is less well elucidated. BCLX (BCL2L1) is the primary antagonist of BAX in RGCs. We developed a mCherry-BCLX fusion protein, which prevented BAX recruitment and activation to the mitochondria in tissue culture cells exposed to staurosporine. This fusion protein was then packaged into adeno-associated virus serotype 2, which was used to transduce RGCs after intravitreal injection and force its overexpression. Transduced RGCs express mCherry-BCLX throughout their somas and axons along the entire optic tract. In a model of acute optic nerve crush, the transgene prevented the recruitment of a GFP-BAX fusion protein to mitochondria and provided long-term somal protection up to 12 weeks post injury. To test the efficacy in glaucoma, DBA/2J mice were transduced at 5 months of age, just prior to the time they begin to exhibit ocular hypertension. Gene therapy with mCherry-BCLX did not affect the longitudinal history of intraocular pressure elevation compared to naive mice but did robustly attenuate both RGC soma pathology and axonal degeneration in the optic nerve at both 10.5 and 12 months of age. BCLX gene therapy is a promising candidate for glaucoma therapy.
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http://dx.doi.org/10.1038/s41419-021-04068-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355227PMC
August 2021

The Influence of Mitochondrial Dynamics and Function on Retinal Ganglion Cell Susceptibility in Optic Nerve Disease.

Cells 2021 06 25;10(7). Epub 2021 Jun 25.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.

The important roles of mitochondrial function and dysfunction in the process of neurodegeneration are widely acknowledged. Retinal ganglion cells (RGCs) appear to be a highly vulnerable neuronal cell type in the central nervous system with respect to mitochondrial dysfunction but the actual reasons for this are still incompletely understood. These cells have a unique circumstance where unmyelinated axons must bend nearly 90° to exit the eye and then cross a translaminar pressure gradient before becoming myelinated in the optic nerve. This region, the optic nerve head, contains some of the highest density of mitochondria present in these cells. Glaucoma represents a perfect storm of events occurring at this location, with a combination of changes in the translaminar pressure gradient and reassignment of the metabolic support functions of supporting glia, which appears to apply increased metabolic stress to the RGC axons leading to a failure of axonal transport mechanisms. However, RGCs themselves are also extremely sensitive to genetic mutations, particularly in genes affecting mitochondrial dynamics and mitochondrial clearance. These mutations, which systemically affect the mitochondria in every cell, often lead to an optic neuropathy as the sole pathologic defect in affected patients. This review summarizes knowledge of mitochondrial structure and function, the known energy demands of neurons in general, and places these in the context of normal and pathological characteristics of mitochondria attributed to RGCs.
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http://dx.doi.org/10.3390/cells10071593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306483PMC
June 2021

The effects of a mitochondrial targeted peptide (elamipretide/SS31) on BAX recruitment and activation during apoptosis.

BMC Res Notes 2021 May 22;14(1):198. Epub 2021 May 22.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, 571A Medical Sciences, 1300 University Avenue, Madison, WI, 53706, USA.

Objective: Elamipretide (SS31) is a mitochondria-targeted peptide that has reported functions of stabilizing mitochondrial cristae structure and improving mitochondrial bioenergetics. Several studies have documented cell protective features of this peptide, including impairment of intrinsic apoptosis by inhibiting the recruitment and activation of the pro-apoptotic BAX protein. We used live-cell imaging of ARPE-19 cells expressing fluorescently labeled BAX, cytochrome c, and a mitochondrial marker to investigate the effect of elamipretide on the kinetics of BAX recruitment, mitochondrial outer membrane permeabilization (as a function of cytochrome c release), and mitochondrial fragmentation, respectively.

Result: In nucleofected and plated ARPE-19 cells, elamipretide accelerated the formation of larger mitochondria. In the presence of the apoptotic stimulator, staurosporine, cells treated with elamipretide exhibited moderately slower rates of BAX recruitment. Peptide treatment, however, did not significantly delay the onset of BAX recruitment or the final total amount of BAX that was recruited. Additionally, elamipretide showed no impairment or delay of cytochrome c release or mitochondrial fragmentation, two events associated with normal BAX activation during cell death. These results indicate that the protective effect of elamipretide is not at the level of BAX activity to induce pro-apoptotic mitochondrial dysfunction after the initiation of staurosporine-induced apoptosis.
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http://dx.doi.org/10.1186/s13104-021-05613-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141144PMC
May 2021

Characteristics of intracellular propagation of mitochondrial BAX recruitment during apoptosis.

Apoptosis 2021 02 11;26(1-2):132-145. Epub 2021 Jan 11.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, 1300 University Ave, Madison, WI, 53706, USA.

Recent advancements in live cell imaging technologies have identified the phenomenon of intracellular propagation of late apoptotic events, such as cytochrome c release and caspase activation. The mechanism, prevalence, and speed of apoptosis propagation remain unclear. Additionally, no studies have demonstrated propagation of the pro-apoptotic protein, BAX. To evaluate the role of BAX in intracellular apoptotic propagation, we used high speed live-cell imaging to visualize fluorescently tagged-BAX recruitment to mitochondria in four immortalized cell lines. We show that propagation of mitochondrial BAX recruitment occurs in parallel to cytochrome c and SMAC/Diablo release and is affected by cellular morphology, such that cells with processes are more likely to exhibit propagation. The initiation of propagation events is most prevalent in the distal tips of processes, while the rate of propagation is influenced by the 2-dimensional width of the process. Propagation was rarely observed in the cell soma, which exhibited near synchronous recruitment of BAX. Propagation velocity is not affected by mitochondrial volume in segments of processes, but is negatively affected by mitochondrial density. There was no evidence of a propagating wave of increased levels of intracellular calcium ions. Alternatively, we did observe a uniform increase in superoxide build-up in cellular mitochondria, which was released as a propagating wave simultaneously with the propagating recruitment of BAX to the mitochondrial outer membrane.
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http://dx.doi.org/10.1007/s10495-020-01654-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082518PMC
February 2021

Targeting HDAC3 in the DBA/2J spontaneous mouse model of glaucoma.

Exp Eye Res 2020 11 21;200:108244. Epub 2020 Sep 21.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, United States; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, United States.

High intraocular pressure (IOP) is the most common risk factor associated with glaucoma in humans. While lowering IOP is effective at reducing the rate of retinal ganglion cell (RGC) loss, to date, no treatment exists to directly preserve these cells affected by damage to the optic nerve. Recently, histone deacetylase-3 (HDAC3) has become a potential therapeutic target because it plays an important role in the early nuclear atrophic events that precede RGC death. Conditional knockout or inhibition of HDAC3 prevents histone deacetylation, heterochromatin formation, apoptosis, and eventual RGC loss following acute optic nerve injury. Using these approaches to repress HDAC3 activity, we tested whether targeting HDAC3 protects RGCs from ganglion cell-specific BRN3A expression loss, total somatic cell loss, and optic nerve degeneration in the DBA/2J mouse model of spontaneous glaucoma. Targeted ablation of Hdac3 activity did not protect RGCs from axonal degeneration or somatic cell death in the DBA/2J mouse model of glaucoma. However, inhibition of HDAC3 activity using RGFP966 conferred mild protection against somatic cell loss in the ganglion cell layer in aged DBA/2J mice. Further experimentation is necessary to determine whether other class I HDACs may serve as potential therapeutic targets in chronic models of glaucoma.
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http://dx.doi.org/10.1016/j.exer.2020.108244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8344090PMC
November 2020

BAX-Depleted Retinal Ganglion Cells Survive and Become Quiescent Following Optic Nerve Damage.

Mol Neurobiol 2020 Feb 31;57(2):1070-1084. Epub 2019 Oct 31.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI, USA.

Removal of the Bax gene from mice completely protects the somas of retinal ganglion cells (RGCs) from apoptosis following optic nerve injury. This makes BAX a promising therapeutic target to prevent neurodegeneration. In this study, Bax mice were used to test the hypothesis that lowering the quantity of BAX in RGCs would delay apoptosis following optic nerve injury. RGCs were damaged by performing optic nerve crush (ONC) and then immunostaining for phospho-cJUN, and quantitative PCR were used to monitor the status of the BAX activation mechanism in the months following injury. The apoptotic susceptibility of injured cells was directly tested by virally introducing GFP-BAX into Bax RGCs after injury. The competency of quiescent RGCs to reactivate their BAX activation mechanism was tested by intravitreal injection of the JNK pathway agonist, anisomycin. Twenty-four weeks after ONC, Bax mice had significantly less cell loss in their RGC layer than Bax mice 3 weeks after ONC. Bax and Bax RGCs exhibited similar patterns of nuclear phospho-cJUN accumulation immediately after ONC, which persisted in Bax RGCs for up to 7 weeks before abating. The transcriptional activation of BAX-activating genes was similar in Bax and Bax RGCs following ONC. Intriguingly, cells deactivated their BAX activation mechanism between 7 and 12 weeks after crush. Introduction of GFP-BAX into Bax cells at 4 weeks after ONC showed that these cells had a nearly normal capacity to activate this protein, but this capacity was lost 8 weeks after crush. Collectively, these data suggest that 8-12 weeks after crush, damaged cells no longer displayed increased susceptibility to BAX activation relative to their naïve counterparts. In this same timeframe, retinal glial activation and the signaling of the pro-apoptotic JNK pathway also abated. Quiescent RGCs did not show a timely reactivation of their JNK pathway following intravitreal injection with anisomycin. These findings demonstrate that lowering the quantity of BAX in RGCs is neuroprotective after acute injury. Damaged RGCs enter a quiescent state months after injury and are no longer responsive to an apoptotic stimulus. Quiescent RGCs will require rejuvenation to reacquire functionality.
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http://dx.doi.org/10.1007/s12035-019-01783-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035206PMC
February 2020

Meta-analysis of transcriptomic changes in optic nerve injury and neurodegenerative models reveals a fundamental response to injury throughout the central nervous system.

Mol Vis 2017 24;23:987-1005. Epub 2017 Dec 24.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI.

Purpose: Injury to the central nervous system (CNS) leads to transcriptional changes that effect tissue function and govern the process of neurodegeneration. Numerous microarray and RNA-Seq studies have been performed to identify these transcriptional changes in the retina following optic nerve injury and elsewhere in the CNS following a variety of insults. We reasoned that conserved transcriptional changes between injury paradigms would be important contributors to the neurodegenerative process. Therefore, we compared the expression results from heterogeneous studies of optic nerve injury and neurodegenerative models.

Methods: Expression data was collected from the Gene Expression Omnibus. A uniform method for normalizing expression data and detecting differentially expressed (DE) genes was used to compare the transcriptomes from models of acute optic nerve injury (AONI), chronic optic nerve injury (CONI) and brain neurodegeneration. DE genes were split into genes that were more or less prevalent in the injured condition than the control condition (enriched and depleted, respectively) and transformed into their human orthologs so that transcriptomes from different species could be compared. Biologic significance of shared genes was assessed by analyzing lists of shared genes for gene ontology (GO) term over-representation and for representation in KEGG pathways.

Results: There was significant overlap of enriched DE genes between transcriptomes of AONI, CONI and neurodegeneration studies even though the overall concordance between datasets was low. The depleted DE genes identified between AONI and CONI models were significantly overlapping, but this significance did not extend to comparisons between optic nerve injury models and neurodegeneration studies. The GO terms overrepresented among the enriched genes shared between AONI, CONI and neurodegeneration studies were related to innate immune processes like the complement system and interferon signaling. KEGG pathway analysis revealed that transcriptional alteration between JAK-STAT, PI3K-AKT and TNF signaling, among others, were conserved between all models that were analyzed.

Conclusions: There is a conserved transcriptional response to injury in the CNS. This transcriptional response is driven by the activation of the innate immune system and several regulatory pathways. Understanding the cellular origin of these pathways and the pathological consequences of their activation is essential for understanding and treating neurodegenerative disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757855PMC
May 2018

Targeting HDAC3 Activity with RGFP966 Protects Against Retinal Ganglion Cell Nuclear Atrophy and Apoptosis After Optic Nerve Injury.

J Ocul Pharmacol Ther 2018 04 6;34(3):260-273. Epub 2017 Dec 6.

1 Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison , Madison, Wisconsin.

Purpose: HDAC3 regulates nuclear atrophy as an early response to axonal injury in retinal ganglion cells (RGCs) following optic nerve crush (ONC). Since conditional knockout of Hdac3 prevents nuclear atrophy post ONC, HDAC3 selective inhibition with RGFP966 through localized and systemic dosing of RGFP966 is necessary for application to acute and chronic models of optic nerve injury.

Methods: C57BL/6 mice were injected intravitreally with 1-10 μM RGFP966 immediately following ONC, and retinas were analyzed at 5, 7, and 14 days for metrics of nuclear atrophy and cell loss. Mice were similarly assessed after intraperitoneal (IP) injections with RGFP966 doses of 2-10 mg/kg, and eyes were harvested at 5, 14, and 28 days after ONC. H&E and BrdU staining were used to analyze toxicity to off-target tissues after 14 days of daily treatment with RGFP966.

Results: A single intravitreal injection of RGFP966 prevented histone deacetylation, heterochromatin formation, apoptosis, and DNA damage at 5 and 7 days post ONC. After IP injection, RGFP966 bioavailability in the retina reached peak concentration within 1 h after injection and then rapidly declined. A single IP injection of 2-10 mg/kg RGFP966, significantly prevented histone deacetylation. Repeated IP injections of 2 mg/kg RGFP966 over the course of 2 and 4 weeks post ONC prevented RGC loss. There were no significant toxic or antiproliferative effects to off-target tissues in mice treated daily for 14 days with RGFP966.

Conclusion: Inhibition of HDAC3 activity with systemic dosing of RGFP966 prevents apoptosis-related histone deacetylation and attenuates RGC loss after acute optic nerve injury.
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http://dx.doi.org/10.1089/jop.2017.0059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963665PMC
April 2018

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Invest Ophthalmol Vis Sci 2017 12;58(14):6091-6104

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States.

Purpose: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome.

Methods: Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types.

Results: Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve.

Conclusions: Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.
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http://dx.doi.org/10.1167/iovs.17-22634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716181PMC
December 2017

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

PLoS One 2017 7;12(9):e0184434. Epub 2017 Sep 7.

Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184434PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589231PMC
October 2017

Negative regulators of angiogenesis: important targets for treatment of exudative AMD.

Clin Sci (Lond) 2017 Aug 5;131(15):1763-1780. Epub 2017 Jul 5.

Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, U.S.A.

Angiogenesis contributes to the pathogenesis of many diseases including exudative age-related macular degeneration (AMD). It is normally kept in check by a tightly balanced production of pro- and anti-angiogenic factors. The up-regulation of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), is intimately linked to the pathogenesis of exudative AMD, and its antagonism has been effectively targeted for treatment. However, very little is known about potential changes in expression of anti-angiogenic factors and the role they play in choroidal vascular homeostasis and neovascularization associated with AMD. Here, we will discuss the important role of thrombospondins and pigment epithelium-derived factor, two major endogenous inhibitors of angiogenesis, in retinal and choroidal vascular homeostasis and their potential alterations during AMD and choroidal neovascularization (CNV). We will review the cell autonomous function of these proteins in retinal and choroidal vascular cells. We will also discuss the potential targeting of these molecules and use of their mimetic peptides for therapeutic development for exudative AMD.
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http://dx.doi.org/10.1042/CS20170066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016847PMC
August 2017

BAX to basics: How the BCL2 gene family controls the death of retinal ganglion cells.

Prog Retin Eye Res 2017 03 4;57:1-25. Epub 2017 Jan 4.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA. Electronic address:

Retinal ganglion cell (RGC) death is the principal consequence of injury to the optic nerve. For several decades, we have understood that the RGC death process was executed by apoptosis, suggesting that there may be ways to therapeutically intervene in this cell death program and provide a more direct treatment to the cells and tissues affected in diseases like glaucoma. A major part of this endeavor has been to elucidate the molecular biological pathways active in RGCs from the point of axonal injury to the point of irreversible cell death. A major component of this process is the complex interaction of members of the BCL2 gene family. Three distinct family members of proteins orchestrate the most critical junction in the apoptotic program of RGCs, culminating in the activation of pro-apoptotic BAX. Once active, BAX causes irreparable damage to mitochondria, while precipitating downstream events that finish off a dying ganglion cell. This review is divided into two major parts. First, we summarize the extent of knowledge of how BCL2 gene family proteins interact to facilitate the activation and function of BAX. This area of investigation has rapidly changed over the last few years and has yielded a dramatically different mechanistic understanding of how the intrinsic apoptotic program is run in mammalian cells. Second, we provided a comprehensive analysis of nearly two decades of investigation of the role of BAX in the process of RGC death, much of which has provided many important insights into the overall pathophysiology of diseases like glaucoma.
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http://dx.doi.org/10.1016/j.preteyeres.2017.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350025PMC
March 2017

An intraocular drug delivery system using targeted nanocarriers attenuates retinal ganglion cell degeneration.

J Control Release 2017 02 4;247:153-166. Epub 2017 Jan 4.

Department of Surgery, 5151 Wisconsin Institute for Medical Research, University of Wisconsin-Madison, 1111 Highland Ave, Madison, WI 53705, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA. Electronic address:

Glaucoma is a common blinding disease characterized by loss of retinal ganglion cells (RGCs). To date, there is no clinically available treatment directly targeting RGCs. We aim to develop an RGC-targeted intraocular drug delivery system using unimolecular micelle nanoparticles (unimNPs) to prevent RGC loss. The unimNPs were formed by single/individual multi-arm star amphiphilic block copolymer poly(amidoamine)-polyvalerolactone-poly(ethylene glycol) (PAMAM-PVL-PEG). While the hydrophobic PAMAM-PVL core can encapsulate hydrophobic drugs, the hydrophilic PEG shell provides excellent water dispersity. We conjugated unimNPs with the cholera toxin B domain (CTB) for RGC-targeting and with Cy5.5 for unimNP-tracing. To exploit RGC-protective sigma-1 receptor (S1R), we loaded unimNPs with an endogenous S1R agonist dehydroepiandrosterone (DHEA) as an FDA-approved model drug. These unimNPs produced a steady DHEA release in vitro for over two months at pH7.4. We then co-injected (mice, intraocular) unimNPs with the glutamate analog N-methyl-d-aspartate (NMDA), which is excito-toxic and induces RGC death. The CTB-conjugated unimNPs (i.e., targeted NPs) accumulated at the RGC layer and effectively preserved RGCs at least for 14days, whereas the unimNPs without CTB (i.e., non-targeted NPs) showed neither accumulation at nor protection of NMDA-treated RGCs. Consistent with S1R functions, targeted NPs relative to non-targeted NPs showed markedly better inhibitory effects on apoptosis and oxidative/inflammatory stresses in the RGC layer. Hence, the DHEA-loaded, CTB-conjugated unimNPs represent an RGC/S1R dual-targeted nanoplatform that generates an efficacious template for further development of a sustainable intraocular drug delivery system to protect RGCs, which may be applicable to treatments directed at glaucomatous pathology.
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http://dx.doi.org/10.1016/j.jconrel.2016.12.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323250PMC
February 2017

Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways.

J Neuroinflammation 2016 Apr 28;13(1):93. Epub 2016 Apr 28.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, 571A Medical Sciences-1300 University Ave, Madison, WI, 53706, USA.

Background: Retinal ganglion cell (RGC) soma death is a consequence of optic nerve damage, including in optic neuropathies like glaucoma. The activation of the innate immune network in the retina after nerve damage has been linked to RGC pathology. Since the eye is immune privileged, innate immune functions are the responsibility of the glia, specifically the microglia, astrocytes, and Müller cells that populate the retina. Glial activation, leading to the production of inflammatory cytokines, is a hallmark feature of retinal injury resulting from optic nerve damage and purported to elicit secondary degeneration of RGC somas.

Methods: A mouse model of optic nerve crush (ONC) was used to study retinal glial activation responses. RGC apoptosis was blocked using Bax-deficient mice. Glial activation responses were monitored by quantitative PCR and immunofluorescent labeling in retinal sections of activation markers. ATP signaling pathways were interrogated using P2X receptor agonists and antagonists and Pannexin 1 (Panx1)-deficient mice with RGC-specific deletion.

Results: ONC induced activation of both macroglia and microglia in the retina, and both these responses were dramatically muted if RGC death was blocked by deletion of the Bax gene. Macroglial, but not microglial, activation was modulated by purinergic receptor activation. Release of ATP after optic nerve damage was not mediated by PANX1 channels in RGCs.

Conclusions: RGC death in response to ONC plays a principal stimulatory role in the retinal glial activation response.
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http://dx.doi.org/10.1186/s12974-016-0558-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850653PMC
April 2016

Role of HDACs in optic nerve damage-induced nuclear atrophy of retinal ganglion cells.

Neurosci Lett 2016 06 28;625:11-5. Epub 2015 Dec 28.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA. Electronic address:

Optic neuropathies are characterized by retinal ganglion cell (RGC) death, resulting in the loss of vision. In glaucoma, the most common optic neuropathy, RGC death is initiated by axonal damage, and can be modeled by inducing acute axonal trauma through procedures such as optic nerve crush (ONC) or optic nerve axotomy. One of the early events of RGC death is nuclear atrophy, and is comprised of RGC-specific gene silencing, histone deacetylation, heterochromatin formation, and nuclear shrinkage. These early events appear to be principally regulated by epigenetic mechanisms involving histone deacetylation. Class I histone deacetylases HDACs 1, 2, and 3 are known to play important roles in the process of early nuclear atrophy in RGCs, and studies using both inhibitors and genetic ablation of Hdacs also reveal a critical role in the cell death process. Select inhibitors, such as those being developed for cancer therapy, may also provide a viable secondary treatment option for optic neuropathies.
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http://dx.doi.org/10.1016/j.neulet.2015.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125391PMC
June 2016

The Long-Term Effects on the Retina Damaged by Optic Nerve Axotomy.

Invest Ophthalmol Vis Sci 2015 Sep;56(10):6113

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http://dx.doi.org/10.1167/iovs.15-17997DOI Listing
September 2015

Neuroinflammation in Glaucoma and Optic Nerve Damage.

Prog Mol Biol Transl Sci 2015 10;134:343-63. Epub 2015 Jul 10.

Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA. Electronic address:

Glaucoma is a group of optic neuropathies characterized by the degeneration of retinal ganglion cell axons and somas, ultimately preventing light signals in the retina from reaching the brain. Glaucoma is a leading cause of blindness in the world, and treatment options for patients remain limited and minimally efficacious. A number of mechanisms have been linked to glaucomatous pathophysiology. A leading role is now attributed to neuroinflammatory conditions generated by the resident innate immune cells in the optic nerve and retina. Since the eye is immune privileged, the adaptation of these innate immune cells, termed glia, is crucial following trauma. In this chapter, we discuss the mechanisms associated with normal glial function in a healthy eye, and how changes in glial activation can contribute to the process of glaucomatous neurodegeneration in both the optic nerve and retina.
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http://dx.doi.org/10.1016/bs.pmbts.2015.06.010DOI Listing
February 2016

Tools and resources for analyzing gene expression changes in glaucomatous neurodegeneration.

Exp Eye Res 2015 Dec 19;141:99-110. Epub 2015 May 19.

Department of Biological Sciences, University of Wisconsin - Whitewater, Whitewater, WI, USA.

Evaluating gene expression changes presents one of the most powerful interrogative approaches to study the molecular, biochemical, and cellular pathways associated with glaucomatous disease pathology. Technologies to study gene expression profiles in glaucoma are wide ranging. Qualitative techniques provide the power of localizing expression changes to individual cells, but are not robust to evaluate differences in expression changes. Alternatively, quantitative changes provide a high level of stringency to quantify changes in gene expression. Additionally, advances in high throughput analysis and bioinformatics have dramatically improved the number of individual genes that can be evaluated in a single experiment, while dramatically reducing amounts of input tissue/starting material. Together, gene expression profiling and proteomics have yielded new insights on the roles of neuroinflammation, the complement cascade, and metabolic shutdown as important players in the pathology of the optic nerve head and retina in this disease.
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http://dx.doi.org/10.1016/j.exer.2015.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628862PMC
December 2015

Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush.

J Neuroinflammation 2014 Nov 19;11:194. Epub 2014 Nov 19.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, 571A MSC - 1300 University Ave., Madison, WI, 53706, USA.

Background: Glaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete.

Methods: RGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα (-/-) mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice.

Results: Optic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα (-/-) mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB.

Conclusion: Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.
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http://dx.doi.org/10.1186/s12974-014-0194-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245774PMC
November 2014

Histone deacetylase 3 (HDAC3) plays an important role in retinal ganglion cell death after acute optic nerve injury.

Mol Neurodegener 2014 Sep 28;9:39. Epub 2014 Sep 28.

Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, 6640 MSC - 1300 University Ave, Madison, WI 53706, USA.

Background: Optic nerve damage initiates a series of early atrophic events in retinal ganglion cells (RGCs) that precede the BAX-dependent committed step of the intrinsic apoptotic program. Nuclear atrophy, including global histone deacetylation, heterochromatin formation, shrinkage and collapse of nuclear structure, and the silencing of normal gene expression, comprise an important obstacle to overcome in therapeutic approaches to preserve neuronal function. Several studies have implicated histone deacetylases (HDACs) in the early stages of neuronal cell death, including RGCs. Importantly, these neurons exhibit nuclear translocation of HDAC3 shortly after optic nerve damage. Additionally, HDAC3 activity has been reported to be selectively toxic to neurons.

Results: RGC-specific conditional knockout of Hdac3 was achieved by transducing the RGCs of Hdac3fl/fl mice with an adeno-associated virus serotype 2 carrying CRE recombinase and GFP (AAV2-Cre/GFP). Controls included similar viral transduction of Rosa26fl/fl reporter mice. Optic nerve crush (ONC) was then performed on eyes. The ablation of Hdac3 in RGCs resulted in significant amelioration of characteristics of ONC-induced nuclear atrophy such as H4 deacetylation, heterochromatin formation, and the loss of nuclear structure. RGC death was also significantly reduced. Interestingly, loss of Hdac3 expression did not lead to protection against RGC-specific gene silencing after ONC, although this effect was achieved using the broad spectrum inhibitor, Trichostatin A.

Conclusion: Although other HDACs may be responsible for gene expression changes in RGCs, our results indicate a critical role for HDAC3 in nuclear atrophy in RGC apoptosis following axonal injury. This study provides a framework for studying the roles of other prevalent retinal HDACs in neuronal death as a result of axonal injury.
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http://dx.doi.org/10.1186/1750-1326-9-39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190472PMC
September 2014

Spink2 modulates apoptotic susceptibility and is a candidate gene in the Rgcs1 QTL that affects retinal ganglion cell death after optic nerve damage.

PLoS One 2014 3;9(4):e93564. Epub 2014 Apr 3.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States of America.

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093564PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3974755PMC
February 2015

Evaluation of the percentage of ganglion cells in the ganglion cell layer of the rodent retina.

Mol Vis 2013 27;19:1387-96. Epub 2013 Jun 27.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI, USA.

Purpose: Retinal ganglion cells comprise a percentage of the neurons actually residing in the ganglion cell layer (GCL) of the rodent retina. This estimate is useful to extrapolate ganglion cell loss in models of optic nerve disease, but the values reported in the literature are highly variable depending on the methods used to obtain them.

Methods: We tested three retrograde labeling methods and two immunostaining methods to calculate ganglion cell number in the mouse retina (C57BL/6). Additionally, a double-stain retrograde staining method was used to label rats (Long-Evans). The number of total neurons was estimated using a nuclear stain and selecting for nuclei that met specific criteria. Cholinergic amacrine cells were identified using transgenic mice expressing Tomato fluorescent protein. Total neurons and total ganglion cell numbers were measured in microscopic fields of 10(4) µm(2) to determine the percentage of neurons comprising ganglion cells in each field.

Results: Historical estimates of the percentage of ganglion cells in the mouse GCL range from 36.1% to 67.5% depending on the method used. Experimentally, retrograde labeling methods yielded a combined estimate of 50.3% in mice. A retrograde method also yielded a value of 50.21% for rat retinas. Immunolabeling estimates were higher at 64.8%. Immunolabeling may introduce overestimates, however, with non-specific labeling effects, or ectopic expression of antigens in neurons other than ganglion cells.

Conclusions: Since immunolabeling methods may overestimate ganglion cell numbers, we conclude that 50%, which is consistently derived from retrograde labeling methods, is a reliable estimate of the ganglion cells in the neuronal population of the GCL.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695759PMC
September 2013

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Proc Natl Acad Sci U S A 2013 Mar 19;110(10):4045-50. Epub 2013 Feb 19.

Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.
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http://dx.doi.org/10.1073/pnas.1211284110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593842PMC
March 2013

Nuclear atrophy of retinal ganglion cells precedes the bax-dependent stage of apoptosis.

Invest Ophthalmol Vis Sci 2013 Mar 11;54(3):1805-15. Epub 2013 Mar 11.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53706, USA.

Purpose: Retinal ganglion cells atrophy during the execution of the intrinsic apoptotic program. This process, which has been termed the apoptotic volume decrease (AVD) in other cell types, has not been well-characterized in ganglion cells.

Methods: Acute optic nerve crush was used to examine neuronal atrophy in the ganglion cell layer in wild-type and Bax-deficient mice. Nuclear size was measured from retinal wholemounts. Heterochromatin formation was assessed using transmission electron microscopy, whereas histone H4 acetylation was monitored using immunofluoresence. Ganglion cell and retinal transcript abundance was measured using quantitative PCR.

Results: Nuclear and soma sizes linearly correlated in both control and damaged retinas. Cells in wild-type mice exhibited nuclear atrophy within 1 day after optic nerve damage. Three days after crush, nuclear atrophy was restricted to ganglion cells identified by retrograde labeling, while amacrine cells also exhibited some atrophy by 5 days. Similar kinetics of nuclear atrophy were observed in cells deficient for the essential proapoptotic gene Bax. Bax-deficient cells also exhibited other nuclear changes common in wild-type cells, including the deacetylation of histones, formation of heterochromatin, and the silencing of ganglion cell-specific gene expression.

Conclusions: Retinal ganglion cell somas and nuclei undergo the AVD in response to optic nerve damage. Atrophy is rapid and precedes the Bax-dependent committed step of the intrinsic apoptotic pathway.
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http://dx.doi.org/10.1167/iovs.11-9310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626519PMC
March 2013

The effect of glial fibrillary acidic protein expression on neurite outgrowth from retinal explants in a permissive environment.

BMC Res Notes 2012 Dec 22;5:693. Epub 2012 Dec 22.

Departments of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53706, USA.

Background: Increased expression of glial fibrillary acidic protein (GFAP) within macroglia is commonly seen as a hallmark of glial activation after damage within the central nervous system, including the retina. The increased expression of GFAP in glia is also considered part of the pathologically inhibitory environment for regeneration of axons from damaged neurons. Recent studies have raised the possibility that reactive gliosis and increased GFAP cannot automatically be assumed to be negative events for the surrounding neurons and that the context of the reactive gliosis is critical to whether neurons benefit or suffer. We utilized transgenic mice expressing a range of Gfap to titrate the amount of GFAP in retinal explants to investigate the relationship between GFAP concentration and the regenerative potential of retinal ganglion cells.

Findings: Explants from Gfap-/- and Gfap+/- mice did not have increased neurite outgrowth compared with Gfap+/+ or Gfap over-expressing mice as would be expected if GFAP was detrimental to axon regeneration. In fact, Gfap over-expressing explants had the most neurite outgrowth when treated with a neurite stimulatory media. Transmission electron microscopy revealed that neurites formed bundles, which were surrounded by larger cellular processes that were GFAP positive indicating a close association between growing axons and glial cells in this regeneration paradigm.

Conclusions: We postulate that glial cells with increased Gfap expression support the elongation of new neurites from retinal ganglion cells possibly by providing a scaffold for outgrowth.
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http://dx.doi.org/10.1186/1756-0500-5-693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3544725PMC
December 2012

The cell and molecular biology of glaucoma: mechanisms of retinal ganglion cell death.

Invest Ophthalmol Vis Sci 2012 May 4;53(5):2476-81. Epub 2012 May 4.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.

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http://dx.doi.org/10.1167/iovs.12-9483hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990459PMC
May 2012

Under pressure: cellular and molecular responses during glaucoma, a common neurodegeneration with axonopathy.

Annu Rev Neurosci 2012 12;35:153-79. Epub 2012 Apr 12.

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.

Glaucoma is a complex neurodegenerative disorder that is expected to affect 80 million people by the end of this decade. Retinal ganglion cells (RGCs) are the most affected cell type and progressively degenerate over the course of the disease. RGC axons exit the eye and enter the optic nerve by passing through the optic nerve head (ONH). The ONH is an important site of initial damage in glaucoma. Higher intraocular pressure (IOP) is an important risk factor for glaucoma, but the molecular links between elevated IOP and axon damage in the ONH are poorly defined. In this review and focusing primarily on the ONH, we discuss recent studies that have contributed to understanding the etiology and pathogenesis of glaucoma. We also identify areas that require further investigation and focus on mechanisms identified in other neurodegenerations that may contribute to RGC dysfunction and demise in glaucoma.
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http://dx.doi.org/10.1146/annurev.neuro.051508.135728DOI Listing
November 2012
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