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

ABSTRACT: 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.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.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.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.

SUBMITTER: Janssen KT

PROVIDER: S-EPMC3626519 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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

Janssen Katherine T KT Mac Nair Caitlin E CE Dietz Joel A JA Schlamp Cassandra L CL Nickells Robert W RW

Investigative ophthalmology & visual science 20130311 3

<h4>Purpose</h4>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.<h4>Methods</h4>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 el ...[more]

PMID: 23422829

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Project description: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.

Project description:The BCL-2 (B-cell lymphoma-2) family of proteins contributes to mitochondrial-based apoptosis in models of neurodegeneration, including glaucomatous optic neuropathy (glaucoma), which degrades the retinal ganglion cell (RGC) axonal projection to the visual brain. Glaucoma is commonly associated with increased sensitivity to intraocular pressure (IOP) and involves a proximal program that leads to RGC dendritic pruning and a distal program that underlies axonopathy in the optic projection. While genetic deletion of the Bcl2-associated X protein (Bax-/-) prolongs RGC body survival in models of glaucoma and optic nerve trauma, axonopathy persists, thus raising the question of whether dendrites and the RGC light response are protected. Here, we used an inducible model of glaucoma in Bax-/- mice to determine if Bax contributes to RGC dendritic degeneration. We performed whole-cell recordings and dye filling in RGCs signaling light onset (αON-Sustained) and offset (αOFF-Sustained). We recovered RGC dendritic morphologies by confocal microscopy and analyzed dendritic arbor complexity and size. Additionally, we assessed RGC axon function by measuring anterograde axon transport of cholera toxin subunit B to the superior colliculus and behavioral spatial frequency threshold (i.e., spatial acuity). We found 1 month of IOP elevation did not cause significant RGC death in either WT or Bax-/- retinas. However, IOP elevation reduced dendritic arbor complexity of WT αON-Sustained and αOFF-Sustained RGCs. In the absence of Bax, αON- and αOFF-Sustained RGC dendritic arbors remained intact following IOP elevation. In addition to dendrites, neuroprotection by Bax-/- generalized to αON-and αOFF-Sustained RGC light- and current-evoked responses. Both anterograde axon transport and spatial acuity declined during IOP elevation in WT and Bax-/- mice. Collectively, our results indicate Bax contributes to RGC dendritic degeneration and distinguishes the proximal and distal neurodegenerative programs involved during the progression of glaucoma.

Project description:Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterized by the preferential loss of retinal ganglion cells (RGCs), resulting in optic nerve degeneration and progressive bilateral central vision loss. More than 60% of genetically confirmed patients with DOA carry variants in the nuclear OPA1 gene, which encodes for a ubiquitously expressed, mitochondrial GTPase protein. OPA1 has diverse functions within the mitochondrial network, facilitating inner membrane fusion and cristae modelling, regulating mitochondrial DNA maintenance and coordinating mitochondrial bioenergetics. There are currently no licensed disease-modifying therapies for DOA and the disease mechanisms driving RGC degeneration are poorly understood. Here, we describe the generation of isogenic, heterozygous OPA1 null induced pluripotent stem cell (iPSC) (OPA1+/-) through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of a control cell line, in conjunction with the generation of DOA patient-derived iPSC carrying OPA1 variants, namely, the c.2708_2711delTTAG variant (DOA iPSC), and previously reported missense variant iPSC line (c.1334G>A, DOA plus [DOA]+ iPSC) and CRISPR/Cas9 corrected controls. A two-dimensional (2D) differentiation protocol was used to study the effect of OPA1 variants on iPSC-RGC differentiation and mitochondrial function. OPA1+/-, DOA and DOA+ iPSC showed no differentiation deficit compared to control iPSC lines, exhibiting comparable expression of all relevant markers at each stage of differentiation. OPA1+/- and OPA1 variant iPSC-RGCs exhibited impaired mitochondrial homeostasis, with reduced bioenergetic output and compromised mitochondrial DNA maintenance. These data highlight mitochondrial deficits associated with OPA1 dysfunction in human iPSC-RGCs, and establish a platform to study disease mechanisms that contribute to RGC loss in DOA, as well as potential therapeutic interventions.

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

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

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