Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model

Lei Fang,Peter Meyer,David Goldblum,Albert Neutzner,Josef Flammer,Charles Hemion,Stephan Frank,Selim Orgül,Ian A Trounce

doi:10.1371/journal.pone.0052637

Abstract

PurposeTo study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.MethodsRGC5 cells transfected with expression constructs for MARCH5, MARCH5H43W, Dpr1K38A or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.ResultsExposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5H43W or inactive DrpK38A, mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5H43W or Drp1K38A did significantly delay cell death.ConclusionPreventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.

Highlights

Death of retinal ganglion cells (RGCs) is responsible for vision loss in glaucoma patients
Elevated intraocular pressure (IOP) is involved in RGC death associated with high-tension glaucoma (HTG) [1], while vascular dysregulation and associated ischemiareperfusion injury is linked to normal-tension glaucoma (NTG) [2]
While the mitochondrial network in cells kept under ambient pressure displayed normal tubular mitochondrial morphology, in about 60% of cells exposed to elevated pressure mitochondria switched to a fragmented phenotype

Summary

Introduction

Death of retinal ganglion cells (RGCs) is responsible for vision loss in glaucoma patients. The exact mechanisms causing the demise of RGCs are still under investigation. Different triggers in the various forms of glaucoma probably lead to the observed neurodegenerative process. Elevated intraocular pressure (IOP) is involved in RGC death associated with high-tension glaucoma (HTG) [1], while vascular dysregulation and associated ischemiareperfusion injury is linked to normal-tension glaucoma (NTG) [2]. Irrespective of the actual trigger and the glaucoma subtype, at its heart, glaucoma is a slowly progressing neurodegenerative disorder. RGC5 cells were used as cellular model. These cells are murine neuronal precursor cells and display certain features such as the expression of specific neuronal marker upon differentiation with various compounds [3]

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