Homocysteine-Mediated Modulation of Mitochondrial Dynamics in Retinal Ganglion Cells (RGCs)

Abstract

Purpose: : Moderate elevation in plasma homocysteine (hyperhomocysteinemia) has been implicated in RGC death during glaucoma. Previously we utilized a mouse model of moderate hyperhomocysteinemia (cbs^+/-) to examine retinal morphology and found a ~20% loss of RGCs (Ganapathy et al, 2009). Microarray analysis of cbs^+/- neural retinas revealed altered expression of Opa1 and Fis1, proteins known to regulate mitochondrial dynamics. The present study examined the effect of excess homocysteine on mitochondrial dynamics in RGCs.

Methods: : Cbs^+/- mice maintained on a high methionine diet (0.5% methionine in drinking water) were used for this study. The effect of hyperhomocysteinemia on RGC mitochondria was examined in vivo through gene and protein expression analysis of Opa1 and Fis1 in cbs^+/- neural retinas. Mitochondria within RGC axons underwent systematic ultrastructural analysis to measure area, length, and width, and the distance between the mitochondria and axon wall. To determine whether direct exposure to homocysteine would induce altered mitochondrial dynamics, primary mouse RGCs were cultured and treated with homocysteine and assessed for Opa1 and Fis1 protein.

Results: : Transcriptional expression of all Opa1 isoforms and both transcript variants of Fis1 was elevated in cbs^+/- mice. Opa1 protein was elevated to 191.00 ± 26.40 % and Fis1 to 226.20 ± 4.57 % of wild type. Mitochondria in cbs^+/- retinas were smaller via all parameters studied (area: 0.317±0.012µm² versus 0.424±0.022µm²) and located closer to the axon wall. Opa1 was elevated to 173.2±5.1% after 12h and Fis1 to 216.9±9.5% after 3h of homocysteine treatment to RGCs.

Conclusions: : This study provides the first evidence that homocysteine-induced RGC loss involves dysregulation of mitochondrial dynamics, both in vivo and in vitro. Neurodegenerative diseases that have been associated with hyperhomocysteinemia, including glaucoma and Alzheimer’s disease, have implicated increased mitochondrial fission in the pathogenesis of neuronal death. The finding that excess homocysteine alters the shape and distribution of RGC mitochondria by altering the expression of Opa1 and Fis1 suggests dysregulation of mitochondrial dynamics as a novel mechanism of homocysteine toxicity to neurons.

Keywords: mitochondria • ganglion cells • apoptosis/cell death