Homocysteine-Mediated Modulation of Mitochondrial Dynamics in Retinal Ganglion Cells

Homocysteine is a non-proteinogenic amino acid that is an intermediate in methionine and cysteine metabolism. Severe elevations in plasma homocysteine (hyperhomocysteinemia) are caused by homozygous mutations in the regulatory enzymes involved in homocysteine metabolism; these enzymes include methionine synthase, methylene tetrahydrofolate reductase, and cystathionine β-synthase.1 Moderate hyperhomocysteinemia occurs more commonly and is caused by heterozygous mutations in these regulatory enzymes or by nutritional deficiencies in the vitamins folate, B12, and B6. Recently, several clinical studies reported a correlation between elevated plasma homocysteine and retinal degenerative disorders, including open-angle glaucoma, maculopathy, and diabetic retinopathy.2–9 Our laboratory has investigated extensively the potential of homocysteine to induce toxicity to the retina using both in vitro and in vivo models.10–13 Our earliest in vivo study documented that intravitreal injection of a high level of homocysteine (200 μM) led to abundant cell death in the ganglion cell layer.13 To specifically analyze the effect of endogenous elevation of homocysteine on the retina, our laboratory recently used a mutant mouse model of hyperhomocysteinemia developed in the laboratory of Nobuyo Maeda.14 The mouse harbors a deletion of the gene encoding cystathionine β-synthase (cbs), an enzyme required for the conversion of homocysteine to cysteine. Using the heterozygous mutant mouse (cbs+/−) as a model of moderate hyperhomocysteinemia (∼4- to 7-fold elevation in plasma homocysteine), we found that ganglion cell viability was decreased by approximately 20%; even greater neuronal death was observed in mice with higher levels of this amino acid.10 These studies described for the first time that endogenous elevation in plasma homocysteine induces ganglion cell loss. To investigate potential mechanisms of homocysteine-induced ganglion cell toxicity, we performed microarray analysis on neural retinas of cbs+/− mice. The expression of numerous genes was altered in these mice; two genes of particular interest whose expression levels were changed in the neural retina of the cbs+/− mouse were opa1 and fis1.10 Opa1 and Fis1 proteins are involved in mitochondrial fission and fusion; Opa1 is a modulator of mitochondrial fusion, and Fis1 is the rate-limiting protein in mitochondrial fission.15 Mitochondria are the primary energy-producing organelles and are dynamic in that they require a balance of fission and fusion processes to function properly.15 Excessive fission results in mitochondrial fragmentation and eventual cellular apoptosis, whereas excessive fusion results in elongated mitochondria and inhibition of adequate energy production. Given that retinal ganglion cells are neurons that are highly active, as evidenced by a high metabolic rate, they are especially sensitive to alterations in mitochondrial dynamics.16,17 Deficiencies in mitochondrial fission and fusion processes have been implicated in the pathogenesis of several neurodegenerative disorders, including glaucoma, a disease characterized specifically by retinal ganglion cell loss.16,18–20 In the present study, we test the hypothesis that elevation in homocysteine impairs the balance of mitochondrial fission and fusion in retinal ganglion cells, resulting in excessive mitochondrial fission and cellular apoptosis.