This study provides the first evidence that homocysteine-induced ganglion cell loss involves the dysregulation of mitochondrial dynamics, both in vivo and in vitro. The present data suggest increased mitochondrial fission as a novel mechanism of homocysteine toxicity to neurons. Of particular relevance are glaucoma and Alzheimer's disease, neurodegenerative diseases that are associated with hyperhomocysteinemia and, more recently, have implicated increased mitochondrial fission in their pathogeneses.
These experiments provide compelling evidence that homocysteine induces retinal ganglion cell toxicity through direct NMDA receptor stimulation and implicate, for the first time, the induction of oxidative stress as a potent mechanism of homocysteine-mediated ganglion cell death.
FRalpha and PCFT are expressed in retinal Müller cells and colocalize in the endosomal compartment, suggesting that the two proteins may work coordinately to mediate folate uptake. The unexpected finding of RFC expression and activity in cultured Müller cells may reflect the upregulation of this protein under proliferative conditions.
PURPOSE
Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine.
METHODS
RGC-5 cells were differentiated 24 h using 316 nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 µM) and to high levels of homocysteine (1 mM), glutamate (1 mM) and oxidative stress (25 µM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10 mM) homocysteine was also examined.
RESULTS
Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24 h differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine-differentiated RGC-5 cells with 50, 100 or 250µM homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in non-homocysteine-treated, staurosporine-differentiated cells. The 1 mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18 h treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%.
CONCLUSIONS
RGC-5 cells differentiated for 24 h with 316 nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non- staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.
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