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Andrei Surguchov, Victor Sharov, Irina G. Surgucheva; New Mechanisms of Glaucoma Pathology Spreading. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3845.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucomatous vision loss results from the progressive degeneration of ON axons and the death of RGC. The accumulation and deposition of γ-synuclein (γ-syn) in the optic nerve, both in axonal spheroids and within astrocytes is a new hallmark of glaucoma. Since aberrant forms of γ-syn are associated with pathological manifestations of glaucoma, this ocular disease might be considered as γ-synucleinopathy. Here we investigate the mechanisms of γ-syn secretion from neuronal cells and internalization by glial cells leading to its intracellular accumulation.
Clones of SH-SY5Y cells overexpressing human γ-syn and γ-syn-GFP were generated. After 48h of growth cell extracts (CE) and conditioned media (CM) were collected and analyzed by Western blotting. CM was further incubated with glial cells, which were subsequently examined by immunofluorescent and confocal microscopy to reveal γ-syn internalization and intracellular localization. Exosomes were isolated from CM by differential centrifugation or precipitation by ExoQuick (System Biosciences, Inc.). The following immortalized cell cultures were used as recipients of γ-syn: neuroblastoma cell line SH-SY5Y, glioblastoma/ astrocytoma U87 and A7 astrocytes from the optic nerve.
Both monomeric γ-syn and γ-syn-GFP-fusion protein are secreted into CM in the form of exosomes. Inhibition of proteasomal activity differentially affects the level of secreted γ-syn aggregation. γ-Syn without a GFP tag is secreted predominantly as a monomer both in the presence and absence of proteasome inhibitor MG132. γ-Syn fused to GFP forms aggregates in the presence of MG132 with molecular weight 60-100 KDa. The secretion of γ-syn-GFP from SH-SY-5Y is significantly reduced in the presence of inhibitors of exosome biogenesis GW4869 (68%±5% inhibition) and methyl-β-cyclodextrin (MBC, 92±3% inhibition). MBC decreased monomer γ-syn secretion by 34±4%. Accordingly, the amount of exosome marker CD63 was also reduced in the presence of both inhibitors. The average reduction of CD63 by GW4869 was 46.2±5%, by MBC 73.6±6%. Finally, we found that γ-syn secreted into CM can be internalized by glial cells. According to the Western blot analysis, this process is accompanied by the alteration of γ-syn aggregation pattern which may be explained by changes in protein conformation and/or post-translational modifications.
Our studies revealed exosomal secretion of γ-syn from neuronal cells into CM and its internalization by glial cells as the first steps leading to intracellular accumulation of aberrant γ-syn.
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