Abstract
Purpose::
Since the cellular and molecular mechanisms by which retinal ganglion cells (RGCs) degenerate during glaucoma are not well understood, the purpose of this study was to examine molecular changes occurring within RGC cells that coincide with axonal transport dysfunction within the DBA/2J mouse.
Methods::
Using single and double in situ hybridization in retina sections and whole-mounts, the expression of the RGC-specific gene γ-synuclein was examined in young and old DBA/2J and C57/BL6 mice, and compared to both other RGC mRNAs and proteins, as well as to the mRNA for the amacrine cell marker GAD-67. Immunohistochemical and fluorogold retrograde labeling in flat-mounted retinas were used in combination with γ-synuclein in situ hybridization to correlate various molecular changes within RGCs to each other and to glia reactivity, microglia activation and axonal transport dysfunction.
Results::
In situ hybridization studies showed that glaucomatous DBA/2J retinas have significant loss of γ-synuclein expressing RGCs as well as a significant down-regulation of this gene within many of the remaining RGCs. The γ-synuclein down-regulation coincided with the decline of other RGC mRNAs such as neurofilaments and the transcription factor Brn3b. In contrast, no changes were evident in the expression and number of GAD-67 expressing displaced amacrine cells found in the RGC-layer. The γ-synuclein down-regulation within the retina was non-uniform, and increased GFAP immunolabeling in astrocytes and Müller cells was found specifically in regions where γ-synuclein expression was decreased or completely absent. RGCs with impaired axonal transport, as defined by the cytosolic accumulation of phosphorylated neurofilaments (pNF) in their cell bodies and/or reduced or absent fluorogold retrograde labeling, were shown to have among the lowest levels of γ-synuclein mRNA. Finally, activated microglial cells were observed in the proximity of pNF immunopositive RGCs, suggesting that these cells with the lowest levels of γ -synuclein expression represent the final stages of RGC degeneration.
Conclusions::
These results show that RGC gene expression programs are specifically down-regulated prior to cell death. Since this down-regulation in gene expression is correlated with two signs of axon transport impairment, somatic accumulation of axonal proteins and impaired fluorogold transport, we propose that axon transport impairment and down-regulation of gene expression are causally related.
Keywords: gene/expression • ganglion cells • retinal degenerations: cell biology