Abstract
Purpose: :
The rearrangement of cytoskeletal elements in any cellular remodeling is calcium-dependent. Ca release from internal stores, mediated by ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) represents the hub for intracellular Ca homeostasis in both neurons and glia. Our aim was to characterize the relationship between Ca stores and the intense reorganization of neuronal-glial interfaces in glaucoma.
Methods: :
Expression of calcium signaling proteins together with RGC and glial markers were examined in age-matched retinas from wild type C57BL/6, DBA/2J and DBA/2J-Gpnmb+ mice using quantitative RT-PCR, in situ hybridization, immunohistochemistry (IHC) and calcium imaging methods. The severity of glaucoma was assessed in PPD-labeled nerves using an established grading scheme. Eyes were classified with no or early (less than 10% axons damage), moderate (10-50% axons damage) or severe (more than 50% axons damage) glaucoma.
Results: :
As glaucoma severity increased, there was an upregulation of astrocyte (Gfap; 5.2 ± 1.2 -fold in moderate; 10.6 ± 2.5 -fold in severe glaucoma) and microglial (Iba1 and Cd11) markers and downregulation of RGC markers (Brn3a and Vglut2; Vglut2 changed to 0.93 ± 0.19 in moderate; 0.13 ± 0.04 in severe glaucoma). There was a marked upregulation of RyR1 receptors in DBA/2J retinas (7.95 ± 2.56 -fold for moderate glaucoma; 6.94 ± 1.07 -fold for severe respectively). In situ hybridization and IHC revealed RyR1 expression in all retinal layers, with prominent signals in the ONL, sublamina b of the IPL and the GCL. Endfeet of Müller cells, putative astrocyte processes at the ILM and at the optic nerve head were strongly labeled by RyR1 antibodies. RyR2 was localized to GCL and proximal INL; no significant differences between controls and DBA/2J retinas were observed with respect to RyR2 isoform expression/localization (1.0 ± 0.2 in moderate; 1.5 ± 0.4 -fold in severe glaucoma). IP3R1 was localized to RGC layer and the proximal INL and was significantly more upregulated in severe (1.5 ± 0.2 -fold) and moderate (2.1 ± 0.2 -fold) glaucoma. IP3R2 was localized to RGCs and in the inner retina. Expression of IP3R2 was decreased 0.38 ± 0.02 -fold in moderate and 0.44 ± 0.08 -fold in severe glaucoma.
Conclusions: :
Severe glaucoma is associated with upregulation of RyR1 and IP3R1 genes in glial cells and with downregulation of IP3R2 in RGCs. This data suggests that cytoskeletal rearrangements in DBA/2J glaucomatous remodeling may be driven in part by increased Ca release from intracellular store compartments.
Keywords: degenerations/dystrophies