Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1−/− double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1−/− double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB and CB), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB and CB receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l-Aspartate (L-ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212-2 (WIN, a CB/CB agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca levels evoked by ATP, but had no effect in Ca shifts in neuronal cells activated by KCl. Finally, WIN inhibited [H]-GABA release induced by KCl or L-ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation.

Serum response factor (SRF) controls the expression of muscle contraction and motility genes in mural cells (MCs) of the vasculature. In the retina, MC-SRF is important for correct angiogenesis during development and the continuing maintenance of the vascular tone. The purpose of this study was to provide further insights into the effects of MC SRF deficiency on the vasculature and function of the mature retina in SrfiMCKO mice that carry a MC-specific deletion of Srf. Retinal morphology and vascular integrity were analyzed in vivo via scanning laser ophthalmoscopy (SLO), angiography, and optical coherence tomography (OCT). Retinal function was evaluated with full-field electroretinography (ERG). We found that retinal blood vessels of these mutants exhibited different degrees of morphological and functional alterations. With increasing severity, we found vascular bulging, the formation of arteriovenous (AV) anastomoses, and ultimately, a retinal detachment (RD). The associated irregular retinal blood pressure and flow distribution eventually induced hypoxia, indicated by a negative ERG waveform shape. Further, the high frequency of interocular differences in the phenotype of individual SrfiMCKO mice points to a secondary nature of these developments far downstream of the genetic defect and rather dependent on the local retinal context.

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