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Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP.

ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells (RGCs), we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.

SUBMITTER: Cheng HH

PROVIDER: S-EPMC5998634 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP.

Cheng Huan-Huan HH Ye Hui H Peng Rui-Ping RP Deng Juan J Ding Yong Y

Neural regeneration research 20180501 5

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells (RGCs), we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 10 ...[more]

PMID: 29863024

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Project description:Human Müller glia with stem cell characteristics (hMGSCs) have been shown to improve retinal function upon transplantation into rat models of retinal ganglion cell (RGC) depletion. However, their translational potential may depend upon successful engraftment and improvement of retinal function in experimental models with anatomical and functional features resembling those of the human eye. We investigated the effect of allogeneic transplantation of feline Müller glia with the ability to differentiate into cells expressing RGC markers, following ablation of RGCs by N-methyl-d-aspartate (NMDA). Unlike previous observations in the rat, transplantation of hMGSC-derived RGCs into the feline vitreous formed aggregates and elicited a severe inflammatory response without improving visual function. In contrast, allogeneic transplantation of feline MGSC (fMGSC)-derived RGCs into the vitrectomized eye improved the scotopic threshold response (STR) of the electroretinogram (ERG). Despite causing functional improvement, the cells did not attach onto the retina and formed aggregates on peripheral vitreous remnants, suggesting that vitreous may constitute a barrier for cell attachment onto the retina. This was confirmed by observations that cellular scaffolds of compressed collagen and enriched preparations of fMGSC-derived RGCs facilitated cell attachment. Although cells did not migrate into the RGC layer or the optic nerve, they significantly improved the STR and the photopic negative response of the ERG, indicative of increased RGC function. These results suggest that MGSCs have a neuroprotective ability that promotes partial recovery of impaired RGC function and indicate that cell attachment onto the retina may be necessary for transplanted cells to confer neuroprotection to the retina. Significance: Müller glia with stem cell characteristics are present in the adult human retina, but they do not have regenerative ability. These cells, however, have potential for development of cell therapies to treat retinal disease. Using a feline model of retinal ganglion cell (RGC) depletion, cell grafting methods to improve RGC function have been developed. Using cellular scaffolds, allogeneic transplantation of Müller glia-derived RGC promoted cell attachment onto the retina and enhanced retinal function, as judged by improvement of the photopic negative and scotopic threshold responses of the electroretinogram. The results suggest that the improvement of RGC function observed may be ascribed to the neuroprotective ability of these cells and indicate that attachment of the transplanted cells onto the retina is required to promote effective neuroprotection.

Project description:Cyclosporin A (CsA) inhibits the opening of the mitochondrial permeability transition pore (MPTP) by interacting with cyclophilin D (CypD) and ameliorates neuronal cell death in the central nervous system against ischemic injury. However, the molecular mechanisms underlying CypD/MPTP opening-mediated cell death in ischemic retinal injury induced by acute intraocular pressure (IOP) elevation remain unknown. We observed the first direct evidence that acute IOP elevation significantly upregulated CypD protein expression in ischemic retina at 12?h. However, CsA prevented the upregulation of CypD protein expression and promoted retinal ganglion cell (RGC) survival against ischemic injury. Moreover, CsA blocked apoptotic cell death by decreasing cleaved caspase-3 protein expression in ischemic retina. Of interest, although the expression level of Bcl-xL protein did not show a significant change in ischemic retina treated with vehicle or CsA at 12?h, ischemic damage induced the reduction of Bcl-xL immunoreactivity in RGCs. More importantly, CsA preserved Bcl-xL immunoreactivity in RGCs of ischemic retina. In parallel, acute IOP elevation significantly increased phosphorylated Bad (pBad) at Ser112 protein expression in ischemic retina at 12?h. However, CsA significantly preserved pBad protein expression in ischemic retina. Finally, acute IOP elevation significantly increased mitochondrial transcription factor A (Tfam) protein expression in ischemic retina at 12?h. However, CsA significantly preserved Tfam protein expression in ischemic retina. Studies on mitochondrial DNA (mtDNA) content in ischemic retina showed that there were no statistically significant differences in mtDNA content among control and ischemic groups treated with vehicle or CsA. Therefore, these results provide evidence that the activation of CypD-mediated MPTP opening is associated with the apoptotic pathway and the mitochondrial alteration in RGC death of ischemic retinal injury. On the basis of these observations, our findings suggest that CsA-mediated CypD inhibition may provide a promising therapeutic potential for protecting RGCs against ischemic injury-mediated mitochondrial dysfunction.

Project description:High pressure is the most important factor inducing retinal ganglion cell (RGC) apoptosis. However, the underlying mechanisms remain obscure. The present study investigated the effects of different levels of hydrostatic pressure (HP) on RGCs and the potential mechanisms involved. Primary cultured rat RGCs were exposed to five levels of HP (0, 20, 40, 60 and 80 mmHg) for 24 h. Morphological changes in RGCs were observed. The viability and apoptosis rate of RGCs were detected using a Cell Counting Kit?8 assay and Annexin V?fluorescein isothiocyanate/propidium iodide flow cytometry, respectively. Western blotting, reverse transcription?quantitative polymerase chain reaction and immunofluorescence were used to detect the expression and mRNA levels of nerve growth factor (NGF), protein kinase B (AKT), apoptosis signal?regulating kinase 1 (ASK1), forkhead box O1 (FoxO1) and cAMP response element binding protein (CREB). In the 0? and 20?mmHg groups, there were no apoptotic morphological changes. In the 40 mmHg group, parts of the cell were shrunken or disrupted. In the 60 mmHg group, neurite extension was weakened and parts of the cells were disintegrating or dying. In the 80 mmHg group, the internal structures of the cells were not visible at all. The apoptosis rates of RGCs were significantly higher and the viability rates significantly lower under 40, 60 and 80 mmHg compared with under 0 or 20 mmHg (all P<0.01). The expression and mRNA levels of NGF, AKT and CREB decreased in a dose?dependent manner in the 40?, 60? and 80?mmHg groups (all P<0.05), but those of ASK1 and FoxO1 increased in a dose?dependent manner (all P<0.05). Interestingly, the alterations to the expression and mRNA levels of CREB were significantly larger compared with the changes in ASK1 or FoxO1 in the 40?, 60? and 80?mmHg groups (all P<0.01). The results of the present study demonstrate that elevated HP of 40, 60 or 80 mmHg reduces viability and induces apoptosis in RGCs, which may occur through effects on the NGF/ASK1/FoxO1 and NGF/AKT/CREB pathways, of which the latter is more strongly affected.

Dataset Information

Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP.

Publications

Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP.

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