p58IPK Is an Endogenous Neuroprotectant for Retinal Ganglion Cells

Abstract

p58^IPK is an endoplasmic reticulum (ER)-resident chaperone playing a critical role in facilitating protein folding and protein homeostasis. Previously, we have demonstrated that p58^IPK is expressed broadly in retinal neurons including retinal ganglion cells (RGCs) and loss of p58^IPK results in age-related RGC degeneration. In the present study, we investigate the role of p58^IPK in neuroprotection by in vitro and in vivo studies using primary RGC culture and two well-established disease-relevant RGC injury models: retinal ischemia/reperfusion (I/R) and microbead-induced ocular hypertension. Our results demonstrate that in both in vivo models, p58^{IPK -/-} mice exhibit significantly increased RGC loss compared to wild type (WT) mice. In vitro, p58^IPK-deficient RGCs show reduced viability and are more susceptible to cell death induced by the ER stress inducer tunicamycin (TM). Overexpression of p58^IPK by adeno-associated virus (AAV) significantly diminishes TM-induced cell death in both WT and p58^{IPK -/-} RGCs. Interestingly, we find that loss of p58^IPK leads to reduced mRNA expression, but not the protein level, of mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor that resides in the ER. Treatment with recombinant MANF protein protects R28 retinal neural cells and mouse retinal explants from TM-induced cell death. Taken together, our study suggests that p58^IPK functions as an endogenous neuroprotectant for RGCs. The mechanisms underlying p58^IPK's neuroprotective action and the potential interactions between p58^IPK and MANF warrant future investigation.

Keywords: X-box binding protein 1; ocular hypertension; p58IPK; retinal ganglion cells; retinal ischemia/reperfusion.

Figure 1

Loss of p58^IPK exacerbates retinal ganglion cell (RGC) cell death in retinal ischemia/reperfusion (I/R) and microbead-induced ocular hypertension. (A) Retinal cryosections (20 μM) from 2-month-old wild type (WT) and p58^{IPK −/−} mice labeled with primary antibodies against various retinal markers and a fluorescent secondary antibody (red). Anti-Pax6 labels amacrine cells and some RGCs; anti-PKCα labels rod bipolar cells and their dendrites as well as a subset of amacrine cells; anti-Ribeye labels ribbon synapses between bipolar cells and photoreceptors in the OPL, as well as synapses between amacrine cells and RGCs in the IPL; anti-calretinin labels three synaptic lamina within the IPL. There is no clear difference between WT and p58^{IPK −/−} retina in labeling for any of these markers. All sections are counterstained with DAPI (blue) to label nuclei. n = 3 mice per group, scale bar = 50 μm. (B) Retinal whole mounts from WT and p58^{IPK −/−} mice at 7 days after retinal I/R were stained with RGC marker, Tuj1. Representative images are shown on the left. Graph shows that the percentage of RGC loss, as determined from wholemount retinal Tuj1 staining, is significantly higher in p58^{IPK −/−} mice compared to WT. Mean ± SD, n = 5 mice. *p < 0.05 (Student’s t-test). Scale bar = 100 μm. (C) Graph depicts the average intraocular pressure (IOP) of sham control eyes and microbead-injected eyes in WT (+/+) and p58^{IPK −/−} mice at the time points as indicated. (D) Retinal whole mounts from WT and p58^{IPK −/−} mice at 14 days after microbead injection were stained with RGC marker, Tuj1. Representative images are shown on the left. Bar graph shows the percentage of RGC loss is significantly greater in p58^{IPK −/−} mice compared to WT. Mean ± SD, n = 4 mice. *p < 0.05 (Student’s t-test).

Figure 2

Overexpressing p58^IPK by adeno-associated virus (AAV) protects RGCs from endoplasmic reticulum (ER) stress-induced cell death. (A) Cultured primary mouse RGCs isolated from p58^{IPK −/−} and WT retinas were infected with AAV-GFP or AAV-p58^IPK 24 h prior to a 16 h-treatment with tunicamycin (TM; 1 μg/ml) or control. Cells are labeled with the live/dead cytotoxicity assay (calcein-AM to label live cells (green) and ethidium homodimer-1 to label dead cells (red)). Representative images of live/dead assay are shown above. Graph depicts the survival percentage of RGCs from WT and p58^{IPK −/−} mice. Under control conditions, p58^{IPK −/−} RGCs infected with AAV-GFP have a significantly lower survival percentage than the matched WT group. RGCs from both WT and p58^{IPK −/−} mice infected with AAV-GFP show a significant decrease in survival percentage after TM treatment compared to control treatment. The survival percentage is significantly increased for RGCs infected with AAV-p58^IPK, compared to infection with AAV-GFP for both WT and p58^{IPK −/−} mice. Mean ± SD, n = 3 independent experiments. *p < 0.05 (one-way ANOVA with Bonferroni post hoc test). Scale bar = 80 μm. (B) Quantitative RT-PCR (qPCR; top) using total RNA from whole retina from 2-month-old WT and p58^{IPK −/−} mice. Two sets of independent primers specific for mesencephalic astrocyte-derived neurotrophic factor (MANF) reveal a mean significant 50% reduction in mRNA level in p58^{IPK −/−} retina compared to WT. All qPCR data are normalized to 18 s. Mean ± SD, n = 3 mice per genotype. *p < 0.05 (Student’s t-test). Western blot analysis (bottom) of whole retina lysate from WT or p58^{IPK −/−} mice reveal that the level of MANF protein is not statistically different for whole retina (n = 6 mice per group). (C) R28 cells in culture exposed to 16-h pre-treatment with 50 ng/ml MANF or vehicle, followed by 5 μg/ml TM or control treatment for 24 h. Cells are labeled with the live/dead cytotoxicity assay as in (A). Quantification of the survival of R28 cells reveals that approximately 60% of R28 cells survive TM treatment in the absence of MANF. With MANF pre-treatment the survival percentage is significantly increased to over 85%. Mean ± SD, n = 4 independent experiments; *p < 0.05, **p < 0.01 (one-way ANOVA with Bonferroni post hoc test). Scale bar = 100 μm.