PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future

Sigma 1 receptor regulates ERK activation and promotes survival of optic nerve head astrocytes

<div><p>The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death.</p></div

S1R knockdown in HeLa cells and ONHAs.

<p>HeLa cells were transfected with human scrambled siRNA or with human S1R siRNA. Western blot analysis of S1R levels at 3 days (A) and 6 days (B) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (C, D). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control *p<0.05, **p<0.01. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (E) and 6 days (F) after scrambled or S1R siRNA transfection. Viability was significantly decreased in S1R siRNA-transfected HeLa cells compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant HeLa cell death compared to non-transfection control (G, H). Data were analyzed using t-test. Significantly different from control ****p<0.0001. Experiments were performed in quadruplicate and repeated 3 times. ONHAs were transfected with rat scrambled siRNA or with rat S1R siRNA. Western blot analysis of S1R levels at 3 days (I) and 6 days (J) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (K, L). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control: **p<0.01, ***p<0.001. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (M) and 6 days (N) after scrambled or S1R siRNA transfection. Viability was not significantly changed in S1R siRNA-transfected ONHAs compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant ONHA death compared to non-transfection control (O, P). Experiments were performed in quadruplicate and repeated 3 times.</p

Knockdown of S1R within ONHAs blocks the (+)-pentazocine-mediated suppression of ERK1/2 phosphorylation.

<p>Phophorylation of ERK was detected after 3 days (A) and 6 days (C) following transfection of scrambled or S1R siRNA in ONHA. At 3 days (A) and 6 days (C) following S1R siRNA transfection, pERK was increased compared to scrambled siRNA control. (B, D) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from S1R siRNA transfected cells versus scrambled siRNA transfected control cells. Data were analyzed using t-test. Significantly different from control: *p<0.05. Experiments were repeated 3 times. (E) Effect of PTZ on H₂O₂–exposed S1R siRNA transfected ONHA. 5 days after S1R siRNA transfection, ONHA were incubated with 100μM H₂O₂ at 37°C for 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by cotreatment). Western blot analysis is shown. (F) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from H₂O₂ exposed, S1R siRNA transfected cells versus control non-H₂O₂ exposed, S1R siRNA transfected cells. Data were analyzed using two-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons.</p

Diabetes and overexpression of proNGF cause retinal neurodegeneration via activation of RhoA pathway.

Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotocin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75(NTR), cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75(NTR) and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75(NTR) expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways

ProNGF selectively activates RhoA kinase activation in vivo and in RGC cultures.

<p><b>A.</b> Pull-down assay of rat retinal lysate showed 2.3-fold increase in the expression of active Rho in diabetic rats as compared with the controls (n = 4–5). <b>B.</b> Pull-down assay of rat retinal lysate showed 1.7-fold increase in active RhoA expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 5). <b>C.</b> Pull-down assay of RGC-5 lysate showed 1.6-fold increase in RhoA expression in RGC-5 cells treated with proNGF as compared with the controls (n = 4). Treatment of rats or RGC-5 with Y27632 blocked RhoA activation proNGF-treated samples but not the control groups. <b>D.</b> Pull-down assay of RGC-5 showing that treatment of RGC-5 cells with TNF-α did not increase RhoA activation as compared with the control group (n = 4). <b>E.</b> Statistical analysis showing overexpression of proNGF in healthy retina induced 3-fold increase in TNF-α mRNA expression as compared with the control group. <b>F.</b> Statistical analysis of G-LISA showing 1.7-fold increase of RhoA in primary RGC cultures treated with proNGF as compared with the control. TNF-α caused modest increase in RhoA activation (20%) as compared with the controls. These effects were reduced by treatment with Y27632. * = significant difference as compared with the control group at p<0.05.</p