Inhibiting Rho kinase blocked proNGF and diabetes-induced retinal neurodegeneration.

<p><b>A,B.</b> Representative images and statistical analysis of TUNEL-HRP-positive cells counted in each retina flat-mount showing ∼9-fold increased number of cell death in retinas from 5 weeks diabetic rats as compared with the controls (n = 4–5). <b>C.</b> Statistical analysis of total number of TUNEL-HRP-positive cells counted in each retina showing ∼6-fold increase of cell death in retinas that overexpress proNGF as compared with the GFP controls (n = 4–5). Treatment with the selective Rho kinase inhibitor Y27632 blocked these effects in diabetic and proNGF overexpression and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control; D, diabetic; Y, Y27632.</p

Diagram depicting the proposed role of proNGF/p75^NTR in diabetic retinopathy.

<p>Retinal neurodegeneration is thought to be induced via direct activation of RhoA kinase in RGC and paracrine inflammatory action in response to increases in proNGF.</p

Inhibiting Rho kinase blocked diabetes- and proNGF-induced apoptotic markers expression.

<p><b>A,C.</b> WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in rats electroporated with proNGF as compared with the controls (n = 4–5). <b>B,D.</b> WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 cells treated with proNGF as compared with the controls (n = 4). <b>E.</b> WB analysis showing 2.1- and 1.6-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 treated with proNGF as compared with the controls. Treatment of rats or RGC-5 with Y27632 blocked all these effects in rats and media treated with proNGF and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control.</p

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

Diabetes and overexpression of proNGF induced expression of p75^NTR in vivo and in vitro.

<p><b>A.</b> WB analysis showing 1.9-fold increase in the expression of p75^NTR in diabetic rats as compared with the controls (n = 4–6). <b>B.</b> WB analysis of rat retinal lysate showed significant increase in p75^NTR expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 4). <b>C.</b> Representative images of rat retina sections showing prominent immunolocalization of p75^NTR in GCL and INL in proNGF overexpressing retinas as compared with GFP-controls (400× magnification). <b>D.</b> Representative images of rat retina sections showing colocalization between p75^NTR in the ganglion cell layer (green) and the specific neuronal marker Thy-1 (red) in the upper pannel or with the specific RGC marker Brn-3a (red)in the lower pannel (400× magnification). <b>E.</b> Western blot analysis shwoing 1.6-fold increase in the expression of p75^NTR in RGC-5 cells treated with proNGF as compared with the controls (n = 4). <b>F.</b> Real-time PCR analysis showing that proNGF induced p75^NTR mRNA expression in freshly isolated primary RGC as compared with the control group. Samples of primary RGC cultures were pooled from 4-different cultures. Treatment with Y27632 significantly reduced p75^NTR expression <i>in vivo</i> and <i>in vitro</i>. G. Representative images showing colocalization (yellow arrow heads) of RGC that expressed p75^NTR (red) and the specific RGC marker Brn3a (green) in isolated mixed neuronal cultures from adult mice. * = significant difference as compared with the rest of the groups at p<0.05. C, control; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. C, control.</p

Inhibiting RhoA blocked proNGF-induced death in primary RGC cultures.

<p><b>A,B.</b> Representative images and statistical analysis showing ∼5-fold increase in TUNEL-positive cells in RGC-5 cells in response to mutant proNGF (50 ng/ml) (200× magnification). <b>C,D.</b> Representative images and statistical analysis showing ∼10-fold increase in TUNEL-positive cells in primary RGC cultures in response to mutant proNGF (50 ng/ml) (200× magnification). Co-treatment with Y27632 (1 µM) protected RGC in proNGF-treated group but had no effect on control cells. * = significant difference as compared with the rest of the groups at p<0.05 (n = 4). C, control; Y, Y27632.</p