Diabetes and Overexpression of proNGF Cause Retinal Neurodegeneration via Activation of RhoA Pathway

Abstract

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 streptozotosin 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, p75NTR, 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 p75NTR 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 p75NTR expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.

Neuropeptides, Trophic Factors, and Other Substances Providing Morphofunctional and Metabolic Protection in Experimental Models of Diabetic Retinopathy

“…The direct apoptotic effect of proNGF on RGCs delineates the apoptotic role of RhoA activation in retinal neurodegeneration. The significant activation of RhoA/p38 mitogen-activated protein kinase (MAPK) pathway causes neuronal death with increasing phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK in response to overexpression of proNGF in the diabetic rat retina (Al-Gayyar et al, 2013). …”

Nerve growth factor in diabetic retinopathy: beyond neurons

“…In addition to alterations in TrkA function, expression of the p75 NTR receptor is increased in diabetic retinas, which combined with increased proNGF expression, favors activation of proapoptotic and proinflammatory pathways [30,32,33]. In the diabetic retina, this shift toward proNGF/p75 NTR signaling is accompanied by neurodegeneration, evidenced by death of RGC, vascular dysfunction and activation of RhoA/p38MAPK, a common pathway implicated in both neuronal death and vascular permeability [29,31,54–59]. The action of proNGF/p75 NTR does not depend on additional factors present in the diabetic milieu, as evidenced by studies in which overexpression of cleavage-resistant proNGF, in otherwise healthy rodent retinas, causes increased neuronal cell death and vascular permeability [33,54,60].…”

“…Our work demonstrated that proNGF-induced apoptosis in primary RGC cultures is accompanied by increased p75 NTR expression, and Rho kinase-dependent activation of RhoA, p38MAPK and JNK [54], all participants in apoptotic pathways downstream of the p75 NTR receptor [52,55,56,67,68]. Additionally, in both diabetic rats and in a rat model overexpressing proNGF, the p75 NTR receptor and apoptotic markers, cleaved poly (ADP-ribose) polymerase and caspase-3, are also increased in a Rho kinase-dependent manner [54]. Collectively, these results suggest a mechanism of retinal neurodegeneration triggered by proNGF/p75 NTR activation of RhoA and p38MAPK/JNK apoptotic pathways in RGC.…”

“…However, this mechanism is not without controversy. Although the p75 NTR receptor directly mediates neuronal death in the developing retina, there is disagreement whether the p75 NTR receptor is expressed by RGCs in the mature retina [54,69–72]. Another pathway by which proNGF can cause ganglion cell death involves paracrine effect of proNGF/p75 NTR -mediated secretion of TNF-α by Müller cells [33,72,73].…”

“…One possibility is that proNGF/p75 NTR -mediated activation of RhoA leads to degradation of tight junction proteins and increased permeability. RhoA is a molecular switch known to be activated in the diabetic retina, and activation of RhoA/p38MAPK is a common pathway implicated in both neuronal death and vascular permeability [29,31,54–59]. One question yet to be answered is what relationship does proNGF/p75 NTR -mediated permeability have with VEGF?…”