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.
Highlights
Our recent studies showed significant accumulation of proNGF that was positively correlated with accelerated retinal neurodegeneration in models of diabetes [1,2]
Since ganglion cell layer (GCL) contains a mixed population of retinal ganglion cells and displaced amacrine cells, RGCs were labeled and counted using Brn3a antibody (Fig. 2B) and normalized to retina length
The main findings of the current study are: 1) Overexpression of the proNGF mimics diabetes action resulting in retinal neurodegeneration in vivo and in vitro, 2) Inhibiting Rho kinase exerted neuroprotective effects by inhibiting p75NTR expression, inhibiting inflammation and activation of JNK/p38MAPK in response to proNGF or diabetes (Fig. 8)
Summary
Our recent studies showed significant accumulation of proNGF that was positively correlated with accelerated retinal neurodegeneration in models of diabetes [1,2]. Our studies demonstrated a mechanism by which diabetes-induced peroxynitrite impairs the activity and expression of MMP-7, an extracellular enzyme involved in maturation of NGF leading to accumulation of the proNGF [2]. In non-diabetic models, overexpression of p75NTR has been shown to constitutively activate RhoA leading to neuronal death via activation of p38MAPK pathway [8,9,10,11,12]. RhoA is a major small GTP-binding protein that acts as a molecular switch to play either a pro-death or pro-survival role in the nervous system depending on both the type of neuron and the particular neurodegenerative insult involved (for review [16]). Prior report showed that activation of RhoA can directly induce neuronal death in excitotoxic model [9]. Whether RhoA activation can induce retinal neurodegeneration in response to proNGF or diabetic insult remains unexplored
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