Epicatechin blocks pro-nerve growth factor (proNGF)-mediated retinal neurodegeneration via inhibition of p75 neurotrophin receptor expression in a rat model of diabetes [corrected].
ABSTRACT Accumulation of pro-nerve growth factor (NGF), the pro form of NGF, has been detected in neurodegenerative diseases. However, the role of proNGF in the diabetic retina and the molecular mechanisms by which proNGF causes retinal neurodegeneration remain unknown. The aim of this study was to elucidate the role of proNGF in neuroglial activation and to examine the neuroprotective effects of epicatechin, a selective inhibitor of tyrosine nitration, in an experimental rat model of diabetes.
Expression of proNGF and its receptors was examined in retinas from streptozotocin-induced diabetic rats, and in retinal Müller and retinal ganglion cells (RGCs). RGC death was assessed by TUNEL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in diabetic retinas and cell culture. Nitrotyrosine was determined using Slot-blot. Activation of the tyrosine kinase A (TrkA) receptor and p38 mitogen-activated protein kinase (p38MAPK) was assessed by western blot.
Diabetes-induced peroxynitrite impaired phosphorylation of TrkA-Y490 via tyrosine nitration, activated glial cells and increased expression of proNGF and its receptor, p75 neurotrophin receptor (p75(NTR)), in vivo and in Müller cells. These effects were associated with activation of p38MAPK, cleaved poly-(ADP-ribose) polymerase and RGC death. Treatment of diabetic animals with epicatechin (100 mg kg(-1) day(-1), orally) blocked these effects and restored neuronal survival. Co-cultures of RGCs with conditioned medium of activated Müller cells significantly reduced RGC viability (44%). Silencing expression of p75(NTR) by use of small interfering RNA protected against high glucose- and proNGF-induced apoptosis in RGC cultures.
Diabetes-induced peroxynitrite stimulates p75(NTR) and proNGF expression in Müller cells. It also impairs TrkA receptor phosphorylation and activates the p75(NTR) apoptotic pathway in RGCs, leading to neuronal cell death. These effects were blocked by epicatechin, a safe dietary supplement, suggesting its potential therapeutic use in diabetic patients.
- SourceAvailable from: Mohammed M Al-Gayyar[show abstract] [hide abstract]
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, 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.PLoS ONE 01/2013; 8(1):e54692. · 3.73 Impact Factor
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ABSTRACT: Aims The purpose of this study was to investigate the antinociceptive effect of epicatechin as well as the possible mechanisms of action in diabetic rats. Main methods Rats were injected with streptozotocin to produce hyperglycemia. The formalin test was used to assess the nociceptive activity. Key findings Acute pre-treatment with epicatechin (0.03-30mg/kg, i.p.) prevented formalin-induced nociception in diabetic rats. Furthermore, daily or every other day treatment for 2weeks with epicatechin (0.03-30mg/kg, i.p.) also prevented formalin-induced nociception in diabetic rats. Acute epicatechin-induced antinociception was prevented by L-NAME (N(ω)-nitro-L-arginine methyl ester hydrochloride, 1-10mg/kg, non-selective nitric oxide synthesis inhibitor), 7-nitroindazole (0.1-1mg/kg, selective neuronal nitric oxide synthesis inhibitor), ODQ (1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one, 0.2-2mg/kg, guanylyl cyclase inhibitor) or glibenclamide (1-10mg/kg, ATP-sensitive K(+) channel blocker). Moreover, epicatechin (3mg/kg)-induced antinociception was fully prevented by methiothepin (0.1-1mg/kg, serotonergic receptor antagonist), WAY-100635 (0.03-0.3mg/kg, selective 5-HT1A receptor antagonist) or SB-224289 (0.03-0.3mg/kg, selective 5-HT1B receptor antagonist). In contrast, BRL-15572 (0.03-0.3mg/kg, selective 5-HT1D receptor antagonist) only slightly prevented the antinociceptive effect of epicatechin. Naloxone (0.1-1mg/kg, opioid antagonist) did not modify epicatechin´s effect. Significance Data suggest the involvement of the nitric oxide-cyclic GMP-K(+) channels pathway as well as activation of 5-HT1A and 5HT1B, and at a lesser extent, 5-HT1D, but not opioid, receptors in the antinociceptive effect of epicatechin in diabetic rats. Our data suggest that acute or chronic treatment with epicatechin may prove to be effective to treat nociceptive hypersensitivity in diabetic patients.Life sciences 09/2013; · 2.56 Impact Factor
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ABSTRACT: Neurotrophins can regulate opposing functions that result in cell survival or apoptosis, depending on which form of the protein is secreted and which receptor and signaling pathway is activated. We have recently developed a transgenic model in which inducible and patchy Muller cell ablation leads to photoreceptor degeneration. This study aimed to examine the roles of mature neurotrophin-3 (NT3), pro-NT3 and p75 neurotrophin receptor (P75NTR) in photoreceptor degeneration in this model. Transgenic mice received tamoxifen to induce Muller cell ablation. Changes in the status of Muller and microglia cells as well as expression of mature NT3, pro-NT3 and P75NTR were examined by immunohistochemistry and Western blot analysis. Recombinant mature NT3 and an antibody neutralizing 75NTR were injected intravitreally 3 and 6 days after Muller cell ablation to examine their effects on photoreceptor degeneration and microglial activation. We found that patchy loss of Muller cells was associated with activation of surviving Muller cells and microglial cells, concurrently with reduced expression of mature NT3 and upregulation of pro-NT3 and P75NTR. Intravitreal injection of mature NT3 and a neutralizing antibody to P75NTR, either alone or in combination, attenuated photoreceptor degeneration and the beneficial effect was associated with inhibition of microglial activation. Our data suggest that Muller cell ablation alters the balance between the protective and deleterious effects of mature NT3 and pro-NT3. Modulation of the neuroprotective action of mature NT3 and pro-apoptotic pro-NT3/P75NTR signaling may represent a novel pharmacological strategy for photoreceptor protection in retinal disease.Journal of Neuroinflammation 11/2013; 10(1):137. · 4.35 Impact Factor