Sang-Soo Kang

Resveratrol inhibits neuronal apoptosis and elevated Ca2+/calmodulin-dependent protein kinase II activity in diabetic mouse retina

OBJECTIVE This study investigated the effects of resveratrol, a natural polyphenol with neuroprotective properties, on retinal neuronal cell death mediated by diabetes-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). RESEARCH DESIGN AND METHODS Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received buffer. All mice were killed 2 months after the injections, and the extent of neuronal cell death, CaMKII, and phospho-CaMKII protein expression levels and CaMKII kinase activity were examined in the retinas. To assess the role of CaMKII in the death of retinal neurons, a small-interfering RNA (siRNA) or specific inhibitor of CaMKII was injected into the right vitreous humor, and vehicle only was injected into the left vitreous humor, 2 days before death. Resveratrol (20 mg/kg) was administered by oral gavage daily for 4 weeks, beginning 1 month after the fifth injection of either STZ or buffer. RESULTS The death of retinal ganglion cells (RGCs), CaMKII, phospho-CaMKII protein levels, and CaMKII activity were all greatly increased in the retinas of diabetic mice compared with controls, 2 months after induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive signals co-localized with CaMKII- and phospho-CaMKII immunoreactive RGCs. However, in addition to CaMKII knockdown and inhibition by siRNA or a specific inhibitor, respectively, resveratrol provided complete protection from diabetes-induced retinal cell death. CONCLUSIONS In the present study, resveratrol prevented diabetes-induced RGC death via CaMKII downregulation, implying that resveratrol may have potential therapeutic applications for prevention of diabetes-induced visual dysfunction.

Protein Kinase C-δ Mediates Neuronal Apoptosis in the Retinas of Diabetic Rats via the Akt Signaling Pathway

OBJECTIVE—Protein kinase C (PKC)-δ, an upstream regulator of the Akt survival pathway, contributes to cellular dysfunction in the pathogenesis of diabetes. Herein, we examined the role of PKC-δ in neuronal apoptosis through Akt in the retinas of diabetic rats. RESEARCH DESIGN AND METHODS—We used retinas from 24- and 35-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) diabetic and Long-Evans Tokushima Otsuka (LETO) nondiabetic rats. To assess whether PKC-δ affects Akt signaling and cell death in OLETF rat retinas, we examined 1) PKC-δ activity and apoptosis; 2) protein levels of phosphatidylinositol 3-kinase (PI 3-kinase) p85, heat shock protein 90 (HSP90), and protein phosphatase 2A (PP2A); 3) Akt phosphorylation; and 4) Akt binding to HSP90 or PP2A in LETO and OLETF retinas in the presence or absence of rottlerin, a highly specific PKC-δ inhibitor, or small interfering RNAs (siRNAs) for PKC-δ and HSP90. RESULTS—In OLETF retinas from 35-week-old rats, ganglion cell death, PKC-δ and PP2A activity, and Akt-PP2A binding were significantly increased and Akt phosphorylation and Akt-HSP90 binding were decreased compared with retinas from 24-week-old OLETF and LETO rats. Rottlerin and PKC-δ siRNA abrogated these effects in OLETF retinas from 35-week-old rats. HSP90 siRNA significantly increased ganglion cell death and Akt-PP2A complexes and markedly decreased HSP90-Akt binding and Akt phosphorylation in LETO retinas from 35-week-old rats compared with those from nontreated LETO rats. CONCLUSIONS—PKC-δ activation contributes to neuro-retinal apoptosis in diabetic rats by inhibiting Akt-mediated signaling pathways.

Expression of 14-3-3 ζ and interaction with protein kinase C in the rat retina in early diabetes

Aims/hypothesisThe present study aimed to investigate the expression levels of and the relationship between 14-3-3 ζ and protein kinase C (PKC) in the retina of early diabetes.MethodsChanges in the expression levels of, and interaction between, 14-3-3 ζ and PKC were investigated by Northern and Western blot analyses, immunoprecipitation and double immunostaining in the retina of diabetic rats after 6 weeks of diabetes. PKC activity was examined using a PKC assay.ResultsIn the diabetic retina, the molecular levels of 14-3-3 ζ were reduced, while those of PKC β and ζ were increased. Direct interaction between 14-3-3 ζ and PKC was markedly decreased in the retina after 6 weeks of diabetes, while PKC activity was increased.Conclusions/interpretationThese findings show that a reduction in 14-3-3 ζ can induce PKC activation, suggesting that this is a main cause of visual dysfunction in the retina during diabetes.

Clusterin regulates transthyretin amyloidosis.

Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.