Hyperglycemic neuropathy and its relevance to mitochondrial function: In vitro investigations on the potential neuroprotective effect of mitochondrial (dys)function and mitophagy/autophagy modulation

Neuropathy is the most prominent complication (approximately in half of patients) of chronic hyperglycemia. Mitochondrial dysfunction and mitophagy/autophagy demodulation have been indicated to play crucial roles in pathogenesis of hyperglycemic neuropathy; however, whether agents that provide these modulatory effects have neuroprotective effects has not investigated. In this context, the aim of this thesis study is to evaluate the neuroprotective effects of selected agents known to provide aforementioned modulations (anethole trithione, urolithin A, rapamycin and their combinations with metformin) against in vitro stress induced by high glucose (HG) in dorsal root ganglion (DRG) neuron cell culture and to provide evidence for their therapeutic potential. DRG neurons from (12 weeks old) C57BL/6J mice were cultured as a cellular model of hyperglycemic neuropathy. The neuroprotective effects of test agents on HG-induced neurons were examined using "cell index" as main parameter with xCELLigence real-time cell analyzer for 70 hours. Statistical analyses were performed by one-way ANOVA and Fisher's LSD post-hoc test, with significance set at p<0.05. HG (50 mM) exposure caused a significant decline in cell indices in DRG neurons with physiological glucose levels. Co-treatment of anethole trithione with HG-induction failed to significantly attenuate HG-induced neurotoxicity at any dose (p>0.05). Co-treatment of urolithin A (10 μM or 30 μM) or rapamycin (0.1 μM or 0.3 μM) with HG-induction significantly prevented HG-induced decrease in cell index (p=0.01, p=0.04, p=0.036 and p=0.008 respectively). Co-treatment of metformin or its combinations with other test agents failed to significantly attenuate HG-induced neurotoxicity (p>0.05). This thesis study indicates unique evidence that urolithin A and rapamycin, which modulate mitophagy/autophagy, have dose-dependent neuroprotective capacity against HG-induced neurotoxicity in murine DRG neurons. Further in vivo preclinical and clinical studies are warranted to address the results of this study and providing evidence on the mediating molecular mechanisms. Keywords: Diabetic Neuropathy, Hyperglycemia, Mitophagy, Mitochondria, Autophagy