Role of autophagy in reducing peripheral neuropathy and diabetic peripheral neuropathy

Abstract

Peripheral neuropathy occurs when there is damage to the central or peripheral nervous system due to injury, disease, diabetes, or drug use. Endoplasmic reticulum (ER) stress is a distinct feature observed in neuropathic pain. Neuropathy is accompanied by changes in the levels of various proteins such as GSK-3β. GSK-3β not only regulates gluconeogenesis but also autophagy thus playing a key role in cellular physiology including proteostasis and autophagy. 6-bromoindirubin-3'-oxime (6-BIO) is an inhibitor of GSK-3β. Inhibition of GSK-3β results in inhibition of mTOR thereby activating autophagy. To understand the effect of ER stress and autophagy in alleviating pain, ER stress induced by tunicamycin was used to elicit pain in these studies. Chronic Diabetic animal model was also used to investigate diabetic peripheral neuropathy. Tunicamycin blocks N-linked glycosylation leading to ER stress. As ER stress is one of the hallmarks of neuropathic pain, we have used intraplantar injection of tunicamycin to induce neuropathic pain. Peripheral neuropathy induced by tunicamycin and that during chronic diabetes was observed to be reduced upon treatment with 6-BIO in rat as well as mice models. To substantiate the involvement of ER stress in pathway intrinsic to neuropathic pain and the role of autophagy in mitigating it, SH-SY5Y neurons were used as a model system. Western blot, RT-PCR, and fluorescence microscopy were deployed to study the effect of 6-BIO in modulating ER stress and autophagy in SH-SY5Y neurons pre-treated with tunicamycin. p-mTOR, is a widely used marker for examining ER stress and autophagy, hence its upregulation denotes occurrence of ER stress, and its down regulation indicates autophagy. It was observed that the level of p-mTOR was increased in SH-SY5Y neuronal cells upon treatment with tunicamycin implying a reduction in autophagy. But when 6-BIO was added to the cells already treated with tunicamycin there was a reduction in the level of p-mTOR showing that 6-BIO increased autophagy in cells already treated with tunicamycin. Additionally, decrease in expression of protein markers like LC-3 and SQSTM1/p62 after treatment with 6-BIO in cells pre-treated with tunicamycin shows that the autophagic flux is increased. Upregulation of markers like Beclin1 (BECN1) and LC3 by RT-PCR confirmed an increase in autophagy upon treatment with 6-BIO in presence of tunicamycin. Further assessment to delineate the type of autophagy that was being upregulated by 6-BIO, use of markers like Cathepsin D (CTSD) for degradative autophagy and Rab8A for secretory autophagy showed that secretory autophagy was increased by tunicamycin, while degradative autophagy was increased and at the same time secretory autophagy was reduced when 6-BIO was given after tunicamycin treatment. This shows that 6-BIO treatment of cells under ER stress leads to the formation of autolysosome leading to the degradation of damaged proteins thereby reducing cellular burden caused by ER stress, thus leading to mitigation of pain. A major contributing factor to the effect of tunicamycin in increasing pain is the increased ER stress. By using markers ER stress (CHOP and GRP78) it was observed that 6-BIO treatment in SH-SY5Y cells could reduce the ER stress caused by tunicamycin. Unregulated ER stress leads to apoptosis of cells which further exacerbates the effect of ER stress. Caspase-3 (a marker for apoptosis) was used to assess the effect of 6-BIO on apoptosis. 6-BIO in presence of tunicamycin reduced the protein expression of caspase-3 when compared to only tunicamycin treated cells. Thus, 6-BIO blocks apoptosis resulting from tunicamycin induced ER stress. c-fos is upregulated during neuronal activity including neuropathic pain hence it was used as a marker to verify the occurrence of neuropathy in these neurons by ER stress and its mitigation by increased autophagic flux. The expression of c-fos was increased when treated with tunicamycin and the addition of 6-BIO in tunicamycin background reduced the expression of c-fos. The change in c-fos confirms the observations of behavioral studies which show that tunicamycin increases neuropathy and treatment with 6-BIO after tunicamycin reduced pain. From reported studies it is known that 6-BIO inhibits the activity GSK-3β which is likely to also affect the mobilization of calcium. Further, calcium is an important secondary messenger in neuronal signaling and its levels are altered in neuropathy and diabetic neuropathy. In the current study it is shown that treatment with tunicamycin increases the mobilization of calcium in SH-SY5Y neuronal cells and DRG neurons due to perturbation of ER homeostasis. But when SH-SY5Y cells and DRG neurons are treated with 6-BIO before or after tunicamycin treatment show that the mobilization of calcium is reduced. Also, a reduction of mitochondrial calcium was seen in SH-SY5Y cells when treated with 6-BIO after treatment with tunicamycin. The reduction of calcium mobilization by 6-BIO in tunicamycin background might help in alleviating pain consistent with earlier studies on the causal role of an increase in calcium in peripheral neuropathy. In summary, 6-BIO helps in reducing neuropathic pain caused by tunicamycin and diabetic neuropathy and it does so by reducing ER stress and apoptosis, increasing autophagy, and preventing calcium mobilization. In appendix chapter preliminary experiments are performed to explore the possibility of treating ER stress induced neuropathy by using an activator of Nrf2 – a transcription factor involved in modulating the neuronal redox state.