Investigation of the role and modulation of autophagy for neuroprotection and nerve regeneration using models of peripheral nerve injury

Resumen

Severe peripheral nerve injury (PNI) cause axonal disruption and may produce retrograde neurodegeneration. Axotomized neurons undergo a series of phenotypic changes at the molecular and cellular levels, some of them called endogenous mechanisms of neuroprotection, that promote neuronal surviva that includes the unfolded protein response (UPR), the heat-shock response, the autophagy pathway, the ubiquitin-proteasome system, chaperone, the endoplasmic reticulum (ER)-associated degradation machinery (ERAD) and the antioxidant defence.The intensity and time course of the neuronal response are mainly influenced by the severity of the injury, distance of the lesion to cell body, type of neuron and age. However, when the injury is proximal to the soma, such in the case of peripheral nerve root avulsion (RA), the endogenous mechanisms of neuroprotection might not be properly activated contributing to neurodegeneration. We reasoned that the correction or potentiation of these mechanisms might be effective for neuroprotection. We first characterize the state of autophagy flux after PNI in vivo and found a blockage of these pathway, alterations in microtubule related proteins and vesicle trafficking proteins at 5-7 days post-injury.Subsequently, we modelize some concomitant events associated with autophagy failure such as cytoskeleton abnormalities in in vitro model. Furthermore, we analyse the time course response of autophagy and cytoskeleton in vitro. These revealed that neurodegeneration might occur due to initial microtubule alteration followed autophagy blockage. These cytoskeleton alterations increase astrogliosis and MN death in vivo. Finally, we explored the role of autophagy potentiation. Time-course analysis of pharmacological autophagy induction using rapamycin revealed to be neuroprotective only as as a pre-treatment before RA injury. In addition, autophagy activation mediated by ATG5 overexpression resulted in a MN preservation accompanied by improved internal trafficking and autophagy flux. Previous data demonstrated neuroprotective capacities mediated by GRP78/BiP overexpression that it has been found downregulated in degenerated MNs after the lesion. Considering its relationship with autophagy, we aimed to clarify the mechanisms of these neuroprotection by proteomic analysis. We discovered that GRP78/BiP overexpression induces the downregulation of mitochondrial proteins by the induction of mitophagy. In this activation of mitophagy by GRP78/BiP is implicate IP3R and PINK1. Finally, considering that an effective therapy after PNI should promote axonal regrowth and nerve regeneration, we explored if autophagy stimulation might be pro-regenerative as well. We did so by overexpressing ATG5 or by genetic and pharmacological activation of SIRT1. We discovered that autophagy mediated by SIRT-1/HIF1α promotes neurite outgrowth in vitro. In addition, autophagy potentiation by ATG5 or SIRT1 overexpression enhances functional recovery and axonal growh after the lesion. Overall, these findings suggested that correction or potentiation of endogenous mechanisms such as autophagy may be an effective therapy to increase the survival of disconnected MNs and enhanced axonal regrowth after the peripheral nerve injuries.