Hsp27 Overexpression Enhances Functional Recovery after Prolonged Denervation and Peripheral Neuropathy

Hsp27超量表達增強對長期去神經支配和周圍神經病變的功能恢復

Student thesis: Doctoral Thesis

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Award date3 Jan 2018

Abstract

Peripheral nerves are fragile and susceptible to physical trauma such as car accidents, traction injury, compression and lacerations. The slow growth rate of peripheral axons limits functional recovery in patients that involve long-distance axonal regeneration. Delay in target muscle reinnervation makes the distal milieu non-permissive for the regenerating axons and the denervated muscle undergoes atrophy. Recovery from peripheral nerve injury highly depends on the formation of functional synapse that keeps the skeletal muscle electrically active, a prerequisite for improved motor function recovery. Previous studies showed that regenerating axons must arrive at the target muscle within a “critical period” of 35 days in mice in order to restore motor function recovery. Our previous studies showed that forced expression of human Hsp27 (hHsp27) in neurons accelerated axonal regeneration and functional recovery in transgenic (Tg) mice after peripheral nerve injury. In this study therapeutic potential of hHsp27 beyond the critical time window was examined by using different chronic denervation models of 37, 45 and 55 days. Various parameters like neurobehavioral, electrophysiology, immunohistochemistry and gene expression studies were performed to assess the recovery during the study period. Sensory and motor functional recovery was significantly improved in hHsp27 Tg mice as compared with the littermate (LM) controls. The number of axons in Tg and LM mice was comparable however higher number of fully innervated neuromuscular junction (NMJ) was observed in hHsp27 Tg mice. Electrophysiological studies showed that muscle electrical activity increased considerably in hHsp27 Tg mice, which in line with the NMJ data. In addition, Plantar muscle mass was significantly higher in hHsp27 Tg mice than the LM controls, which showed reduction of muscle atrophy in Tg mice. Morphological analysis of sciatic nerve showed more number of large diameter myelinated axon and presence of healthy mitochondria at the distal tips of hHsp27 Tg group as compared to higher number of damaged mitochondria in LM group. To further investigate the signaling molecules involved during the accelerated regeneration by hHsp27 we focused on the genes involved in NMJ reinnervation and maintenance. qPCR analysis identified upregulation of several genes such as micro RNA-206, fibroblast growth factor (FGF-2), low density lipoprotein receptor related protein (Lrp4) and agrin in hHsp27 Tg mice at different time points. Next part of this study was designed to validate our observation in an animal model of peripheral neuropathy such as Guillain-Barré syndrome (GBS).

GBS is a fatal autoimmune disease affecting the peripheral nervous system. In GBS, immune cells start attacking the major gangliosides present on the peripheral nerves resulting in severe nerve damage. Several case reports confirmed the GBS symptoms after the infection of Campylobacter jejuni. Recent reports show Zika virus infection also induced GBS symptoms. Majority of the GBS patients experienced neurological symptoms such as paresthesia, muscle weakness, pain and areflexia. We showed that forced overexpression of human (h) Hsp27 could overcome anti-ganglioside mediated nerve regeneration inhibition. Passive transfer of anti-ganglioside antibodies (GD1a/GT1b-2b)/IB7 into hHsp 27 transgenic (Tg) mice and their littermates was done to study their functional recovery. In our chronic animal model (90 days) we observed improved sensory and motor functional recovery in hHsp27 Tg mice as compared to their littermates. Our electromyography and histology data showed that hHsp27 Tg mice demonstrated marked improvement of muscle function in terms of increased compound muscle action potential (CMAP) and NMJ reinnervation. To further investigate we used sub-acute animal model (30 days) where we found increased axon number at distal site in addition to higher innervated NMJ and reduced muscle atrophy in hHsp27 Tg group. All the data were validated in two separate line overexpressing hHsp27. Our future work is to elucidate the molecular pathway of hHsp27 involved in overcoming the inhibitory effect of anti-gangliosides. Overall these findings suggest that HSP27 is a potential molecule to be screened as a drug for peripheral nerve neuropathy.