whole-thesis-leung.pdf (3.94 MB)
The role of metallothionein I/II in promoting axonal sprouting in both central nervous system and peripheral nervous system
thesisposted on 2023-05-26, 03:03 authored by Leung, YK
Metallothioneins are a family of low molecular weight, metal-binding proteins with an unusually high cysteine content. The metallothionein I/II isoforms (hereafter referred to as MT) have been demonstrated extensively in a number of studies to promote axonal outgrowth of a variety of central nervous system (CNS) neurons, both in vivo and in vitro, after traumatic injury. However, the precise mechanisms through which MT interacts with injured neurons to promote their regeneration after injury remains largely unknown. The goal of this thesis is to identify some of these molecular mechanisms in both the injured CNS and peripheral nervous system (PNS). The first part of this thesis aimed to identify how exogenous MT interacts with glia and neurons in the CNS after traumatic injury using the in vivo focal needle-stick injury model. It was found that injection of fluorescently labeled-MT (488-MT) into the injury site led to a decrease in overall injury size compared to vehicle treatment (fluorescentunincorporated dye, AlexaFluor-488). Histological analysis found that 488-MT was present in both neurons as well as reactive microglia surrounding the injury site. The internalization of 488-MT by cortical neurons suggests that the injected 488-MT might interact directly upon injured neurons to promote regenerative axonal sprouting, which would be in accordance with prior studies demonstrating that neuronal internalization is required for MT to promote initial neurite outgrowth and elongation. To identify the signaling pathway through which MT exerts its effect upon neurons, the MT were co-injected with receptor-associated protein (RAP), an antagonist for megalin (a recently identified receptor for MT) into the injured cortex using the same injury model. The results demonstrated that the addition of RAP has blocked the effect of MT in promoting regenerative axonal sprouting after injury, thus confirming that MT acts through LRP receptors-dependent pathways to exert its regenerative effect upon injured cortical neurons. The uptake of MT by microglia led to a further investigation into the direct effect of MT on the microglial response after injury. In vivo data suggested that the injection of 488- MT caused a morphological change in reactive microglia. There was also enhanced physical association between reactive microglia and regeneratively spouting neurons. Using a series of neuron-microglia co-culture and media exchange experiments, it was found that MT could block the inhibitory effect of cytokine-activated microglia had upon neurite outgrowth of cultured cortical neurons. Hence this demonstrates that MT might potentially modulate the microglial response, which indirectly leads to improvement in regenerative axonal sprouting after injury. The second half of this thesis evaluated the potential role of MT in promoting nerve regeneration in the PNS. The rationale for this study relates to recent reports that another neuroprotective protein, transthyretin, can promote neurite outgrowth of dorsal root ganglion (DRG) neurons via interaction with the megalin receptor (also a putative receptor for MT). To investigate the effect of MT in promoting neurite regeneration in the DRG neurons, MT was applied to DRG neuron cultures, which led to an improvement in axonal sprouting after scratch injury. To investigate in further detail the mechanism of action of this effect, a compartmentized in vitro injury model was established, which allowed the precise and restricted application of MT to either the axonal processes or neuronal soma immediately after axonal scratch injury. The length of regenerative axonal sprouts was measured 24 hours after injury, and a 4-fold or 1.4-fold increase in regeneration was observed when MT was administered to either the soma or axonal compartments respectively (fold change relative to vehicle-treated control). Interestingly, this correlated with the expression of megalin, which was restricted primarily to the neuronal soma, with very little megalin labeling observed in axons. Finally, MT was found to promote DRG neuron regeneration via LRP- and MAPK- (mitogen-activated protein kinase-)-dependent pathway. Overall, this thesis has provided new insight to the field in understanding some of the precise mechanisms through which MT promotes neural regeneration in both CNS and PNS. It demonstrated that MT may potentially act directly upon microglia to modulate the extracellular environment in response to brain injury, which may then facilitate neuronal sprouting after injury in the CNS. This thesis also demonstrates that MT promotes peripheral nerve regeneration after injury via a megalin and MAPK-dependent mechanism.
Rights statementCopyright 2011 the author Chapter 4 appears to be the equivalent, or less, of an article published in Cellular and molecular life sciences. The final authenticated version is available online at: https://doi.org/10.1007/s00018-011-0790-7