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The role of metallothionein I/II in promoting axonal sprouting in both central nervous system and peripheral nervous system

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Leung, YK (2011) The role of metallothionein I/II in promoting axonal sprouting in both central nervous system and peripheral nervous system. PhD thesis, University of Tasmania.

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Abstract

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.

Item Type: Thesis (PhD)
Keywords: metallothionein, brain injury, nerve regeneration
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Date Deposited: 20 Apr 2012 05:29
Last Modified: 11 Mar 2016 05:53
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