Abstract
BackgroundThe extracellular environment plays an important role in supporting the regeneration of axons after injury. Metallothionein-II (MTII) is a metal-binding protein known for its neuroprotective effect by directly stimulating the growth of axons after injury. Previous studies have shown that MTII also modulates the response of astrocytes and microglia after injury. However, a detailed analysis describing how MTII modulates the interaction between microglia and neurons is lacking.MethodsWe introduced fluorescently labelled MTII into the cortex at the time of needlestick injury to investigate the cellular uptake of MTII using immunohistochemistry with antibodies against cell-type-specific markers. The role of MTII in modulating the effect of microglia on axon outgrowth following an inflammatory response is further investigated using a co-culture model involving primary rodent microglia pre-treated with TNFα and primary rodent cortical neurons. The axon lengths were assessed 24 h after the plating of the neurons onto treated microglia. We also utilised siRNA to knockdown the expression of LRP1, which allows us to investigate the role of LRP1 receptors in the MTII-mediated effect of microglia on axon outgrowth.ResultsFluorescently labelled MTII was found to be associated with neurons, astrocytes and microglia following injury in vivo. Microglia-neuron co-culture experiments demonstrated that exogenous MTII altered the response of microglia to TNFα. The neurons plated onto the TNFα-stimulated microglia pre-treated with MTII have shown a significantly longer axonal length compare to the TNFα-stimulated microglia without the MTII treatment. This suggested that MTII reduce cytokine-stimulated activation of microglia, which would ordinarily impair neurite outgrowth. This inhibitory effect of MTII on activated microglia was blocked by siRNA-mediated downregulation of LRP1 receptor expression in microglia, suggesting that MTII acts via the LRP1 receptor on microglia.ConclusionsThis study demonstrates that exogenous MTII acts via the LRP1 receptor to alter the inflammatory response of microglia following TNFα stimulation, providing a more supportive environment for axon growth.
Highlights
The extracellular environment plays an important role in supporting the regeneration of axons after injury
Exogenous metallothionein-II is associated with neuron, astrocytes and microglia after needlestick injury Our previous studies have shown that MTII can promote neurite outgrowth of cultured cortical neurons [15], through a mechanism involving Low-density lipoprotein receptor (LRP) receptors and downstream mitogen-activated protein kinase (MAPK) signalling [9, 10]
Role of metallothionein-II in modifying microglial response after injury Given that the uptake of MT488 was observed within microglia and that this was associated with an altered cellular morphology, we examined the effect of exogenous MTII upon reactive microglia, using an in vitro microglia and neuron co-culture model
Summary
The extracellular environment plays an important role in supporting the regeneration of axons after injury. Glial cells play an important role in maintaining the health of neurons and following injury to the central nervous system (CNS) In this regard, astrocytes and microglia contribute actively to the formation of the glial scar, which is strongly inhibitory to axon regeneration [1, 2]. Apart from CSPG, myelinassociated inhibitors such as myelin-associated glycoprotein (MAG), Nogo and Oligodendrocyte myelin glycoprotein (OMgp) are present in the glial environment and have a significant role in inhibiting axon outgrowth as well as plasticity [6, 8, 9, 10] Soluble inhibitory cues such as semaphorins are recognised as having an important contribution to the inhibitory environment after CNS injury [9, 10]. The balance between the inhibitory and promoting factors in the injury site are important in determining the outcome of axon regeneration after injury
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