In this study, we first demonstrated that the majority of the exogenously administered MT
488 and A
488 associated with the Iba1-labelled microglia/macrophages within close proximity to the needlestick injury in the cortex. The association of both the MT
488 and A
488 may be in accordance with the well-described function of microglia to phagocytose foreign material [
18]. Using this fluorescence-labelled protein model, we can only demonstrate the association of MTII to microglia in vivo. A further study that involves injecting unlabelled MTII into a transgenic MT-null mice model will be needed to confirm the fate of the injected MTII via post-mortem immunolabelling. However, from what we observed using the fluorescence-labelled MTII, the microglia that had associated with MT
488 had a ramified phenotype in comparison to those internalising A
488. Previous studies have suggested the presence of two subtypes of microglia in the CNS; however, recent studies have suggested that these two subtypes could potentially be extremes of a spectrum between the neuro-toxic (M1) to neuro-protective roles (M2) that microglia have in response to an extracellular signal [
19]. The changes in microglia phenotype in the presence of MTII observed in this study indicate that MTII plays a role in altering the function of the microglia to a more neuro-protective form rather than a switch in the subtypes. However, further study is needed to clarify the expression of the neuro-protective phenotype markers in the microglia of the brains treated with MT
488. It should be noted in this experiment model, the amount of MTII injected (1 μL of 2 mg/mL of MTII) is not a significant amount compared to the endogenous MTII concentration in human. The concentration of the MTII used in this experiment model is mainly to allow us to study the cellular association of extracellular MTII, which is the main aim of this experiment. A limitation of this study is the use of the Iba-1 antibody, which labelled both microglia and infiltrating peripheral macrophages; this makes it difficult to specify whether the cells that have internalised the A
488 and MT
488 are microglia or infiltrated macrophages or both. To further investigate the direct influence that MTII has on microglia, we utilised a microglia-neuron co-culture model. In this co-culture model, the microglia is pre-conditioned with either TNFα only or TNFα and MTII in serum-free media, which is then replaced with neurobasal media containing 10% serum prior to the plating of cortical neurons; therefore, the addition of the serum-derived MTII will not affect the pre-conditioning on microglia from the treatments, hence the experiment outcome from the co-culture model. Using this model, we have demonstrated that pre-treating the microglia with TNFα (a pro-inflammatory cytokine) led to a decrease in neurite outgrowth and that the addition of MTII abrogated this effect. Hence, this demonstrated that MTII is capable of exerting a specific effect upon TNFα-treated microglia in culture. It has been recently demonstrated that TNFα treatment in primary microglia culture leads to an inflammatory response via suppressing the expression LRP1 [
20]. The activation of LRP1 receptors has been shown in various studies to be involved in decreasing the inflammatory response of microglia. It has also been demonstrated that MTII bind to both LRP1 and LRP2 (also known as megalin) receptors in neurons [
9,
10,
21]. In this study, we hypothesised that MTII bind to LRP1 receptor on microglia which leads to a decrease in the inflammatory response in microglia through its action on LRP1 receptors. To investigate this, we have utilised siRNA to knockdown LRP1 receptors in our microglia and neuron co-cultures. Our results demonstrated that the knockdown of LRP1 receptor attenuates the effect of MTII in promoting neurite outgrowth after TNFα treatment. This suggests that MTII modulates microglia function via the LRP1 receptor.
This is the first report showing that MTII acts through the LRP1 receptor to alter the microglia response. It has been previously reported that LRP1 receptor activation by an Apolipoprotein-E (Apo-E) mimetic peptide also decreases the microglia inflammatory response via a mitogen-activated protein kinase (MAPK) dependent pathway [
22]. Notably, we have reported previously that MTII stimulates the growth of cultured cortical neurons via LRP2 or the megalin receptor [
9,
10]. Collectively, this suggests that LRP signalling could be involved in promoting axon regeneration and neural recovery following traumatic injury via multiple cellular pathways. We have previously reported that MTII modulates the behaviour of reactive astrocytes through a JAK/STAT signalling pathway [
11]. A recent study had also demonstrated the involvement of LRP1 in microglia activation via modulating the JNK and NF-kappaB signalling pathways [
20]. The JAK/STAT, JNK and NF-kappaB have been demonstrated to be important stress kinase pathways involved in microglia inflammatory response [
23]. Thus, these pathways could potentially be the downstream pathway for MTII-LRP1 interaction in microglia; however, this will need to be confirmed in future studies.
In this current study, MTII treatment caused a distinct change in the morphology of microglia. In saline-treated animals, microglia displayed a spherical and amoeboid morphology that is proposed to be associated with a reactive and inflammatory phenotype that inhibits neural regeneration (commonly referred to as the M1 phenotype) [
24]. However, MTII treatment caused a clear difference in microglial morphology, resulting in ramified structures that resemble the M2 phenotype that supports neural regeneration [
24]. Our in vitro studies support the observation that MTII induces a pro-regenerative microglial phenotype in response to TNFα. Collectively, our data clearly demonstrates that MTII activates an LRP1 receptor-dependent pathway in microglia, providing for the first time a mechanistic explanation for the numerous in vivo reports that MTII modulates the microglial response to CNS injury and stress [
12‐
14,
16].