Astrogliosis is a hallmark of central nervous system (CNS) neuroinflammatory disorders such as multiple sclerosis (MS). Astrocytes can play both beneficial and detrimental roles in response to neuroinflammation, thus a detailed understanding of the underlying molecular mechanisms governing astrogliosis might facilitate the development of therapeutic targets. While astrocytes do not express voltage-gated sodium channel (VGSC) Nav1.5 in nonpathological human brain, they exhibit robust upregulation of Nav1.5 within acute and chronic MS lesions. We investigated the contribution of voltage-gated sodium channels to astrogliosis in an in vitro model of mechanical injury to astrocytes. Previous studies have shown that a scratch injury to astrocytes invokes dual mechanisms of migration and proliferation in these cells. Our results demonstrate that wound closure after mechanical injury, involving both migration and proliferation, is attenuated by pharmacological treatment with tetrodotoxin (TTX) and KB-R7943, at a dose that blocks reverse mode of the Na+/Ca2+ exchanger (NCX), and by knockdown of Nav1.5 mRNA. We also show that astrocytes display a robust [Ca2+] transient after mechanical injury and demonstrate that this [Ca2+] response is also attenuated by TTX, KB-R7943, and Nav1.5 mRNA knockdown. This study provides support for a contribution of VGSCs in the pathway leading to astrogliosis.
We present here evidence supporting a contribution of sodium channel Nav1.5 to astrogliosis in an in vitro model of glial mechanical injury. We further implicate fluctuations in [Ca2+] due to reverse operation of NCX, triggered by VGSC activity, as a mechanism by which Nav1.5 contributes to the response of astrocytes to mechanical injury. Our results establish a link between the activity of VGSCs and astrogliosis by way of alterations in [Ca2+] . Here we show, in an in vitro model of mechanical injury to astrocytes, that voltage-gated sodium channel (VGSC) Nav1.5, traditionally viewed as a cardiac sodium channel, contributes to the astrocytic response to the insult via triggering reverse mode of the Na+/Ca2+ exchanger (NCX).
We then investigated the temporal dynamics of astrocytic Nav1.5 channel expression in response to neuroinflammatory pathologies. We examined astrocytes from mice with monophasic and chronic-relapsing experimental autoimmune encephalomyelitis (EAE) by immunohistochemistry to determine whether Nav1.5 is expressed in these cells, and whether the expression correlates with severity of disease and/or phases of relapse and remission. Our results demonstrate that Nav1.5 is upregulated in astrocytes in situ in a temporal manner that correlates with disease severity in both monophasic and chronic-relapsing EAE. Furthermore, in chronic-relapsing EAE, Nav1.5 expression is upregulated during relapses and subsequently attenuated during periods of remission. These observations are consistent with the suggestion that Nav1.5 can play a role in the response of astrocytes to inflammatory pathologies in the CNS and suggest Nav1.5 may be a potential therapeutic target to modulate reactive astrogliosis in vivo.
Finally, we investigated whether Nav1.5 expression in astrocytes plays a role in the pathogenesis of EAE. We created a conditional knockout of Nav1.5 in astrocytes and determined whether this affects the clinical course of EAE, focal macrophage and T cell infiltration, and diffuse activation of astrocytes. We show that deletion of Nav1.5 from astrocytes leads to significantly worsened clinical outcomes in EAE, with increased inflammatory infiltrate in both early and late stages of disease, unexpectedly, in a sex-specific manner. Removal of Nav1.5 in astrocytes leads to increased inflammation in female mice with EAE, including increased astroglial response and infiltration of T cells and phagocytic monocytes. These cellular changes are consistent with more severe EAE clinical scores. Additionally, we found evidence suggesting possible dysregulation of the immune response – particularly regarding infiltrating macrophages and activated microglia – in female Nav1.5 KO mice compared to WT littermate controls. Together, our results show that deletion of Nav1.5 from astrocytes leads to significantly worsened clinical outcomes in EAE, with increased inflammatory infiltrate in both early and late stages of disease, in a sex-specific manner.
|Advisor:||Waxman, Stephen George|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 80/07(E), Dissertation Abstracts International|
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