Connexin 30 Deficiency Attenuates Chronic but Not Acute Phases of Experimental Autoimmune Encephalomyelitis Through Induction of Neuroprotective Microglia

Abstract

Glial connexins (Cxs) form gap junction channels through which a pan-glial network plays key roles in maintaining homeostasis of the central nervous system (CNS). In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), expression of astrocytic Cx43 is lost in acute lesions but upregulated in chronic plaques, while astrocytic Cx30 is very low in normal white matter and changes in its expression have not been convincingly shown. In Cx30 or Cx43 single knockout (KO) mice and even in Cx30/Cx43 double KO mice, acute EAE is unaltered. However, the effects of Cx30/Cx43 deficiency on chronic EAE remains to be elucidated. We aimed to clarify the roles of Cx30 in chronic neuroinflammation by studying EAE induced by myelin oligodendrocyte glycoprotein peptide 35–55 in Cx30 KO mice. We found that Cx30 deficiency improved the clinical symptoms and demyelination of chronic but not acute EAE without influencing CD3+ T cell infiltration. Furthermore, increased ramified microglia in the naïve state and induced earlier and stronger microglial activation in the acute and chronic phases of EAE was observed. These activated microglia had an anti-inflammatory phenotype, as shown by the upregulation of arginase-1 and brain-derived neurotrophic factor and the downregulation of nitric oxide synthase 2. In the naïve state, Cx30 deficiency induced modest enlargement of astrocytic processes in the spinal cord gray matter and a partial reduction of Cx43 expression in the spinal cord white matter. These astrocytes in Cx30 KO mice showed earlier and stronger activation during the acute phase of EAE, with upregulated A2 astrocyte markers and a significant decrease in Cx43 in the chronic phases. Spinal cord neurons and axons were more preserved in Cx30 KO mice than in littermates in the chronic phase of EAE. These findings suggest that Cx30 deficiency increased ramified microglia in the CNS in the naïve state and improved chronic EAE through redirecting microglia toward an anti-inflammatory phenotype, suggesting a hitherto unknown critical role of astrocytic Cx30 in regulating microglial number and functional state.

Microglial Morphometric Parameters Correlate With the Expression Level of IL-1β, and Allow Identifying Different Activated Morphotypes

“…It is known that microglial dynamic conversion from resting to activated is accompanied by a retraction of the cellular processes into the cell body (Kettenmann et al, 2011). The subjective (Davis et al, 1994; Kloss et al, 2001; Norden et al, 2016) and objective (Kozlowski and Weimer, 2012; Davis et al, 2017; Fang et al, 2018) evaluation of the circularity have shown that roundness is directly related to microglial activation. Similarly, in the present study the greater the circularity, the higher the degree of inflammatory activation, a fact that is clearly evidenced in cluster 4.…”

Novel animal model of multiple sclerosis: The glial connexin gap junction as an environmental tuner for neuroinflammation

“…Gene set enrichment analysis showed a decreased proinflammatory cytokine gene set and increased expression of the anti‐inflammatory gene set. The numbers of microglia and astroglia were increased in Cx30 KO mice, but the clinical course was ameliorated because of the anti‐inflammatory activation of glial cells 26 …”

Beneficial and Detrimental Remodeling of Glial Connexin and Pannexin Functions in Rodent Models of Nervous System Diseases

“…These mice were not different either in terms of the degree of immune cell reactivity in the spinal cord, evaluated using ionized calcium binding adaptor molecule 1 (Iba1) immunostaining at the endpoint of clinical evaluation. Interestingly, the global Cx30 deficiency reduced the severity of the chronic phase (8 weeks after the insult) in this model and this effect was correlated with the emergence of an anti-inflammatory phenotype in both spinal cord astrocytes and microglia (Fang et al, 2018). Given that a reduction in the abundance of Cx43 during the chronic phase of experimental autoimmune encephalomyelitis was observed in global Cx30 deficient mice, the neuroprotective effect could be mediated indirectly via astrocytic Cx43.…”