Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease

Abstract

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40–60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0369-5) contains supplementary material, which is available to authorized users.

Regulatory role of oligodendrocyte gap junctions in inflammatory demyelination

“…A major question raised by these studies, and requiring further investigation, is whether the connexin loss leads to a direct reduction of BBB/BSCB integrity, which in turn promotes an increased inflammatory response, or whether loss of connexins promotes a more vigorous intrinsic brain immune response, as indicated by the abnormal cytokine responses in GJ deficient mice, which secondarily causes BBB/BSCB disruption. Previous studies in brains of mice deficient in oligodendrocyte GJs demonstrated activation of immune cells (Georgiou et al, 2017;Markoullis, Sargiannidou, Gardner, et al, 2012;Olympiou et al, 2016;Schiza et al, 2015;Tress et al, 2011) and upregulation of pro-inflammatory genes expressed by both microglia and astrocytes (Wasseff & Scherer, 2015). Some evidence for a pro-inflammatory environment in the CNS resulting from loss of oligodendrocyte connexins has been shown in Cx32/Cx47 double KO mice (Georgiou et al, 2017;Schiza et al, 2015;Wasseff & Scherer, 2015).…”

X linked Charcot-Marie-Tooth disease and multiple sclerosis: emerging evidence for an association

“…However, early studies demonstrated that Cx32 mutants associated with CNS phenotypes were retained in the endoplasmic reticulum and Golgi apparatus of oligodendrocytes, suggesting a toxic gain of function 39. In fact, recent studies have found that under the influence of systemic inflammation transdominant effects of mutant Cx32 on other connexins can disrupt oligodendrocyte gap junctions, although oligodendrocyte dysfunction is further exacerbated by intracellularly retained mutants (eg, Thr55Ile) 40. Nevertheless, other studies have recently observed that even mutations associated with a complete loss of Cx32 can produce CNS dysfunction 41.…”

The role of oligodendrocyte gap junctions in neuroinflammation

“…Furthermore, at least one case with a start codon mutation in GJB1 and documented complete loss of Cx32 expression was also associated with transient CNS manifestations, suggesting that gain-offunction effects are not necessary for the CNS manifestations of CMT1X [62]. Further studies to elucidate the consequences of GJB1 mutations, in particular in the CNS, were subsequently undertaken and discussed below [63,64].…”

“…In order to understand the cellular mechanisms underlying the CNS phenotypes in patients with CMT1X (above), we used a model of systemic inflammation induced by lipopolysaccharide (LSP) injection. We compared the effects of LPS-induced systemic inflammation in Cx32 KO mice expressing the T55I mutant (KO T55I) with WT and Cx32 KO mice [63]. We assumed that dominant effects of the T55I mutant might only become obvious under stress conditions that mimic the metabolic and inflammatory events leading to encephalopathy in CMT1X patients [55][56][57][58].…”