rapid-specific-immunomagnetic-isolation-mouse-primary

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

University of Illinois at Chicago

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.

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

CMT1X, an X-linked inherited neuropathy, is caused by mutations in GJB1, which codes for Cx32, a gap junction protein expressed by Schwann cells and oligodendrocytes. Many GJB1 mutations cause central nervous system (CNS) abnormality in males, including stable subclinical signs and, less often, short-duration episodes characterized by motor difficulties and altered consciousness. However, some mutations have no apparent CNS effects. What distinguishes mutations with and without CNS manifestations has been unclear. Here we studied a total of 14 Cx32 mutations, 10 of which are associated with florid episodic CNS clinical syndromes in addition to peripheral neuropathy. The other 4 mutations exhibit neuropathy without clinical or subclinical CNS abnormalities. These "PNS-only" mutations (Y151C, V181M, R183C and L239I) form gap junction plaques and produce levels of junctional coupling similar to those for wild-type Cx32. In contrast, mutants with CNS manifestations (F51L, E102del, V139M, R142Q, R142W, R164W T55I, R164Q and C168Y) either form no morphological gap junction plaques or, if they do, produce little or no detectable junctional coupling. Thus, PNS and CNS abnormalities may involve different aspects of connexin function.

Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations.

Connexins are a family of integral membrane proteins most of which form gap junctions and many of which form hemichannels as well. Mutations in at least 9 of the 21 genes encoding human connexin proteins cause human diseases. Mutations in GJB1 (Cx32), expressed in both Schwann cells and oligodendrocytes, cause both a form of inherited peripheral neuropathy and a variety of CNS symptoms. Mutations in GJC2 (Cx47), expressed in oligodendrocytes cause three disorders: a severe early onset dysmyelinating disorder, Pelizaeus-Merzbacher-Like disease (PMLD1 or HLD2); hereditary spastic paraplegia (SPG44), which has a milder phenotype and later onset; and a subclinical leukodystrophy. The clinical phenotypes and genetics associated with each disorder will be reviewed, focusing on features which may provide clues to pathogenesis. In vitro and animal model data which may shed light on these phenotypes will then be discussed along with recent work which may impact on therapeutic approaches for these disorders.

Diseases of connexins expressed in myelinating glia.

Peripheral neurological complications of lymphomas are rare and much less frequent than central complications. Nonetheless, on occasion, systemic non-Hodgkin's lymphoma may directly infiltrate the peripheral nervous system at various levels. This report describes a man with non-Hodgkin's lymphoma and leptomeningeal disease who developed progressive areflexic quadraparesis. Initial electromyography (EMG) was consistent with a polyradiculopathy and a repeat EMG performed 1 month later for worsening symptoms showed evidence of demyelination. The patient expired due to systemic complications of his illness. Autopsy of the sural nerve showed moderately severe distal sensory axonal loss, direct infiltration of the brachial plexus by malignant lymphocytes and demyelination in brachial and lumbar plexus, most prominent in areas of neoplastic infiltration. Based on this patient's course and pathology, we suggest that widespread demyelination may accompany neurolymphomatosis and the clinical presentation may be indistinguishable from an acute demyelinating neuropathy.

Article	Year
Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease. Acta neuropathologica communications\| PubMed ID: 27585976	2016
Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations. Scientific reports\| PubMed ID: 28071741	2017
Diseases of connexins expressed in myelinating glia. Neuroscience letters\| PubMed ID: 28545922	2017
Acute demyelinating neuropathy in a patient with neurolymphomatosis. BMJ case reports\| PubMed ID: 29507018	2018

Charles K. Abrams

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