A novel microRNA-132-sirtuin-1 axis underlies aberrant B-cell cytokine regulation in patients with relapsing-remitting multiple sclerosis [corrected].

Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.

Implication of perturbed axoglial apparatus in early pediatric multiple sclerosis.

Cerebrospinal fluid samples collected from children during initial presentation of central nervous system inflammation, who may or may not subsequently be diagnosed as having multiple sclerosis (MS), were subjected to large-scale proteomics screening. Unexpectedly, major compact myelin membrane proteins typically implicated in MS were not detected. However, multiple molecules that localize to the node of Ranvier and the surrounding axoglial apparatus membrane were implicated, indicating perturbed axon-glial interactions in those children destined for diagnosis of MS.

Functional studies of MLC1 mutations in Chinese patients with megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC, MIM# 604004) is an autosomal recessive inherited disease mostly resulting from MLC1 mutations. In this study, we finished the functional analysis of MLC1 mutations identified recently in Chinese patients, including five newly described missense mutations (R22Q, A32V, G73E, A275T, Y278H), one known nonsense mutation (Y198X), and two known missense mutations (S69L, T118M). We found MLC1(wt) was localized to the cell periphery, whereas mutant R22Q, A32V, G73E, S69L and T118M were trapped in the lumen of endoplasmic reticulum (ER) when we transfected the wild-type and mutant MLC1 in U373MG cells. Compared to wild type, the mutant G73E, T118M, Y198X and A275T transcript decreased and all mutants except R22Q had lower protein expression in transfected U373MG cells. Therefore, we propose that all these eight MLC1 mutations had functional effect either on their protein/mRNA expression, or on their intracellular protein localization, or both.

Misalignment of PLP/DM20 transmembrane domains determines protein misfolding in Pelizaeus-Merzbacher disease.

A large number of genetic diseases have been associated with truncated or misfolded membrane proteins trapped in the endoplasmic reticulum (ER). In the ER, they activate the unfolded protein response, which can trigger cell death. Hence, a better understanding of protein misfolding features might help in developing novel therapies. Here, we have studied the molecular basis of Pelizaeus-Merzbacher disease, a leukodystrophy defined by mutations of the PLP1 gene and ER retention of two encoded tetraspan myelin proteins, PLP and DM20. In mouse oligodendroglial cells, mutant isoforms of PLP/DM20 with fewer than all four transmembrane (TM) domains are fully ER retained. Surprisingly, a truncated PLP with only two N-terminal TM domains shows normal cell-surface expression when coexpressed with a second truncated PLP harboring the two C-terminal TM domains. This striking ability to properly self-align the TM domains is disease relevant, as shown for the smaller splice isoform DM20. Here, the increased length of TM domain 3 allows for compensation of the effect of several PLP1 point mutations that impose a conformational constraint onto the adjacent extracellular loop region. We conclude that an important determinant in the quality control of polytopic membrane proteins is the free alignment of their TM domains.

A common mechanism of PLP/DM20 misfolding causes cysteine-mediated endoplasmic reticulum retention in oligodendrocytes and Pelizaeus-Merzbacher disease.

A large number of mutations in the human PLP1 gene lead to abnormal myelination and oligodendrocyte death in Pelizaeus-Merzbacher disease (PMD). Here we show that a major subgroup of PMD mutations that map into the extracellular loop region of PLP/DM20 leads to the failure of oligodendrocytes to form the correct intramolecular disulfide bridges. This leads to abnormal protein cross-links and endoplasmic reticulum retention and activates the unfolded protein response. Importantly, surface expression of mutant PLP/DM20 can be restored and the unfolded protein response can be reverted by the removal of two cysteines. Thus, covalent protein cross-links emerge as a cause, rather than as a consequence, of endoplasmic reticulum retention.

Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites.

During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth.