Subcellular trafficking of proteolipid protein (PLP/DM20) and novel mechanisms of ER retention in Pelizaeus-Merzbacher disease

Missense mutations that predict the misfolding of membrane proteins have been associated with a number of neurogenetic diseases. However, it is not known how apparently minor changes in the amino acid sequence of an extracellular loop or a transmembrane domain lead to complete ER retention with complex loss- and gain-of-function effects. I have chosen PLP/DM20, a highly conserved and abundant tetraspan myelin protein, associated with Pelizaeus-Merzbacher disease (PMD), as a model system. By expressing wildtype and mutant PLP isoforms in glial cells, surprising molecular properties became apparent, including the ability to self-assemble from two truncated PLP polypeptides, and to form conformation sensitive epitope that become masked as the protein matures in the ER. With respect to human disease, it was possible to identify a novel molecular mechanism by which missense mutations cause ER retention of misfolded PLP. Unexpectedly, pairs of cysteines within an extracellular loop of PLP/DM20 play a critical role. Multiple disease-causing mutations require the presence of cysteines such that misfolded PLP/DM20 is efficiently retained in the ER. Replacing cysteines by serine completely prevents ER retention and restores normal trafficking of mutant PLP/DM20. This demonstrates a novel pathological mechanism by which missense mutations greatly reduce the efficiency of intramolecular disulfide bridging. When exposed by misfolding to the ER lumen, unpaired cysteines engage in alternative oxidations that lead to abnormal intermolecular crosslinks. Since extracellular cysteines are a feature of many membrane proteins, this novel pathomechanism is likely to contribute to a diverse group of genetic diseases. To monitor the expression and subcellular trafficking of PLP in vivo, a transgenic knock-in mouse in being generated that will express a PLP-EGFP fusion protein under control of the endogenous promoter. In an attempt to develop a cure for Pelizaeus-Merzbacher disease (PMD), we treated a genuine animal model (rumpshaker mice) with Turmeric. The active constituent of this herbal drug (Curcumin) is a non-toxic Ca2+ adenosine triphosphatase pump inhibitor, and known to release membrane proteins from ER retention. In a pilot experiment, we extended the lifespan of rumpshaker mice from 20 to 60 days. These promising data suggest that a therapeutic strategy should be developed for PMD, using turmeric and our in vitro and in vivo models

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

Cellular response to micropatterned growth promoting and inhibitory substrates

BACKGROUND: Normal development and the response to injury both require cell growth, migration and morphological remodeling, guided by a complex local landscape of permissive and inhibitory cues. A standard approach for studying by such cues is to culture cells on uniform substrates containing known concentrations of these molecules, however this method fails to represent the molecular complexity of the natural growth environment. RESULTS: To mimic the local complexity of environmental conditions in vitro, we used a contact micropatterning technique to examine cell growth and differentiation on patterned substrates printed with the commonly studied growth permissive and inhibitory substrates, poly-L-lysine (PLL) and myelin, respectively. We show that micropatterning of PLL can be used to direct adherence and axonal outgrowth of hippocampal and cortical neurons as well as other cells with diverse morphologies like Oli-neu oligodendrocyte progenitor cell lines and fibroblast-like COS7 cells in culture. Surprisingly, COS7 cells exhibited a preference for low concentration (1 pg/mL) PLL zones over adjacent zones printed with high concentrations (1 mg/mL). We demonstrate that micropatterning is also useful for studying factors that inhibit growth as it can direct cells to grow along straight lines that are easy to quantify. Furthermore, we provide the first demonstration of microcontact printing of myelin-associated proteins and show that they impair process outgrowth from Oli-neu oligodendrocyte precursor cells. CONCLUSION: We conclude that microcontact printing is an efficient and reproducible method for patterning proteins and brain-derived myelin on glass surfaces in order to study the effects of the microenvironment on cell growth and morphogenesis

Regulation of miRNA 219 and miRNA Clusters 338 and 17-92 in Oligodendrocytes

MicroRNAs (miRs) regulate diverse molecular and cellular processes including oligodendrocyte (OL) precursor cell (OPC) proliferation and differentiation in rodents. However, the role of miRs in human OPCs is poorly understood. To identify miRs that may regulate these processes in humans, we isolated OL lineage cells from human white matter and analyzed their miR profile. Using endpoint RT-PCR assays and quantitative real-time PCR, we demonstrate that miR-219, miR-338, and miR-17-92 are enriched in human white matter and expressed in acutely isolated human OLs. In addition, we report the expression of closely related miRs (miR-219-1-3p, miR-219-2-3p, miR-1250, miR-657, miR-3065-5p, miR-3065-3p) in both rodent and human OLs. Our findings demonstrate that miRs implicated in rodent OPC proliferation and differentiation are regulated in human OLs and may regulate myelination program in humans. Thus, these miRs should be recognized as potential therapeutic targets in demyelinating disorders

TMEM10 Promotes Oligodendrocyte Differentiation and is Expressed by Oligodendrocytes in Human Remyelinating Multiple Sclerosis Plaques.

Oligodendrocyte precursor cells (OPCs) differentiate during postnatal development into myelin-forming oligodendrocytes, in a process distinguished by substantial changes in morphology and the onset of myelin gene expression. A mammalian-specific CNS myelin gene, tmem10, also called Opalin, encodes a type 1 transmembrane protein that is highly upregulated during early stages of OPC differentiation; however, a function for TMEM10 has not yet been identified. Here, consistent with previous studies, we detect TMEM10 protein in mouse brain beginning at ~P10 and show that protein levels continue to increase as oligodendrocytes differentiate and myelinate axons in vivo. We show that constitutive TMEM10 overexpression in the Oli-neu oligodendroglial cell line promotes the expression of the myelin-associated genes MAG, CNP and CGT, whereas TMEM10 knock down in primary OPCs reduces CNP mRNA expression and decreases the percentage of MBP-positive oligodendrocytes that differentiate in vitro. Ectopic TMEM10 expression evokes an increase in process extension and branching, and blocking endogenous TMEM10 expression results in oligodendrocytes with abnormal cell morphology. These findings may have implications for human demyelinating disorders, as oligodendrocytes expressing TMEM10 are detected in human remyelinating multiple sclerosis lesions. Together, our findings provide evidence that TMEM10 promotes oligodendrocyte terminal differentiation and may represent a novel target to promote remyelination in demyelinating disorders

Interaction of PLP with GFP-MAL2 in the Human Oligodendroglial Cell Line HOG

The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP), the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP)-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested