Oscillatory calcium release and sustained store-operated oscillatory calcium signaling prevents differentiation of human oligodendrocyte progenitor cells.

Endogenous remyelination in demyelinating diseases such as multiple sclerosis is contingent upon the successful differentiation of oligodendrocyte progenitor cells (OPCs). Signaling via the Gαq-coupled muscarinic receptor (M1/3R) inhibits human OPC differentiation and impairs endogenous remyelination in experimental models. We hypothesized that calcium release following Gαq-coupled receptor (GqR) activation directly regulates human OPC (hOPC) cell fate. In this study, we show that specific GqR agonists activating muscarinic and metabotropic glutamate receptors induce characteristic oscillatory calcium release in hOPCs and that these agonists similarly block hOPC maturation in vitro. Both agonists induce calcium release from endoplasmic reticulum (ER) stores and store operated calcium entry (SOCE) likely via STIM/ORAI-based channels. siRNA mediated knockdown (KD) of obligate calcium sensors STIM1 and STIM2 decreased the magnitude of muscarinic agonist induced oscillatory calcium release and attenuated SOCE in hOPCs. In addition, STIM2 expression was necessary to maintain the frequency of calcium oscillations and STIM2 KD reduced spontaneous OPC differentiation. Furthermore, STIM2 siRNA prevented the effects of muscarinic agonist treatment on OPC differentiation suggesting that SOCE is necessary for the anti-differentiative action of muscarinic receptor-dependent signaling. Finally, using a gain-of-function approach with an optogenetic STIM lentivirus, we demonstrate that independent activation of SOCE was sufficient to significantly block hOPC differentiation and this occurred in a frequency dependent manner while increasing hOPC proliferation. These findings suggest that intracellular calcium oscillations directly regulate hOPC fate and that modulation of calcium oscillation frequency may overcome inhibitory Gαq-coupled signaling that impairs myelin repair.

Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following experimental demyelination.

Chronic demyelination in the human CNS is characterized by an inhibitory microenvironment that impairs recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) leading to failed remyelination and axonal atrophy. By network-based transcriptomics, we identified sulfatase 2 (Sulf2) mRNA in activated human primary OPCs. Sulf2, an extracellular endosulfatase, modulates the signaling microenvironment by editing the pattern of sulfation on heparan sulfate proteoglycans. We found that Sulf2 was increased in demyelinating lesions in multiple sclerosis and was actively secreted by human OPCs. In experimental demyelination, elevated OPC Sulf1/2 expression directly impaired progenitor recruitment and subsequent generation of oligodendrocytes thereby limiting remyelination. Sulf1/2 potentiates the inhibitory microenvironment by promoting BMP and WNT signaling in OPCs. Importantly, pharmacological sulfatase inhibition using PI-88 accelerated oligodendrocyte recruitment and remyelination by blocking OPC-expressed sulfatases. Our findings define an important inhibitory role of Sulf1/2 and highlight the potential for modulation of the heparanome in the treatment of chronic demyelinating disease.

Muscarinic Receptor M3R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells.

Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGFαR+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease.SIGNIFICANCE STATEMENT The identification of drug targets aimed at improving remyelination in patients with demyelination disease is a key step in development of effective regenerative therapies to treat diseases, such as multiple sclerosis. Muscarinic receptor antagonists have been identified as effective potentiators of remyelination, but the receptor subtypes that mediate these receptors are unclear. In this study, we show that genetic M3R ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, M3R represents an attractive target for induced remyelination in human disease.

Network-Based Genomic Analysis of Human Oligodendrocyte Progenitor Differentiation.

Impaired human oligodendrocyte progenitor cell (hOPC) differentiation likely contributes to failed remyelination in multiple sclerosis. The characterization of molecular pathways that regulate hOPC differentiation will provide means to induce remyelination. In this study, we determined the gene expression profile of PDGFαR+ hOPCs during initial oligodendrocyte commitment. Weighted gene coexpression network analysis was used to define progenitor and differentiation-specific gene expression modules and functionally important hub genes. These modules were compared with rodent OPC and oligodendrocyte data to determine the extent of species conservation. These analyses identified G-protein ß4 (GNB4), which was associated with hOPC commitment. Lentiviral GNB4 overexpression rapidly induced human oligodendrocyte differentiation. Following xenograft in hypomyelinating shiverer/rag2 mice, GNB4 overexpression augmented myelin synthesis and the ability of hOPCs to ensheath host axons, establishing GNB4 as functionally important in human myelination. As such, network analysis of hOPC gene expression accurately predicts genes that influence human oligodendrocyte differentiation in vivo.