Identification of VHY/Dusp15 as a regulator of oligodendrocyte differentiation through a systematic genomics approach.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by a progressive loss of myelin and a failure of oligodendrocyte (OL)-mediated remyelination, particularly in the progressive phases of the disease. An improved understanding of the signaling mechanisms that control differentiation of OL precursors may lead to the identification of new therapeutic targets for remyelination in MS. About 100 mammalian Protein Tyrosine Phosphatases (PTPs) are known, many of which are involved in signaling both in health and disease. We have undertaken a systematic genomic approach to evaluate PTP gene activity in multiple sclerosis autopsies and in related in vivo and in vitro models of the disease. This effort led to the identification of Dusp15/VHY, a PTP previously believed to be expressed only in testis, as being transcriptionally regulated during OL differentiation and in MS lesions. Subsequent RNA interference studies revealed that Dusp15/VHY is a key regulator of OL differentiation. Finally, we identified PDGFR-beta and SNX6 as novel and specific Dusp15 substrates, providing an indication as to how this PTP might exert control over OL differentiation.

Sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 are functionally upregulated on astrocytes under pro-inflammatory conditions.

BACKGROUND: Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P(3)) signaling and SphK1 in activated rat astrocytes has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: Using immunohistochemistry we observed the upregulation of S1P(3) and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P(3) and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P(3) agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P(3) upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P(3) signaling compared to S1P(1) signaling was demonstrated by the use of selective S1P(3) or S1P(1) agonists. CONCLUSION/SIGNIFICANCE: In summary, our data demonstrate that the SphK1/S1P(3) signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.

Chemical inducers and transcriptional markers of oligodendrocyte differentiation.

Oligodendrocytes generate and maintain myelin, which is essential for axonal function and protection of the mammalian central nervous system. To advance our molecular understanding of differentiation by these cells, we screened libraries of pharmacologically active compounds and identified inducers of differentiation of Oli-neu, a stable cell line of mouse oligodendrocyte precursors (OPCs). We identified four broad classes of inducers, namely, forskolin/cAMP (protein kinase A activators), steroids (glucocorticoids and retinoic acid), ErbB2 inhibitors, and nucleoside analogs, and confirmed the activity of these compounds on rat primary oligodendrocyte precursors and mixed cortical cultures. We also analyzed transcriptional responses in the chemically induced mouse and rat OPC differentiation processes and compared these with earlier studies. We confirm the view that ErbB2 is a natural signaling component that is required for OPC proliferation, whereas ErbB2 inhibition or genetic knockdown results in OPC differentiation.

Live imaging of remyelination after antibody-mediated demyelination in an ex-vivo model for immune mediated CNS damage.

Mononuclear cell infiltrates, deposits of immunoglobulin and complement as well as demyelination and axonal damage are neuropathological hallmarks of Multiple Sclerosis (MS) lesions. An involvement of antibodies is further suggested by the presence of oligoclonal immunoglobulins in the cerebrospinal fluid of almost all MS patients. However, which mechanisms are most relevant for de- and remyelination and axonal loss in MS lesions is poorly understood. To characterize the regenerative abilities of demyelinated CNS tissue, we utilized murine organotypic cerebellar slice cultures expressing GFP in oligodendrocytes. The addition of a demyelinating monoclonal antibody specific for myelin oligodendrocyte glycoprotein and complement induced complete myelin destruction and oligodendrocyte loss, as demonstrated by confocal live imaging and staining for different myelin proteins. After removal of antibodies and complement we visualized the stages of remyelination, presumably originating from proliferating oligodendrocyte precursor cells and guided by morphologically intact appearing axons. Allowing for the detailed live imaging of de- and remyelination in an ex vivo situation closely resembling the three dimensional cytoarchitecture of the CNS, we provide a useful experimental system for the evaluation of new therapeutic strategies to enhance remyelination and repair in MS.

Convergent functional genomics of oligodendrocyte differentiation identifies multiple autoinhibitory signaling circuits.

Inadequate remyelination of brain white matter lesions has been associated with a failure of oligodendrocyte precursors to differentiate into mature, myelin-producing cells. In order to better understand which genes play a critical role in oligodendrocyte differentiation, we performed time-dependent, genome-wide gene expression studies of mouse Oli-neu cells as they differentiate into process-forming and myelin basic protein-producing cells, following treatment with three different agents. Our data indicate that different inducers activate distinct pathways that ultimately converge into the completely differentiated state, where regulated gene sets overlap maximally. In order to also gain insight into the functional role of genes that are regulated in this process, we silenced 88 of these genes using small interfering RNA and identified multiple repressors of spontaneous differentiation of Oli-neu, most of which were confirmed in rat primary oligodendrocyte precursors cells. Among these repressors were CNP, a well-known myelin constituent, and three phosphatases, each known to negatively control mitogen-activated protein kinase cascades. We show that a novel inhibitor for one of the identified genes, dual-specificity phosphatase DUSP10/MKP5, was also capable of inducing oligodendrocyte differentiation in primary oligodendrocyte precursors. Oligodendrocytic differentiation feedback loops may therefore yield pharmacological targets to treat disease related to dysfunctional myelin deposition.