Identification of VHY/Dusp15 as a Regulator of Oligodendrocyte Differentiation through a Systematic Genomics Approach

<div><p>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 <em>in vivo</em> and <em>in vitro</em> 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.</p> </div

VHY/Dusp15 expression during Olineu differentiation.

a,b<p>Data extracted from in-house microarray analysis of transcriptional changes in olineu differentiation induced with different classes of chemical inducers. MBP production is qualitatively reported and reflects the original quantitative data obtained by western blot analysis.</p>*<p><i>P</i><0.05;</p>**<p><i>P</i><0.001.</p><p>For more details please refer to the original publications ^{a, b}.</p

Ten-point standardized rating scale for EAE clinical score monitoring.

<p>The final score for each animal is determined by the addition of all the above mentioned categories. Note: Score  =  15 for dead animal.</p

Disease course of mice undergoing EAE after immunization with rat MOG35-55.

<p>*Mice were immunized <i>s.c.</i> with rat MOG ₃₅₋₅₅. Pertussis toxin was administered <i>i.p.</i> at day 0 and day 2. 12 animals per group were included in this study. Neurological impairment was monitored using a ten-point standardized rating scale and expressed as Mean ± Standard Deviation. Spinal cord and cerebellum were taken at day14 (D14), day17 (D17) and day28 (D28) in MOG-induced (n = 4) and SHAM mice (n = 4). The body weight was monitored over the disease time course and the body weight loss ± SEM was reported. Disease onset occurred at day 10 and reached a maximum at day 22.</p

PTPs the most strongly modulated during EAE in mice spinal cord and cerebellum.

<p>Number of PTP genes significantly modulated during the EAE time course in the spinal cord and in the cerebellum has been monitored and represented in two graphics. The number of PTP genes modulated increases dramatically over time. At day 28, the number of PTP genes modulated decreases until a basal level in cerebellum but remains high in the spinal cord. The highest fold changes in gene expression versus Sham animals have been reported in the table. Most of these PTPs have already been described in inflammatpry processes. Statistical analysis were performed using student <i>t-</i>test.</p

Identification of VHY potential substrates using phospho-peptides arrays.

<p>Each graphic correspond to the results expressed as optical density in arbitrary unites (a.u.) arising from two different arrays representing 720 different phospho-peptides. An arbitrary threshold allowed for the selection of the three phospho-peptides hits of each array namely (1) PDGFR-β (Platelet-derived Growth Factor Receptor beta); MK13 (MAPKinase 13/p38MAPKdelta); ATF2 (Activating Transcription Factor 2); SNX6 (Sorting Nexin 6); IF (Intrinsic factor); ErbB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3). Arrays were ran at pH6 with an enzyme concentration of 2 ng/mL.</p

Clinical data of MS and control autopsies included in the study.

*<p><i>CWM</i>: Control white matter; <i>CGM</i>: Control gray matter; <i>NAWM</i>: Normal appearing white matter; <i>WML</i>: White matter lesioned; <i>NAGM</i>: Normal appearing gray matter; <i>GML</i>: Gray matter lesioned.</p

Expression of OL markers and PTP genes over time in fetal mouse mixed cortical cultures.

<p><b>A</b>. mRNA expression of myelin markers CNP, MBP and PLP and the OPC marker cspg4 measured by qPCR. Results are expressed as fold change ± SEM corresponding to the <i>ratio</i> between % of gene expression <i>vs</i> HKGs at day <i>in vitro</i> (DIV) 8, 12 or 15 and % of gene expression <i>vs</i> HKGs at DIV5. Myelin markers expression increases during culture maturation, reflecting OPCs spontaneous differentiation over time. <b>B</b>. The modulation of the expression of the PTP gene family was monitored at DIV5, 8, 12 and 15 and expressed as fold increase <i>vs</i> DIV5. 39 PTPs were found to be modulated at least 2 fold in mouse mixed cortical culture. PTPs included in the final restrictive set are highlighted on the graphic.</p

Schematic overview of the experimental approach.

<p>Schematic overview of the experimental approach.</p

Fold-changes in mRNA expression for selected PTPs in several MS-related samples.

<p>Fold-changes in mRNA expression for selected PTPs in several MS-related samples.</p