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

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

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

Schematic overview of the experimental approach.

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

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

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

Phosphatase activity of GST-tagged full length Dusp15/VHY.

<p>A. Dusp15 phosphatase activity was assessed using the DiFMUP (6,8-difluoro-4-methyumbelliferyl phosphate) assay at the experimentally optimal enzyme concentration of 4 ng/mL. B. Optimal pH activity (pH 6) was determined by testing a pH range from pH 3 to 8. C. and D. Activity of Dusp15/VHY on phospho-peptides substrates corresponding respectively to pY₁₁₉ and pY₇₇₁ dephosphorylation sites of SNX6 (NED(pY₁₁₉)AGYIIPPAP) and PDGFR-β (IESSN(pY₇₇₁)MAPYD). VHY/Dusp15 was used at 4 ng/mL at pH6 and activity was detected using the Malachite Green phosphate detection assay. OD, Optical Density at 620 nm. Dissociation constant (Km) was calculated as the substrate concentration needed to reach V_max/2 and expressed as Mean ± S_EM of three different experiments.</p

Correlation chart between MBP expression and dusp15 expression over a time course of differentiation in olineu.

<p>Values expressed as fold induction <i>versus</i> undifferentiated controls (starting cultures) and correspond to the Mean ± SD of two different experiments (n = 2). Dusp15/VHY expression increases with time and correlates with MBP expression during the first steps of Oli-neu differentiation then Dusp15 expression reaches a maximum at 15 h prior to the MBP expression peak occurring at 24 h.</p