Developmental expression and function analysis of protein tyrosine phosphatase receptor type D in oligodendrocyte myelination

Abstract

Receptor protein tyrosine phosphatases (RPTPs) are extensively expressed in the central nervous system (CNS), and have distinct spatial and temporal patterns in different cell types during development. Previous studies have demonstrated possible roles for RPTPs in axon outgrowth, guidance, and synaptogenesis. In the present study, our results revealed that protein tyrosine phosphatase, receptor type D (PTPRD) was initially expressed in mature neurons in embryonic CNS, and later in oligodendroglial cells at postnatal stages when oligodendrocytes undergo active axonal myelination process. In PTPRD mutants, oligodendrocyte differentiation was normal and a transient myelination delay occurred at early postnatal stages, indicating the contribution of PTPRD to the initiation of axonal myelination. Our results also showed that the remyelination process was not affected in the absence of PTPRD function after a cuprizone-induced demyelination in adult animals.

Keywords: axonal myelination; corpus callosum; cuprizone; spinal cord.

Figure 1

Spatiotemporal expression of PTPRD in the developing spinal cord. Spinal cord sections from E18.5 (A,A′), P3 (B,B′), P7 (C,C′), P15 (D,D′) and P30 (E,E′) wild-type animals were subject to in situ RNA hybridization (ISH) with PTPRD riboprobes. Arrows show the PTPRD+ cells in the white matter. A′-E′, higher magnification of the ventrolateral region of A–E. Scale bars: 100 μm.

Figure 2

Selective expression of PTPRD in differentiated oligodendrocyte lineage. A–C, P7 wild-type mouse spinal cord and corpus callosum sections were subjected to PTPRD ISH followed by anti-APC immunohistochemical staining. Double positive cells are represented by blue arrows; PTPRD positive only motor neurons are indicated by red arrowheads (B). D–F, P4 spinal cords from wild-type (D), Olig1−/− (E) and CNP^+/cre;Nkx2.2^fl/fl (F) were hybridized with PTPRD riboprobe. PTPRD expression in the white matter region was dramatically reduced in Olig1−/− and CNP^+/cre;Nkx2.2^fl/fl mutants. Scale bar: 100 μm.

Figure 3

Normal oligodendrocyte differentiation and myelin gene expression in PTPRD mutants. Traverse spinal cord sections from P7 wild-type (A, B, C, D) and PTPRD knock-out (A′, B′, C′, D′) littermates were subjected to immunostaining with anti-Olig2 (A, A′) or anti-APC (B, B′), and in situ hybridization with MBP (C, C′) or PLP (D, D′). Scale bar: 50 μm. E, Western immunoblotting of postnatal day 7, 14 and 2 months spinal cord extracts from wild-type and PTPRD mutants with anti-MBP and anti-β-actin antibodies. Comparable MBP expression was detected in PTPRD knock-out mice as compared to wild-type littermates.

Figure 4

Normal differentiation and maturation of oligodendrocytes in PTPRD mutant mice. Traverse spinal cords sections from P4 wild-type (A, B) and PTPRD knock-out (A′, B′) littermates were immunostained with anti-Olig2 (A, A′), anti-APC (B, B′). Similarly, spinal cords sections from P14 wild-type (C, D, E) and PTPRD knock-out (C′, D′, E′) were subjected to immunostaining with anti-Olig2 (C, C′) or anti-APC (D, D′), and in situ hybridization with PLP (E, E′). No significant difference is detected between wild-type and knock-out mutants. Scale bar: 50 μm.

Figure 5

Axon myelination defects in the spinal cords of PTPRD mutants. A–C′, EM analysis of myelinated fibers in spinal cord sections from wild type and PTPRD mutant mice at P4 (A-A′), P7 (B-B′) and P14 (C-C′). D, Percentage of myelinated axons per micrograph in the wild type and mutant mice at different developmental stages (n=3). E, g-ratio of fibers in the wild-type and mutant mice at various stages. Data were represented as Mean±S.E.M. *, p < 0.05. **, p < 0.01. n.s., not significant. Scale bar: 2 μm.

Figure 6

Electrophysiological and behavior test in PTPRD mutants. A, B, Latencies and amplitudes of the tcMMEP recordings from adult wild-type and PTPRD mutants. C, Beam walking assay. Data were represented as Mean±S.D., n=4 animals. n.s., not significant.

Figure 7

Elevated expression of PTPRD in regenerated oligodendrocytes during remyelination. In situ hybridization with PTPRD probe in the corpus callosum in cuprizone-treated mice during demyelination (A) and remyelination (B). Scale bar: 50 μm.

Figure 8

Similar OPC differentiation and myelin gene expression in PTPRD mutants during remyelination. Immunostaining of anti-APC (B-B′), in situ hybridization with MBP (E-E′) or PLP probes (H-H′) showed severe demyelination of the corpus callosum (delimited by dotted lines) in wild-type (B, E, H) and PTPRD mutant (B′, E′, H′) mice after 6 weeks cuprizone treatment. Compared with wild-type mice, similar expression of APC (C,C′), MBP (F,F′), and PLP (I,I′) was observed in the corpus callosum area of PTPRD mutant mice after recovery for 1 week. Scale bar: 50 μm.