PIKE is essential for oligodendroglia development and CNS myelination

Abstract

Oligodendrocyte (OL) differentiation and myelin development are complex events regulated by numerous signal transduction factors. Here, we report that phosphoinositide-3 kinase enhancer L (PIKE-L) is required for OL development and myelination. PIKE-L expression is up-regulated when oligodendrocyte progenitor cells commit to differentiation. Conversely, depleting phosphoinositide-3 kinase enhancer (PIKE) expression by shRNA prevents oligodendrocyte progenitor cell differentiation. In both conventional PIKE knockout (PIKE(-/-)) and OL-specific PIKE knockout mice, the number of OLs is reduced in the corpus callosum. PIKE(-/-) OLs also display defects when forming myelin sheath on neuronal axons during neonatal development, which is partially rescued when PTEN is ablated. In addition, Akt/mTOR signaling is impaired in OL-enriched tissues of the PIKE(-/-) mutant, leading to reduced expression of critical proteins for myelin development and hypomyelination. Moreover, myelin repair of lysolecithin-induced lesions is delayed in PIKE(-/-) brain. Thus, PIKE plays pivotal roles to advance OL development and myelinogenesis through Akt/mTOR activation.

Fig. 1.

Expression of PIKE-L in OLs is a developmentally regulated process. (A) Expression analysis of PIKE-L in various brain regions. Brains from wild-type (P7) and PIKE^−/− mice were collected and dissected into different parts. The amount of total PIKE-L protein was examined using immunoblotting. (B) Expression of PIKE-L in OLs of CC of P30 mouse. (Scale bar, 20 μm.) (C) Increased expression of PIKE-L in differentiating OPC. Cultured rat OPCs at different differentiation days were stained with antibodies that recognize the C terminus of PIKE-L or MBP. (Scale bar, 20 μm.) (D) Expression of PIKE-L is elevated in developing optic nerve. Optic nerves collected from C57BL/6 mice of different ages were homogenized, and the amount of PIKE-L, Akt, and tubulin was examined using immunoblotting. Akt phosphorylation on S473 was also determined. (E) Quantification of Akt phosphorylation and PIKE-L expression shown in D (***P < 0.001 vs. P10, one-way ANOVA; n = 3). (F) OPCs isolated from rat were infected with control adenovirus or shPIKE-expressing adenovirus during OL differentiation. The number of O4⁺ cells was visualized by IF staining and quantified (***P < 0.001, Student t test; n = 6). (G) OPCs isolated from rat were infected with control adenovirus or shPIKE-expressing adenovirus during OL differentiation. The number of O1⁺ cells was visualized by IF staining and quantified (***P < 0.001, Student t test; n = 6). (H) PIKE-L depletion causes aberrant morphology in mature OLs. (Scale bar, 20 μm.) (I) Quantitation of dendritic length of cultured OLs after adenovirus infection (***P < 0.001, Student t test; n = 4).

Fig. 2.

The number of mature OLs and Akt/mTOR signaling are reduced in PIKE-ablated brain. (A) PIKE^−/− brain contains fewer mature OLs. Brains from wild-type and PIKE^−/− mice (P16) were collected, and the mature OLs in CC were visualized using IF staining against CC1. (Scale bar, 20 μm.) (B) Quantification of CC1⁺ cell in PIKE^−/− CC (*P < 0.05, Student t test; n = 3). (C) PLP-GFP:PIKE^−/− brain contains fewer mature OLs in CC. Brains of PLP-GFP:PIKE^+/+ and PLP-GFP:PIKE^−/− mice (P21) were collected, and the OLs in the CC were visualized and counted using confocal microscopy. (Scale bar, 50 μm.) (D) Quantification of GFP⁺ cells in the CC of PLP-GFP:PIKE^+/+ and PLP-GFP:PIKE^−/− mice (*P < 0.05, ***P < 0.001, Student t test; n = 3). (E) Optic nerves from wild-type (^+/+) and PIKE^−/− (^−/−) mice of different ages (P16 and P23) were collected and homogenized. Expression of Akt, mTOR, QK5, QK6, QK7, PIKE-L, and tubulin was examined by immunoblotting. Phosphorylations of Akt and mTOR were also determined. (F) Quantification of the band intensity shown in E (solid bars, PIKE^+/+; open bars, PIKE^−/−; *P < 0.05, **P < 0.01, ***P < 0.001, Student t test; n = 3). (G) OPKO mice contain fewer OLs in the CC. Mature OLs in CC were visualized using IF staining against CC1. (Scale bar, 50 μm.) (H) Quantification of CC1⁺ cells in the CC of control and OPKO mice (*P < 0.05, Student t test; n = 4). (I) Reduced MBP expression and Akt/mTOR phosphorylations in OPKO mice. Optic nerves and the olfactory bulbs from the control and OPKO mice after tamoxifen administration were collected. Expression of MBP, Akt, mTOR, and tubulin was then examined using immunoblotting. Phosphorylations of Akt and mTOR were also determined. (J) Quantification of the MBP protein amount shown in I (*P < 0.05, Student t test; n = 3).

Fig. 3.

PIKE^−/− axons are hypomyelinated. (A) Defective expression of MBP in the optic nerve of PIKE^−/− mice. Optic nerves from wild-type (^+/+) and PIKE^−/− (^−/−) mice of different ages (P16 and P23) were collected. Expression of MBP, CNP, PIKE-L, and tubulin were then examined using immunoblotting. (B) Quantification of the protein amount shown in A (solid bars, PIKE^+/+; open bars, PIKE^−/−; *P < 0.05, Student t test; n = 3). (C) IF staining of MBP in the CC of wild-type, PIKE^−/−, and PIKE^−/−PTEN^−/− double-knockout mice (P7). (Scale bar, 50 μm.) (D) Defective MBP expression and Akt phosphorylation in PIKE^−/− mice could be rescued by PTEN ablation. Optic nerves from wild-type, PIKE^−/−, and PIKE^−/−PTEN^−/− double-knockout mice (P23) were collected. Expression of MBP, PTEN, Akt, PIKE-L, and tubulin were then examined using immunoblotting. Phosphorylation of Akt was also tested. (E) Quantification of the protein amount shown in D (*P < 0.05, one-way ANOVA; n = 3). (F) Mature OLs in the CC of wild-type, PIKE^−/−, and PIKE^−/−PTEN^−/− mice (P23) were visualized using IF staining against CC1. (Scale bar, 20 μm.) (G) Quantification of CC1⁺ cells in CC of wild-type, PIKE^−/−, and PIKE^−/−PTEN^−/− mice (*P < 0.05, one-way ANOVA). (H) Representative electron micrograph of CC in cross-section from wild-type and PIKE^−/− mice at P16. (Scale bar, 20 μm.) (I) Axon diameter of wild-type (^+/+) and PIKE^−/− (^−/−) neurons in the CC (n = 6). (J) Histogram representing the G-ratio of wild-type (^+/+) and PIKE^−/− (^−/−) axons (***P < 0.001, Student t test; n = 6).

Fig. 4.

Delayed remyelination in PIKE^−/− brain. (A) Representative coronal brain slices of PLP-GFP:PIKE^+/+ and PLP-GFP:PIKE^−/− mice (2-mo-old) collected at 5 (D5) and 21 (D21) days after LL injection. Damaged area is marked by the arrow. (Scale bar, 500 μm.) (B) SMI 32 staining in LL-injected CC after 5 (D5) and 21 (D21) days. (Scale bar, 50 μm.) (C) Number of GFP⁺ OLs in the damaged CC after LL injection (*P < 0.05, Student t test; n = 3). (D) Ki67 staining of the demyelinated CC in PLP-GFP:PIKE^+/+ and PLP-GFP:PIKE^−/− brains 5 d after LL injection. Cells that show positive Ki67 signals are indicated with arrows and are magnified in the Insets. (Scale bar, 50 μm.) (E) Number of Ki67⁺ cells in the CC and SVZ after LL injection (***P < 0.05, Student t test; n = 3). (F) IF staining of MBP in the LL-damaged CC. (Scale bar, 50 μm.)