TAPP1 Represses the Differentiation of Oligodendrocyte and its Deficiency Accelerates Myelin Regeneration after Demyelinating Injuries

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Myelin, the lipid-rich insulation that supports the integrity of axons, enables rapid conduction of nerve impulses and information flow to distant brain areas [1]. Oligodendrocytes (OLs) are glial cells that myelinate axons with specialized lipid membrane extensions [2]. OL progenitor cells (OPCs) arise from neural stem cells [3], and undergo proliferation before terminal differentiation and eventual myelination. Impairment at any stage of OL development can affect myelin formation. Dysfunction of OLs and CNS myelin causes devastating neurological disorders represented by multiple sclerosis [4]. In addition, emerging evidence has revealed deficits in OLs and myelin in psychiatric disorders such as schizophrenia [5]. Delineating the mechanisms that underlie OL differentiation is crucial to understand myelin formation, and is a critical prerequisite for the development of novel strategies against myelin-associated disorders.

In the past decades, accumulating evidence has demonstrated that both extracellular signals and/or intracellular factors play crucial roles in governing OL differentiation and myelination [6]. Of these, many (e.g. myelin regulatory factor) are initially expressed in white-matter OLs at the myelinogenic stage and their expression is required for CNS myelination [7]. Recently, we discovered a pleckstrin homology domain-containing adapter protein - TAPP1 - that is selectively expressed in differentiating or pre-myelinating OLs. In vitro studies have revealed that it functions as a repressor of OL differentiation as it suppresses OPC differentiation in culture [8]. However, the in vivo role of TAPP1 in OL development and axonal myelination is currently unknown. In the present study, we aimed to characterize the function of TAPP1 in myelin development and regeneration using genetic approaches and a chemically-induced axonal demyelination model.

To determine the role of TAPP1 in OL differentiation and myelin development, we crossed TRE2-TAPP1-FLAG transgenic mice with PLP-tTA knock-in mice [9]. In the resulting double-transgenic (DTG) mice, the TAPP1 fused in-frame with the FLAG sequence at its C-terminus was induced in the OLs that initially expressed Plp (Fig. 1A). As illustrated in the postnatal day 7 (P7) spinal cord, FLAG was observed in white-matter OLs (28.25% ± 1.017%) in the ventral spinal cord (Fig. 1B, C), indicating the successful induction of exogenous TAPP1 protein under the Plp promoter. Double immunostaining demonstrated that all FLAG-positive cells were co-labeled with the general OL marker OLIG2 (22.85% ± 0.2497% of OLIG²⁺ cells were FLAG⁺), but not with the immature OPC marker PDGFRa, as expected for pre-myelinating OLs (Fig. 1D–E′). The effects of TAPP1 overexpression in myelin development were investigated by the expression of mature OL markers such as CC1, MBP, and Plp. We found that immunostaining for CC1 and MBP was significantly decreased in the P7 transgenic spinal cord (Fig. 1F–G′, M). This result was further verified by the marked decrease in Mbp and Plp transcription as revealed by RNA in situ hybridization (Fig. 1H–I′). Quantitative analyses showed a dramatic decrease in the number of mature Plp⁺ OLs in the white matter of DTG spinal tissue (the Plp signal was clearly seen in individual cell bodies) (Fig. 1N). In addition, we examined the proliferation and apoptosis of OLs and found no apparent differences between control and DTG mice (Fig. 1J–K′, O). In addition, no phospho-Erk1/2 was expression was detected in DTG tissue (Fig. 1L, L′). Together, these results demonstrated that, during the myelinogenic stage, excessive TAPP1 protein represses OL differentiation and myelin gene expression and inhibits Erk phosphorylation. These findings are consistent with our previous report that TAPP1 is expressed in differentiating OLs and over-expression of TAPP1 in cultured OPCs inhibits their terminal differentiation [8]. It is conceivable that TAPP1 expression at the perinatal stage prevents the premature differentiation of OLs and rapid down-regulation thereafter is necessary for the rapid synthesis of myelin proteins in mature OLs that form myelin sheaths.

Fig. 1.

Induced TAPP1 expression in pre-myelinating OLs attenuates their differentiation. A Schematic of the generation of Plp^tTA/+; TRE2-TAPP1-FLAG mice for induction of TAPP1 protein in PLP-expressing OLs by Tet-Off system, which did not stop the expression of TAPP1 in the PLP⁺ OLs unless doxycycline was added (arrows, transcription start sites). B Immunostaining for Flag (green) and TAPP1 (red) in spinal cord sections from Plp^tTA/+ [control (CTR)] and Plp^tTA/+; TRE2-TAPP1-FLAG (DTG) mice (arrows, exogenous TAPP1; arrowheads, endogenous TAPP1). C Quantification of Flag⁺ TAPP1⁺ cells among total TAPP1⁺ cells and FLAG⁺ OLIG2⁺ cells among total OLIG2⁺ cells. D, D′ Representative images showing the co-staining of FLAG and OLIG2 in the spinal cord from CTR and DTG mice (arrows, double-positive cells). E, E′ Spinal cord sections from P7 CTR (E) and DTG mice (E′) double-immunostained with anti-FLAG anti-PDGRFa. FLAG⁺ cells are not co-labeled with anti-PDGFRa. F–G′ Double-immunofluorescent labeling with antibodies against CC1 (green) and MBP (red) in P7 spinal cord sections from CTR (F, G) and DTG mice (F′, G′) (scale bars, 50 μm). H-I′ In situ hybridization for Mbp (H, H′) and Plp (I, I′) in spinal cord sections from CTR (H, I) and DTG mice (H′, I′), showing the inhibition of OL differentiation by overexpressing TAPP1 (scale bar, 50 μm). J, J′ Co-staining with anti-OLIG2 and anti-Ki67 for cell proliferation analysis in P7 CTR and DTG mice. K, K′ TUNEL assays showing that overexpressing TAPP1 does not affect OL apoptosis. L, L′ Reduced expression of phospho-Erk1/2 in DTG mice. Representative images of phospho-Erk1/2 staining in CTR tissue are indicated by arrows (scale bar, 50 μm). M Quantitative analysis of images as in F and F′ showing that the number of CC1⁺cells is conspicuously lower in DTG mice. N Quantification of Plp⁺ cells per section in CTR and DTG spinal cord. O Quantification of Ki67⁺ OLIG2⁺ cells among total OLIG2⁺ cells. P Quantification of CC1⁺ p-Erk1/2⁺ cells among total CC1⁺ cells. Mean ± SEM, n = 3.

The findings that TAPP1 is a negative regulator of OL differentiation and myelin gene expression raise the possibility that the differentiation of OLs is influenced by TAPP1 deficiency. However, no apparent differences were found between wild-type and TAPP1 ablation in the spinal cord and corpus callosum at postnatal stages (data not shown), so we then addressed the possibility that ablation of TAPP1 may enhance myelin regeneration after demyelinating injury. We induced focal axonal demyelination by stereotaxic injection of lysophosphatidylcholine (LPC) into the corpus callosum of control and TAPP1-KO mice (JAX Lab, stock No: 007201). LPC was injected at the injury site and analyzed at 7, 14, and 21 days post-injection (dpi), corresponding to the recruitment, differentiation, and remyelination phases, respectively (Fig. 2B). Axonal demyelination was confirmed by the marked reduction of immunofluorescent MBP staining in the corpus callosum of both wild-type and TAPP1-KO mice at 7 dpi (Fig. 2C). Interestingly, the density of differentiated CC1⁺ OLs in the lesion sites was significantly higher in the TAPP1-KO mice than in the wild-type, suggesting the increased differentiation of new OLs in the demyelinating tissue (Fig. 2D, E). At 14 dpi, the expression of both MBP and CC1 in the TAPP1-KO mice was apparently higher than in the control tissues. Similar results were obtained at 21 dpi. Collectively, these results indicated that ablation of TAPP1 elevates myelin repair in demyelinated lesions induced by LPC. We previously demonstrated that knockdown of TAPP1 in cell culture promotes OL differentiation, and Erk (extracellular signal-regulated kinase) 1/2 expression and phosphorylation. The Erk1/2 pathway has been shown to be critical for OL differentiation both in vitro and in vivo. In line with these findings, the level of phospho-Erk1/2 in the spinal cord of DTG mice was down-regulated when TAPP1 was overexpressed (Fig. 1L–L′, P). In contrast, the expression of phospho-Erk1/2 was dramatically higher in the lesioned regions of TAPP1-KO mice at all demyelinating stages than in the wild-type, (Fig. 2F, G), providing strong in vivo evidence that activation of Erk1/2 is actively engaged in myelin repair [10–12]. Thus, it is plausible that the active state of MAPK/ERK signaling induced by ablation of TAPP1 significantly accelerates OL differentiation and myelin formation for remyelination. During the remyelination process, adult OPCs first proliferate and migrate to the lesion site where they differentiate and produce new myelin sheaths [13, 14]. However, the presence of inhibitory factors in the adult injury site may make the differentiation and myelination less efficient than at early embryonic stages [14, 15]. It is conceivable that TAPP1 is one of these repressors and contributes to the limited capacity for remyelination, suggesting that targeting TAPP1 is a potentially beneficial strategy for the treatment of demyelinating disorders.

Fig. 2.

TAPP1 mutation enhances remyelination in LPC-induced demyelinated lesions. A LPC injection site in the corpus callosum of adult brain. B Schedule of histological analyses in the LPC lesion paradigm. C, D LPC lesions wild-type and TAPP1-KO mice stained for MBP (C, red) or CC1 (D, red) at 7, 14, and 21 days post-injection, and counterstained with DAPI (blue). Dashed lines delineate lesion areas. E Density of CC1⁺ cells in wild-type and TAPP1-KO mouse lesioned regions at 7, 14, and 21 dpi. F Phospho-Erk1/2 immunostaining (red) in wild-type and TAPP1-KO brain tissue (red) at 7, 14, and 21 days post-injection of LPC. Tissue counterstained with DAPI (blue). Scale bars, 50 μm. G Statistical analyses of p-Erk1/2⁺ cells in lesioned regions. Mean ± SEM, n = 3.

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All authors claim that there are no conflicts of interest.