Transmembrane Protein 108 Inhibits the Proliferation and Myelination of Oligodendrocyte Lineage Cells in the Corpus Callosum

doi:10.21203/rs.3.rs-942005/v1

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Transmembrane Protein 108 Inhibits the Proliferation and Myelination of Oligodendrocyte Lineage Cells in the Corpus Callosum

https://doi.org/10.21203/rs.3.rs-942005/v1

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published 11 Apr, 2022

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Background: Abnormal white matter is a common neurobiological change in bipolar disorder, and dysregulation of myelination in oligodendrocytes is the cause. Transmembrane protein 108 (Tmem108), as a susceptible gene of bipolar disorder, is expressed higher in oligodendrocyte lineage cells than any other lineage cells in the central nervous system. Moreover, Tmem108 mutant mice exhibit mania-like behaviors, belonging to one of the signs of bipolar disorder. However, it is unknown whether Tmem108 regulates myelination of the oligodendrocytes.

Results: Tmem108 expression in the corpus callosum decreased with the development, and hypermyelination of the corpus callosum was found in Tmem108 mutant mice, accompanying high expression of myelin basic protein. Strikingly, both oligodendrocyte progenitor cell proliferation and oligodendrocyte myelination were enhanced in the mutant mice. Furthermore, the mutant mice exhibited mania-like behavior after acute restraint stress and were susceptible to drug-induced epilepsy.

Conclusions: Tmem108 inhibited oligodendrocyte progenitor cell proliferation and mitigated oligodendrocyte maturation in the corpus callosum, which may also provide a new role of Tmem108 involving bipolar disorder pathogenesis.

Cellular & Molecular Neuroscience

Tmem108

Oligodendrocyte

Myelination

Corpus callosum

Bipolar disorder

Bipolar disorder (BD) is a severe mental disease characterized by manic states being usually interspersed with periods of depression [1], affecting 1%-1.5% of the population [1, 2]. Aberrant white matter microstructure is proposed as a mechanism underlying BD, including the dimension of irritability and widespread increases in radial diffusivity [3–5]. Abnormal white matter connectivity may be associated with BD pathophysiology [4, 6], and elevated rates of white matter hyperintensities are widely observed in BD [3–5].

The CC is the brain's major white matter fiber tract [6, 7], containing most axonal transmissions between the two cerebral hemispheres. The CC develops the last brain structure to complete myelination, which is also a period accompanying the peak onset of BD [8]. Anatomical abnormalities in the CC have been reported in magnetic resonance imaging studies in BD patients, possibly because of altered myelination leading to impaired interhemispheric communication [9]. Changes in area and thickness in the corpus callosum (CC) have been reported in BD, and neuropathological data and imaging suggest possible abnormalities in myelination and glial function [2, 8]. Given the strong genetic underpinnings of both BD and white matter microstructure, such whiter matter aberrations may be a disease marker and an endophenotype of BD [3, 10].

Several genome-wide association studies (GWAS) suggest that Tmem108 is a susceptible gene of BD[11–13], and the relevant single nucleotide polymorphism (SNP) site is not in the coding region of Tmem108, which is speculated that the SNP may affect its expression[13, 14]. Strikingly, the recent GWAS screened BD risk loci in the Han Chinese population, covering 1822 BD patients and 4650 control individuals, and the data was replicated analysis. After finally multiple analyses between Han Chinese and European populations, a new SNP (rs9863544) in BD patients were found, locating in the upstream regulatory region of the Tmem108 gene [13]. Tmem108 expression change may be one of the onset reasons for BD.

Our previous research found that adult neurogenesis is impaired in Tmem108 mutant mice, and manic behavior is found in the mutant mice [15], indicating that Tmem108 also is related to BD. Furthermore, RNA sequencing showed that Tmem108 expression is much higher in newly formed OLs than in other cells in the central nervous system (CNS) [16]. Therefore, these studies indicate that Tmem108 may play an essential role in OLs development and myelination.

In this study, Tmem108 expression in the corpus callosum (CC) was higher in young mice than in adult mice and colocalized with OLs in young mice CC, implying promising function in myelination with the development. Intriguingly, myelin basic protein (MBP) was highly expressed in Tmem108 mutant mice in immunohistochemistry (IHC) staining, and western blots (WB) assay and electron microscopes revealed hypermyelination in the CC of the mutant mice, especially early-onset myelination in small axons. Consistently, the cytological experiment showed that Tmem108 inhibited OL progenitor cell (OPC) proliferation and mitigated the maturation of CC OLs by preventing the myelination of small-diameter axons. One of the possible mechanisms might be the Wnt signaling pathway, partially similar to the regulation in adult neurogenesis [15]. Moreover, Tmem108 mutant mice exhibited manic behavior after acute restraint stress and were susceptible to drug-induced epilepsy. This study disclosed the function of Tmem108 in CC, which may also provide a new role of Tmem108 involving BD pathogenesis via regulating the myelination.

Animals

Tmem108 mutant (Tmem108-LacZ; MMRRC: 032633-UCD) mice were described previously [15, 17, 18]. In brief, the first coding exon of Tmem108 was replaced with the β-galactosidase/neomycin cassette. Tmem108 mutant mice in the paper were Tmem108-LacZ homozygous (Tmem108 -/-). Mice were feed in a room 12-h light/dark cycle, at 22–25℃, with ad libitum access to rodent chow diet and clean water. The experimental protocols were performed according to the "guidelines for the care and use of experimental animals" issued by Nanchang University for research about vertebrate animals. For in vivo experiment, surgery was performed with sodium pentobarbital anesthesia (50 mg/kg, intraperitoneal injection), and efforts were executed to minimize suffering and reduce the animal number. After the terminal experiments, mice were euthanized by carbon dioxide inhalation.

Reagents

X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was purchased from Sigma-Aldrich (B4252, 30 mg/ml for staining); Pilocarpine hydrochloride and scopolamine methyl-bromide were also purchased from Sigma-Aldrich; Other chemicals were purchased from Sangon Biotech (BBI Life Sciences CO. China).

Antibodies information as follows: Rabbit anti-β-Actin antibody (Santa Cruz Biotechnology, sc-1616-R; 1:2000 for blotting); Rat anti-MBP antibody (Millipore, MAB386; 1:2000 for blotting; 1:1000 for staining); Rabbit anti-TMEM108 antibody (1:1000 for blotting) was kindly presented by Dr. J. Liu [19]; Goat anti-rabbit IgG poly-HRP secondary antibody (32260) and Goat anti-rat IgG poly-HRP secondary antibody (31471) were purchased from Thermo Fisher Scientific (1:2000 for blotting); Mouse anti-Ki67 (BD Biosciences, 550609; 1:1000 for staining); Rabbit anti-Olig2 antibody (Millipore, AB9610, 1:1000 for staining); Mouse anti-APC antibody (CC1, Millipore Sigma, MABC200, 1:800 for staining) (Tracy J Yuen 2014 Cell); Alexa Fluor 488 goat anti-rabbit lgG (Thermo Fisher Scientific, A32731; 1:1000 for staining), Alexa Fluor 568 goat anti-mouse lgG (Thermo Fisher Scientific, A-11031; 1:1000 for staining).

Behavioral analysis

For the forced swimming test (FST), mice were forced to swim in a two-liter beaker filled with about fifteen-centimeter-height water for 6 min. A camera monitored mice movements with tracking software (Video freeze version 2.5.5.0, Med Associate Inc.). The immobility in the last four min was obtained for statistical analysis.

The pilocarpine model was conducted according to the previous study [20, 21]. In order to minimize the peripheral side effects, mice were injected with scopolamine methyl-bromide (2 mg/kg mice weight, intraperitoneal injection) 30 min before pilocarpine hydrochloride (dissolved in 0.9% saline, 200 mg/kg mice weight, intraperitoneal injection) treatment. Then, mice were injected with pilocarpine (100 mg/kg mice weight) every 30 min. Behavioral seizure score was according to the criteria by Racine [20]: stage 0, no seizure; stage 1, head nodding; stage 2, sporadic full-body shaking and spasms; stage 3, chronic full-body spasms; stage 4, jumping, shrieking, and falling over; stage 5, violent convulsions, falling over and dying.

Western blots (WB)

WB was conducted as our previous research, and whiter matter (CC and medulla) and gray matter (top layers of cortex) separation were according to the previous study [22]. We homogenized the tissues in lysis buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, leupeptin, and pepstatin A protease inhibitors, in 1×DPBS). The protein was separated by SDS-PAGE and then transferred to a nitrocellulose membrane. After being blocked, the membrane was incubated with primary antibody and HRP-coupled secondary antibody in turn. In the last, the immunoreacted bands were captured by an enhanced chemiluminescence system (Bio RAD), and the band intensities were performed with ImageJ software.

Immunohistochemistry (IHC) staining

The mice brain's coronal sections (30 µm) were prepared by microtome (Leica CM1950) for IHC. After incubated in a citrate buffer for antigen repair, sections were permeabilized with a 20% tween for 20 min. Next, the sections were blocked for 1 h at room temperature and then incubated with primary antibody at 4°C overnight. Afterward, sections were exposed to the secondary antibody for 2 h in the dark at room temperature. Finally, the sections were transferred to the slides and mounted with coverslips. The images were captured with an inverted fluorescence microscope (Olympus FSX100). DAPI was used to identify the cellular nuclei.

Quantitative real-time PCR (qPCR)

Total RNA was isolated from mice brains according to the manufacturer's instructions of TRIzol Reagent (Invitrogen), and complementary DNA (cDNA) was synthesized following the manufacturer's protocol of High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). The qPCR primer sets as below: Tmem108 (5′-CCTGAGCTACTGGAACAATGCC-3′ and 5′-CAGTGTCTCGATAGTCGCCAT TG-3′), and Gapdh (5′-CATCACTGCCACCCAGAAGACTG-3′ and 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′). qPCR was carried out by the StepOnePlus Real-Time PCR system (Applied Biosystems) using the mix. qPCR was performed as described previously [23]. Expression levels of mRNA were normalized to the reference gene Gapdh using a ΔCT method.

X‑gal staining

X-gal is an inert chromogenic substrate for β-gal, and β-gal hydrolyzes X-gal into colorless galactose and 4-chloro-3-brom-indigo, forming an intense blue precipitate. X-gal staining was carried out as our previous study [15, 24]. In brief, the coronal sections of Tmem108 mutant mice were prepared by microtome (Leica CM1950) and permeabilized with a detergent solution (0.01% sodium deoxycholate, 0.02% NP-40, 2 mM MgCl2 in 0.1 M pH 7.4 phosphate buffer) at 4°C. After incubated in staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mg/ml X-gal in detergent solution) overnight at 37°C, the sections were transferred to the slides and mounted with coverslips. Finally, the images were captured with an inverted fluorescence microscope (Olympus FSX100).

Electron Microscopy

For electron microscopy, young (P14) and adult (P60) male mice were perfused through the heart with phosphate buffer (PB 0.1 M, pH 7.4) followed by 2% paraformaldehyde with 0.5% glutaraldehyde in PB. After carefully dissecting the brain, the CC was immediately put into 2.5% glutaraldehyde incubating overnight at 4°C and rinsed in PB 5 min triple times. Then at room temperature, the CC was postfixed in1% osmium tetroxide for 1 h, dehydrated through 30% and 50% ethanol 15 min in turn, and immersed in 70% uranyl acetate saturating ethanol overnight. Afterward, the sample was dehydrated through 80% and 95% ethanol 15 min, in turn, followed by incubation of 100% ethanol 40 min twice. The dehydrated sample was infiltrated in epoxypropane (30 min), epoxypropane:ethoxyline resin (1:1, 2 h), epoxypropane:ethoxyline resin (1:2, 1 h), and ethoxyline resin (Ephon812, 2 h) in a gelatin capsule in turn. Then, the sample was polymerized in an oven at 45°C 12 h and 65°C 48 h. Next, ultra-thin sections (70 nm) of the transversal cut CC axons were prepared on an ultramicrotome (LKB-Nova, Sweden). The sections were transferred onto 100 mesh copper grids followed rinsed with ddH₂O 15 min triple times and stained in lead citrate 15 min followed with ddH₂O 10 min triple times. Finally, the sections on the grids were examined on a transmission electron microscope (JEOL, JEM-2100, Japan). The g-ratio of a myelinated axon was calculated as the axonal diameter and fiber diameter ratio.

Statistical analysis

Values of all data are mean ± SEM (standard error of the mean). Statistical analysis was carried out by GraphPad Prism 6.01. The statistical significance between the mutant and control mice was calculated by two-way ANOVA (analysis of variance) and a two-tailed student t-test. The difference was defined as significant if the p-value < 0.05.

High expression of Tmem108 in OLs of the young mice CC

Tmem108 expression was verified by qPCR and WB (Fig. 1). For the whole brain of wild-type mice, qPCR results showed that Tmem108 had a high expression in the young mice (Fig. 1A), and the protein level of Tmem108 was similar to the mRNA level (Fig. 1B). qPCR and WB were utilized to check Tmem108 expression in different brain areas (Fig. 1C, 1D), including cerebellum (CB), thalamus (THY), hippocampus (HP), corpus callosum (CC), cerebral cortex (CT), striatum (STR), prefrontal cortex (PFC) and olfactory bulb (OB). Though CC and STR seemed to like having a low expression of Tmem108, the results supported Tmem108 expression in both areas.

The highest expression cell type in the mice brain was newly forming OLs (Fig S1) [13]. CC was considered an area with high myelinated axons, and therefore, CC was recruited to explore the role of Tmem108 in the OL. Tmem108 expression decreased in postnatal development in qPCR assay (Fig. 1E). Meanwhile, X-gal staining confirmed that Tmem108 expression was higher in young mice CC than in adult mice CC (Fig. 1F). Co-staining assay suggested that X-gal was remarkably co-stained with OL marker Olig2 of CC in the young mice (Fig. 1G).

Hypermyelination of the CC in Tmem108 mutant mice

Myelin sheath can be observed by transmission electron microscopy, presenting thick, dark closed curves around myelinated axons. In this research, the myelinated axons of the CC from young and adult perfused male mice were examined under an electron microscope. The myelinated axons' ultrastructure was obtained for statistics, and the percentage of the myelinated axons in the total axons was reported. Littermate male mice were used to minimize the background effect from other genes between the mutant mice and the control mice.

Because myelination is increasing with mice's development, it was not surprising that the percentage of the myelinated axons in the adult mice was higher than in the young mice (Fig. 2B, 2F). The g-ratio value of a myelinated axon is defined as the ratio of the axonal diameter and myelinated fiber diameter, considered a typical myelination indicator. g-ratio value of CC in the young mutant mice was lower than the control mice (Fig. 2C). In line with expectation, hypermyelination of the CC in the adult mutant mice was observed (Fig. 2G), similar to the young mutant mice. Moreover, the mean g-ratio values of the CC axons with different diameters in the mice were counted and statistically analyzed (Fig. 2D, 2H). Notably, thin fibers in the myelinated axons of adult mice CC processed a small value of g-ratio (Fig. 2H).

Tmem108 mitigates the maturation of CC OLs by restricting the myelination of small-diameter axons

Enhancement of OL maturation in the mutant mice was also demonstrated by electron microscopy. In the adult mice, myelin sheath was not different between the mutant and the control mice (Fig. 2E, 2K), though their g-ratio was distinct (Fig. 2G). The percentage of diverse diameter fibers in total myelinated axons was investigated (Fig. 2I). Strikingly, nearly half myelinated axons in the mutant mice were thin fibers, with the axon diameter no more than 600 nm (Fig. 2I), and the diameter of myelinated OLs in the mutant mice was smaller than that in the control mice (Fig. 2K).

High expression of MBP in Tmem108 mutant mice

Whole-brain MBP level in postnatal mice was quantified by WB (Fig. 3A), and MBP expression was higher in Tmem108 mutant mice than in the control mice after postnatal. According to the previous study [22], gray matter and white matter were separated, and the latter was considered brain areas with plenty of myelinated axons. WB indicated that gray matter in the mutant mice brain had more MBP protein than in the control brain (Fig. 3B). Consistent with anticipation, MBP in CC of the mutant mice brain was higher than the control mice brain (Fig. 3C). Relative mRNA levels of Mbp also were examined in the postnatal mice brain (Fig S2), in gray matter and whiter matter of adult mice (Fig S2B), and CC of adult mice (Fig S2C). Mbp mRNA level seemed consistent with MBP protein level in the mutant mice.

Although Tmem108 mutant did not alter the CC area (Fig. 3D, 3E), cerebral cortex structure, and the hippocampus construction (Fig S3), MBP fluorescence intensity of the brain CC in Tmem108 mutant mice elevated (Fig. 3D, 3F).

The proliferation of OL increased in the CC of the young mutant mice

Myelination begins relatively late in the development of mice until after birth, and myelin sheaths are first seen at P11. Increased myelination occurs during neonatal development of the mice [25]. We speculated that young mice before P11 might have a high proliferation of OPCs or newly forming OLs. Therefore, the CC of young mice at P7 was co-stained in Ki67 with Olig2 (Fig. 4A). Olig2 positive cell density increased in the mutant mice (Fig. 4B), resulting from the high proliferation of the OPCs or newly forming OLs in the mutant mice, representing by Ki67 and Olig2 double-positive cell density in the mice (Fig. 4C). It indicated that Tmem108 inhibited OPC and newly forming OLs proliferation.

The maturation of CC OLs in Tmem108 mutant mice was enhanced

To investigate the OLs maturation in the Tmem108 mutant mice, we utilized CC1 staining to evaluate the maturation of CC OLs in adult mice (Fig. 5A). Intriguingly, although OL density did not change in adult mice (Fig. 5B), maturation of OLs representing by Olig2 and CC1 double-positive cells increased in the mutant mice (Fig. 5C), implying that Tmem108 mitigated the maturation of CC OLs.

Mania-like behavior and easily induced epilepsy in Tmem108 mutant mice

Tmem108 mutant mice display anti-depression behavior (mania-like) behavior in the previous study [15]. In this study, 24-h restraint stress worsened the mania behavior in FST (Fig. 6B). Over half of Tmem108 mutant mice exhibited severe mania-like behavior during the restraint stress and died of physical exertion (Fig. 6C). Furthermore, after the restraint stress, the remaining mutant mice struggled desperately without rest in FST (Fig. 6B), indicating severe mania-like behavior in the mutant mice. Meanwhile, the control mice could be separated into the mania-like group, depressive group, and resistant group (Fig. 6B).

Due to the similarities and the behavioral manifestation between pathophysiological mechanisms and the chronic seizures for their spontaneous and recurrent characteristics, the pilocarpine injection model is considered a classic experimental protocol to mimick human temporal lobe epilepsy [26]. The pilocarpine model was utilized to evaluate the potential epilepsy of the mutant mice. In order to avoid the peripheral side effects, scopolamine block was injected before the pilocarpine treatment (Fig. 6D). The mice were observed continuously for behavioral seizures after each pilocarpine injection. Tmem108 mutant mice quickly reached seizure stage 5 compared with the control mice (Fig. 6E,6F), indicating Tmem108 involved the occurrence of induced epilepsy.

Abnormal myelin development and the mental diseases

Although SCZ and BD account for 2% − 4% of the world population, the pathogenesis and treatment of SCZ and BD are unclear and unsatisfied [27, 28]. Strikingly, imaging and autopsy studies not only show that abnormal white matter is a common neurobiological change in BD and SCZ patients[4] but also reveal that SCZ patients are accompanying with dysregulation of oligodendrocyte (OL) related processes, such as myelination developmental disorder, abnormal expression of myelination gene and number changes of OLs[29, 30]. However, the molecular between abnormal myelination and the two mental diseases is unclear.

The myelin sheath is composed of bilayer lipids as the frame, with proteins embedded as one of the plasma membranes. Most of the proteins in the myelin sheath are transmembrane proteins, such as MBP and proteolipid protein. In these proteins, MBP accounts for 30% of the total myelin protein in the CNS and is critical for myelination [31, 32]. MBP expression was enhanced in Tmem108 mutant mice via WB and IHC staining, and the mutant mice also exhibited hypermyelination by electron microscopy.

The myelin sheath in mature OL acts as an external insulator for current conduction, facilitating rapid saltatory impulse conduction with reduced axonal diameters. Moreover, myelin also provides essential nutritional support for myelinated neurons. Myelinated fibers are widely distributed in the brain, and myelin sheath is essential for maintaining neural circuits. Accordingly, hypomyelination or hypermyelination of OL deriving from abnormal myelination may be one of the bases of cognitive impairment in SCZ and BD, also relating to poor prognosis [29, 33].

Potential multiple functions of Tmem108 in the CNS

Tmem108, also known as Retrolinkin [19, 34, 35], is located on human chromosome 3q21. GWAS found that TMEM108 is not only related to substance addiction [36], smoking withdrawal [37], and alcohol addiction [38–41], but also is a susceptibility gene of SCZ [11, 12, 14] and BD [11–13].

O'Donovan et al. found that the SNP (rs7624858) mutation in the intron of Tmem108 is related to SCZ [14] and speculated that the site caused Tmem108 to become a susceptibility gene of SCZ by affecting gene expression. Jiao et al. disclosed that Tmem108 mutant mice are impaired in spatial memory, and fear startles contextual memory and is more sensitive in PPI performance [17], a classic and plausible psychophysiological measurement of sensorimotor gating for SCZ in rodents and humans [42, 43].

The nature of the severe mental illness has been debated for more than one century. According to the prevailing manuals, International Classification of Diseases, BD, and schizophrenia reveal striking similarities, and the difference is that sensory gating and cognitive impairments are less pronounced in BD patients [44]. BD and schizophrenia consistently ranked among the leading causes of disability worldwide [45, 46], with similarities across multiple levels, such as overlapping brain structural [47, 48] and shared genetic risk factors [49–52]. BD and schizophrenia are severe psychiatric disorders with high heritability, but to date, unknown etiology, sharing genetic risk factors, and a possible illness mechanism is abnormal myelination [10, 53, 54].

Although Tmem108 mutant impairs adult neurogenesis of the mice, it does not induce depression-like behavior but stirs manic-like behavior, suggesting Tmem108 is higher correlating with BD than depression [15]. Strikingly, one recent GWAS screened BD risk loci in the Han Chinese population, covering 1822 BD patients and 4650 control individuals, and the data was replicated analysis. After finally multiple analyses between Han Chinese and European populations, a new SNP (rs9863544) in BD patients were found, locating in the upstream regulatory region of the Tmem108 gene [13]. Tmem108 expression change may be one of the onset reasons of the related psychiatry diseases.

Researchers have debated whether severe, chronic irritability without episodic mania constitutes a developmental phenotype of BD [5]. Neurobiological models of BD emphasize white matter aberrant development, and white matter microstructure is often described as fractional anisotropy, which is positively associated with the smaller axon diameter and increased axon packing density [5].

The potential molecular mechanism of Tmem108 regulating myelination

In 2014, Zhang Y et al. purified eight representative cell populations in the CNS and generated the RNA transcriptome database for the different types of cells [16]. Intriguingly, Tmem108 is expressed in the neurons as in previous research and has a higher expression in the OL lineage cells than in the neurons, significantly much higher in newly formed OL than in any other cells (Fig. S1). Tmem108 mutant mice had manic-like behavior and were more active than the control group in forced swimming and tail suspension experiments [15]. Moreover, 24-hour restraint exacerbated the manic-like behavior of the mutant mice, and the mutant mice were easily induced epilepsy by pilocarpine, which may be partially related to the abnormal myelination.

Although Tmem108 expression was low in the CC of the mice without detectable by X-gal staining, its expression was high in young wild-type mice and could be colocalized with Olig2 positive cells by utilizing the gene reporter mice. To explore how Tmem108 inhibited proliferation and myelination of OL cells, gene expression with myelination regulation [55] was detected by qPCR. Intriguingly, Tcf4 was also expressed highly besides myelin regulatory factor (Myrf) in all three brain areas of Tmem108 mutant mice (Fig S4). In previous research, Tmem108 was reported to involve adult neurogenesis by the Wnt signaling pathway. In CC, most genes with significantly altering expression were downstream of the Wnt signaling pathway, such as Id2, Id4, Tcf4, and Tcf7l2.

Wnt signaling plays a complicated role in the OL myelination, depending on the final effector in the signaling pathway. Canonical Wnt/β-catenin signaling pathway strongly inhibits differentiation [56–58]. Under Wnt3a treatment, differentiation of OPC is strongly delayed or blocked [58], recruiting Tcf4/Tcf7l2 to β-catenin target genes to promote proliferation [57, 59]. ID2 and ID4 are regarded as the potential targets of Wnt/β-catenin/Tcf4 signals in OL development.

Not surprisingly, β-catenin decrease leads to enhancing the premyelinating OL. However, OL differentiation is not enhanced but reduced in Tcf7l2 knockout mice [56, 59], and differentiation is delayed in β-catenin inactivated mice [60], indicating the complexity of β-catenin /Tcf7l2. The potential mechanism is Tcf7l2 interacting with HDAC1 (Histone deacetylases 1) and HDAC2, which repress the expression of differentiation inhibitors [56]. Thereby, Tcf7l2 acts like a molecular switch, blocking or promoting OL differentiation by associating with the different binding partners [61]. We speculated that Tmem108 regulated proliferation and myelination via the Wnt signaling pathway depending on different effectors, which needs further research in vivo and in vitro.

This study disclosed that Tmem108 inhibited OPC proliferation and mitigated the maturation of CC OLs, which may also provide a new role of Tmem108 as a BD risk gene via regulating myelination.

ANOVA: Analysis of variance; BD: Bipolar disorder; CB: Cerebellum; CC: Corpus callosum; CNS: Central nervous system; CT: Cerebral cortex; GWAS: Genome-wide association study; FST: Forced swimming test; HP: Hippocampus; IHC: Immunohistochemistry; MBP: Myelin basic protein; OB: Olfactory bulb; OL: Oligodendrocyte; OPC: OL progenitor cell; PFC: Prefrontal cortex; PPI: Pre-pulse inhibition test; qPCR: quantitative real-time PCR; SEM: Standard error of the mean; SNP: Single nucleotide polymorphism; STR: Striatum; SCZ: Schizophrenia; Tmem108: Transmembrane protein 108; THY: Thalamus; WB: Western blots; X-gal: 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside.

Acknowledgments

We thank Dr. Baoming Li (Institute of Psychological Sciences, Hangzhou Normal University), Dr. Bingxing Pan, Dr. Erkang Fei, and Dr. Suqi Zou (Institute of Life Science, Nanchang University) for assisting in this project. Thanks to Dr. Jiajia Liu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for providing the TMEM108 antibody and Dr. Shiwen Luo (Medical School of Jiangxi, Nanchang University) and Dr. Huifeng Jiao (School of Basic Medical Science, Nanchang University) for suggestions Tmem108 research.

Author Contributions

S Wang initiated and designed the study. J Yu and X Liao performed a glucose tolerance test, insulin resistance test, behavior test, and qPCR. J Yu and Y Zhong performed the metabolism test. Y Wu and D Lin assisted in the animal test. Y Zhang and D Ren cultured mice. S Zou, X Lai, E Fei, and C Ma analyzed data. H Jiao and M Yan advised on the project. S Wang wrote the manuscript with input from all coauthors. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported partly by grants from China's National Natural Science Foundation (31760276, 31960171, 31460260) and the Jiangxi Natural Science Foundation (20171BAB204019 20192ACB20022).

Availability of Data and Materials

The datasets used or analyzed in our study are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

All experiments involving animals were conducted according to the "guidelines for the care and use of experimental animals" issued by Nanchang University. The Committee on the Ethics of Animal Experiments of the University of Nanchang approved the protocol.

Consent for Publication

Not applicable

Competing Interests

The authors have no conflicts of interest to declare.

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Transmembrane Protein 108 Inhibits the Proliferation and Myelination of Oligodendrocyte Lineage Cells in the Corpus Callosum

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