Tmem117, an oligodendrocyte-enriched regulator of NCX activity, links myelin homeostasis to counterregulation and metabolic health

Significance

This study uncovers a surprising connection between myelin integrity and whole-body metabolism through the transmembrane protein Tmem117, whose molecular mechanism of action was previously unknown. We show that Tmem117 is crucial for maintaining healthy oligodendrocytes and myelin, and its loss in mature oligodendrocytes disrupts myelination, weakens the body’s response to hypoglycemia, and causes long-term, sex-specific metabolic changes. Mechanistically, we show that Tmem117 stabilizes the calcium exchanger NCX1. Our findings reveal that myelin integrity can influence peripheral metabolism—a top–down effect contrary to traditional views—and highlight oligodendrocytes as active players in metabolic regulation. These insights suggest that modulation of the Tmem117–NCX1 axis may represent a potential strategy for influencing oligodendrocyte homeostasis and its subsequent effects on myelin and metabolic health.

Abstract

The counterregulatory response (CRR) to hypoglycemia—critically depending on pancreatic glucagon secretion—is a fundamental, evolutionarily conserved homeostatic mechanism orchestrated by the central nervous system (CNS) to ensure survival during glucose scarcity. Tmem117 was previously identified in a genetic screen as a potential hypothalamic regulator of CRR. Here, we reveal that Tmem117 is enriched in cells of the oligodendrocytic lineage and we characterize the contribution of oligodendrocytic Tmem117 in CRR. We show that depletion of Tmem117 from either all oligodendrocyte lineage cells or only mature oligodendrocytes leads to myelin deficits and male-specific defects in CRR. Furthermore, we reveal that transient, adult-onset depletion of Tmem117 in mature oligodendrocytes is sufficient to induce long-lasting metabolic imbalances in male mice, suggesting that defects in oligodendrocytes and myelin can affect peripheral glucose homeostasis. Mechanistically, we provide insights on the molecular mechanism of action of Tmem117 showing that it regulates intracellular calcium dynamics through its interaction with the sodium-calcium exchanger NCX1. Together, these results redefine our understanding of the cellular contributors to the CRR, highlight the importance of oligodendrocytes in systemic glucose regulation, and position Tmem117 as a promising molecular target for cell-specific manipulation of sodium-calcium exchanger (NCX) activity.

Myelin plays a critical role not only in enabling saltatory action potential conduction but also in providing essential trophic support to axons (1). The integrity of myelin sheaths is maintained by oligodendrocyte lineage cells, which have emerged as key regulators of central nervous system (CNS) homeostasis. Consequently, these cells have become a focal point in research on demyelinating and neurodegenerative diseases (2). Recent advances have underscored the unique biological features of mature myelinating oligodendrocytes, including their high metabolic demands and particular vulnerability to oxidative stress (3).

In parallel, accumulating evidence reveals a surprising degree of plasticity within the oligodendroglial lineage. Oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes can dynamically respond to peripheral metabolic and inflammatory cues, suggesting a bidirectional relationship between CNS glial health and systemic physiology (4, 5). However, this relationship has largely been studied in one direction: how peripheral disruptions, such as inflammation or metabolic imbalance, impact oligodendrocyte function and myelin homeostasis. The converse—whether and how oligodendrocyte homeostasis influences whole-body metabolism—remains an open and underexplored question.

In this study, we investigate the role of Tmem117, a poorly characterized transmembrane protein with a strikingly restricted pattern of expression, notably enriched in oligodendrocytes and a few other specialized cell types—including vasopressin (AVP) neuroendocrine cells, cardiomyocytes, and subsets of epithelial cells from the gut and the kidney. Although its molecular mechanism of action remains unknown, prior work, including our own, has implicated Tmem117 in cellular stress responses (6, 7). Specifically, we previously showed that Tmem117 is required for the survival of vasopressin (AVP)-producing neuroendocrine cells, where its loss leads to ER stress, elevated intracellular calcium levels, and ultimately cell death (7).

Here, we extend this line of investigation to the oligodendrocyte lineage. Using conditional genetic models, we demonstrate that Tmem117 is necessary for maintaining oligodendrocyte homeostasis and myelin integrity. Loss of Tmem117 in oligodendrocytes leads to sex-specific impairments in the counterregulatory response (CRR) to hypoglycemia, as well as long-term metabolic imbalances. Notably, we provide in vitro evidence that this phenotype arises from disruptions in calcium homeostasis, mediated through a functional interaction between Tmem117 and Slc8a1 (NCX1), a sodium-calcium exchanger. We show that Tmem117 promotes NCX1 stability, thereby facilitating calcium extrusion and preventing cytotoxic calcium overload.

Together, our findings identify Tmem117 as a key regulator of calcium homeostasis, provide mechanistic insight into its function via NCX1 stabilization, and reveal a link between CNS myelin health and systemic metabolic regulation.

Results

Tmem117 Is Enriched in Oligodendrocytes.

Tmem117 was previously identified in a genetic screening as a candidate gene for the hypothalamic regulation of the CRR to hypoglycemia, with its expression levels at the hypothalamus being strongly correlated to the amount of glucagon secreted during insulin-induced hypoglycemia (IIH) (8). Our previous work highlighted its important role on CRR regulation through its effect on AVP neuroendocrine cells (7). Except for its expression in AVP neuroendocrine cells, publicly available single-cell RNAseq data from the mouse hypothalamus (9) suggest enrichment of the Tmem117 transcript in oligodendrocyte lineage cells (Fig. 1 A and B). The same enrichment is reported also in datasets extending beyond the hypothalamic area (SI Appendix, Fig. S1 A–C). In the Tabula Muris dataset (10) Tmem117 enriched cells in brain tissue fall within the cluster of oligodendrocytes (SI Appendix, Fig. S1A). In addition, data from mousebrain.org (11) suggest enrichment in mature myelinating oligodendrocytes among others (SI Appendix, Fig. S1 B and C; highlighted by the red arrow).

Fig. 1.

Tmem117 is enriched in oligodendrocytes. (A) UMAP depicting the Tmem117-enriched cells across all cell-type clusters of the mouse hypothalamus (data extracted from HypoMap). (B) Ridge plot depicting the Tmem117 transcript detection levels across all cell-types in the mouse hypothalamus (data extracted from HypoMap). (C) Schematic representation of the approach utilized to verify the detection of Tmem117 in mature oligodendrocytes. (D) Representative images from the mouse brain depicting membrane structures deriving from mature myelinating oligodendrocytes in green (mEGFP) and Tmem117 protein in red (Alexa Fluor 647). Nuclei are labeled with DAPI (white). Magenta and turquoise arrows highlight the somata and processes of AVP neuroendocrine cells, respectively. White arrows highlight Tmem117 positive mature myelinating oligodendrocytes. Scale bars: 1 mm for panel 0 and 10 μm for panels 1 to 12, CPu d: striatum dorsal, CC sm: corpus callosum soma, fi: fimbria of hippocampus, CC ec: corpus callosum external capsule, CPu v: striatum ventral, br: bregma, ic: internal capsule, S2: somatosensory cortex, SON: supraoptic nucleus, sox: supraoptic decussation, LA: lateroanterior hypothalamic nucleus, LH: lateral hypothalamic area, f: fornix.

To test whether the observed enrichment of the transcript is also leading to higher abundance of the Tmem117 protein in oligodendrocytes, we utilized a transgenic mouse line for fluorescent labeling of mature oligodendrocytes and myelin (CNP-mEGFP; JAX:026105) alongside immunostaining against Tmem117 using a KO validated antibody (7) (Fig. 1C). Except from the strong immunofluorescence observed in the somata (Fig. 1D) [(8, 9); magenta arrows] and processes (Fig. 1D) [(8–10); turquoise arrows] of AVP neuroendocrine cells, we also observed a fainter but consistent staining in mature oligodendrocytes in the optic nerve (Fig. 1D) [(8, 9); white arrows]. This staining was consistently observed across several brain areas (Fig. 1D) with majority of Tmem117 positive cells located in white matter tracts in the form of tightly packed “syncytia” (Fig. 1D) (2, 4, 6, 12), but some also identified in sparsely myelinated gray matter areas (Fig. 1D) (5, 7, 10, 11).

These findings establish that Tmem117 is enriched in mature myelinating oligodendrocytes, suggesting a potential, previously unrecognized, role for this protein in oligodendrocyte biology and myelin homeostasis.

Depletion of Tmem117 from the Oligodendrocyte Lineage Leads to Hypomyelination and Male-Specific CRR Deficits.

To study the contribution of oligodendrocytic Tmem117 in CRR we decided to knock-out Tmem117 in all oligodendrocyte lineage cells by crossing our previously generated Tmem117 floxed mouse line (7) with the Olig2-cre mouse line (JAX:025567) (SI Appendix, Fig. S2A).

Both heterozygote Tmem117^fl/+; Olig2-cre^tg/+ (Olig2^TM117KO/+) and homozygote Tmem117^fl/fl; Olig2-cre^tg/+ (Olig2^TM117KO/KO) male mice were noticeably smaller at weaning and until week 8 of age as reflected by their weight gain curves (SI Appendix, Fig. S2B and Movie S1). In contrast, their female littermates were indistinguishable in size and weight gain when compared to control mice not expressing cre recombinase, Tmem117^fl/fl; Olig2-cre^+/+ (Olig2^TM117FL/FL) (SI Appendix, Fig. S2C and Movie S1). Furthermore, collective reexamination of the weaning rates over a duration of 26 mo (445 pups weaned) revealed decreased representation of Olig2^TM117KO male mice (expected: 50%, observed: 38%) with the rates of female Olig2^TM117KO mice remaining within the expected Mendelian ratio (expected: 50%, observed: 45%; SI Appendix, Fig. S2D).

When adult (8-wk-old) Olig2^TM117KO male mice were subjected to an IIH test (SI Appendix, Fig. S2E) their CRR appeared diminished with markedly decreased secretion of glucagon (SI Appendix, Fig. S2F) for hypoglycemic levels comparable to their control Olig2^TM117FL littermates (SI Appendix, Fig. S2G). Correlation analysis of the glycemic levels with plasma glucagon levels further highlights that for comparable levels of glycemia Olig2^TM117KO mice secrete lower amounts of glucagon relative to Olig2^TM117FL controls (SI Appendix, Fig. S2H). In contrast, both hypoglycemia and glucagon secretion were comparable between Olig2^TM117KO and Olig2^TM117FL female mice (SI Appendix, Fig. S2 I and J). Vagal nerve recordings revealed decreased parasympathetic activity in Olig2^TM117KO male mice during IIH (SI Appendix, Fig. S2K).

Furthermore, transmission electron microscopy analysis of the Corpus callosum of Olig2^TM117KO male mice revealed the existence of thinner myelin sheaths (SI Appendix, Figs. S2 L and M1 and M2) and cytoplasmic inclusions suggestive of myelin instability (SI Appendix, Fig. S2 M3–M6).

These data demonstrate that Tmem117 deletion in oligodendrocyte lineage cells leads to growth retardation, defective myelination, and impaired CRR only in male mice, underscoring a sex-dependent vulnerability to oligodendrocyte dysfunction.

Transient Depletion of Tmem117 from Mature Oligodendrocytes in Adult Mice Leads to Male Specific Defects in CRR and Long-Term Metabolic Imbalances.

Since the male-specific phenotype of the Olig2^TM117KO mouse line could be of developmental origin, we next decided to knock-out Tmem117 only in mature oligodendrocytes after the critical waves of CNS myelination.

To this end, we crossed our Tmem117 floxed mouse line (7) with the Plp1-creER^T mouse line (JAX:005975) (Fig. 2A). Initially, a group of male Tmem117^fl/fl; Plp1-creER^T(tg/+) (Plp1^TM117KO) mice and their Tmem117^fl/fl; Plp1-creER^T(+/+) (Plp1^TM117FL) littermate controls were injected intraperitoneally (i.p.) with tamoxifen at P30-P35 to induce recombination of the Tmem117 locus in mature oligodendrocytes and then subjected 4 wk later to the IIH test (Fig. 2B). As shown in Fig. 2C, male Plp1^TM117KO mice present a CRR phenotype similar to that observed previously in Olig2^TM117KO mice, with diminished glucagon secretion for comparable hypoglycemic states (Fig. 2D).

Fig. 2.

Transient depletion of Tmem117 from mature oligodendrocytes in adult male mice leads to defects in CRR and long-term metabolic imbalances. (A) Schematic representation of the transgenic mouse line generation. (B) Schematic representation of the experimental timeline for panels C and D. (C and D) Plasma glucagon concentration (C) and blood glucose (D) from Plp1^TMEM117KO male mice (red) and their Plp1^TMEM117FL control littermates (black) 1 h after i.p. injection of insulin (yellow) or saline (blue) [TAM injections performed at P30-P35; n = 6 to 7 per group; two-way ANOVA RM; P values correspond to Bonferroni’s multiple comparisons test results]. (E) Schematic representation of the experimental timeline for panels F and G. (F and G) Plasma glucagon concentration (F) and blood glucose (G) from Plp1^TMEM117KO male mice (red) and their Plp1^TMEM117FL control littermates (black) 1 h after i.p. injection of insulin (yellow) or saline (blue) (TAM injections performed at P60-P65; n = 9 per group; two-way ANOVA RM; P values correspond to Bonferroni’s multiple comparisons test results). (H) Correlation of glycemia and glucagon secretion (n = 15 to 16 per group; Pearson’s r correlation analysis with comparison of intercepts). (I) Plasma catecholamine concentration from Plp1^TMEM117KO male mice (red) and their Plp1^TMEM117FL control littermates (black) 1 h after i.p. injection of insulin [n = 8 per group; unpaired t test]. (J) Schematic representation of the experimental timeline for panel K. (K) Blood glucose concentration from Plp1^TMEM117KO male mice (red) and their Plp1^TMEM117FL control littermates (black) during a 2-h period following an i.p. insulin injection (n = 10 to 14 per group; two-way ANOVA RM; week 12 to 24 of age). (L) Schematic representation of the experimental timeline for panels M–O. (M) Weight gain curves of male Plp1^TMEM117KO mice (red, orange) and their Plp1^TMEM117FL control littermates (black, gray) on a regular chow diet (red and black, respectively) or on a WD high in fat and sugar (orange and gray, respectively) (n = 10 to 17 per group; three-way ANOVA). (N) Glucose tolerance test responses of male Plp1^TMEM117KO mice (red, orange) and their Plp1^TMEM117FL control littermates (black, gray) on a regular chow diet (red and black, respectively) or on a WD high in fat and sugar (orange and gray, respectively) (n = 7 to 13 per group; three-way ANOVA; week 19 of age). (O) Nonalcoholic steatohepatitis (NASH) scores (mouse-adapted modified Kleiner score) to illustrate the extent of hepatic lipidosis assessed histologically in the liver of male Plp1^TMEM117KO mice (red, orange) and their Plp1^TMEM117FL control littermates (black, gray) on a regular chow diet (red and black, respectively) or on a WD high in fat and sugar (orange and gray, respectively) (n = 7 to 12 per group; two-way ANOVA; P values correspond to Tukey’s multiple comparisons test results; week 19 of age). Lines/bars correspond to mean values and error bars represent ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IIH: insulin induced hypoglycemia, INS: insulin, ITT: insulin tolerance test, SAL: saline, TAM: tamoxifen, WD: western diet.

Then in another group of male mice we induced the recombination in adulthood (P60-65; Fig. 2E) and the effect on glucagon secretion remained the same (Fig. 2 F and G). Correlation analysis of the glycemic levels with plasma glucagon levels reveals a negative correlation for both genotypes, but with a significant difference in their intercepts further highlighting the fact that for glycemic levels comparable to Plp1^TM117FL controls Plp1^TM117KO mice secrete lower amounts of glucagon (Fig. 2H). Quantification of catecholamine levels in the same plasma samples revealed no difference between Plp1^TM117KO mice and their Plp1^TM117FL controls (Fig. 2I).

In contrast, female Plp1^TM117KO mice following exactly the same protocol (P60-65; SI Appendix, Fig. S3A) responded similar to their Plp1^TM117FL littermate controls (SI Appendix, Fig. S3 B and C). Of note is that for comparable levels of hypoglycemia (SI Appendix, Fig. S3E) female Plp1^TM117KO mice in proestrus secreted higher amounts of glucagon (SI Appendix, Fig. S3D), hinting at hormonal modulation of CRR sensitivity.

To evaluate the impact of the decreased glucagon secretion on CRR we performed an insulin tolerance test (ITT) in a separate cohort of male animals at the same timepoint (4 wk after tamoxifen administration; Fig. 2J). As shown in Fig. 2K, male Plp1^TM117KO mice present comparable levels of hypoglycemia to their Plp1^TM117FL littermate controls up to one and a half hours after insulin injection, but afterward they present lower glycemic values compared to control mice, suggesting that the lower secretion of glucagon detected at the 1-h timepoint is impeding the recovery from IIH. In line with this suggestion, female Plp1^TM117KO mice that secrete glucagon in comparable levels to Plp1^TM117FL controls show also comparable glycemic levels for the total duration of the ITT test (SI Appendix, Fig. S3 F and G).

Given the pronounced effect on CRR we wondered whether the transient depletion of Tmem117 from mature oligodendrocytes could have an effect on the long-term metabolic health of the animals. We generated cohorts of male and female mice with depletion in adulthood (P65-P70; Fig. 2L) and monitored their weight gain overtime both under control conditions (chow diet) and upon a metabolic challenge [Western diet (WD) enriched in fat and sugar]. As shown in Fig. 2M, male Plp1^TM117KO mice on regular chow diet gain more weight compared to their Plp1^TM117FL controls. The same effect but with higher variability is also observed for female Plp1^TM117KO mice (SI Appendix, Fig. S3I). A glucose tolerance test performed at the end of the monitoring period (at 19 wk of age) revealed also sex dimorphic effects with male Plp1^TM117KO mice showing higher blood glucose levels in comparison to their Plp1^TM117FL controls, irrespective of the diet (Fig. 2N), and female Plp1^TM117KO mice responding identical to their Plp1^TM117FL control littermates (SI Appendix, Fig. S3J). A full gross and histological postmortem phenotyping of the different mouse groups did not reveal any pathological changes or suggest functional differences in any organ/tissue except for the liver where differences in the extent of hepatocellular lipidosis were observed. These were further investigated and quantified revealing increased steatosis in the mice that received WD. Furthermore, male Plp1^TM117KO mice on chow diet showed increased lipid accumulation when compared to their Plp1^TM117FL chow controls (Fig. 2O and SI Appendix, Fig. S4 and Table S1). On the contrary, female Plp1^TM117KO chow mice and their Plp1^TM117FL chow controls showed similar levels of lipidosis (SI Appendix, Figs. S3K and S4 and Table S1).

These results demonstrate that loss of Tmem117 expression in mature oligodendrocytes triggers CRR defects and systemic metabolic imbalances only in male mice.

Transient Loss of Tmem117 in Mature Oligodendrocytes Triggers Myelin Defects Accompanied by Proteomic Signatures of ER-Stress.

Given the sex-specific phenotype of Plp1^TM117KO mice, we next aimed to investigate if the loss of Tmem117 from mature oligodendrocytes triggers sex dimorphic defects in myelin homeostasis. To investigate the role of oligodendrocytic Tmem117 in myelin homeostasis, we generated a triple transgenic mouse line enabling fluorescent quantification of myelin content by crossing the Plp1^TM117KO mouse line with the CNP-mEGFP mouse line (JAX:026105) (Fig. 3A).

Fig. 3.

Decreased levels of Tmem117 in mature oligodendrocytes of adult male mice lead to myelin defects. (A) Schematic representation of the transgenic mouse line generation and the experimental timeline. (B) Quantification of fluorescently labeled myelin across the whole brain of Plp1^TM117KO/+;CNP-mEGFP male mice (orange) and their Plp1^TM117FL/+;CNP-mEGFP control littermates (black) (n = 3 per group). (C) Representative heatmaps depicting fluorescent intensity per brain region for Plp1^TM117FL/+;CNP-mEGFP control mice (1, 2) and Plp1^TM117KO/+;CNP-mEGFP mice with high (3, 4) or low (5, 6) intensity output. (D) Quantification of fluorescently labeled myelin across several brain regions of Plp1^TM117KO/KO;CNP-mEGFP male mice (red) and their Plp1^TM117FL/FL;CNP-mEGFP control littermates (black) (n = 2 to 5 per group). (E) Representative images of the fluorescently labeled myelin intensity in Plp1^TM117FL/FL;CNP-mEGFP control mice (1) and Plp1^TM117KO/KO;CNP-mEGFP mice with high (2) or low (3) intensity output. (F) Representative images of the fluorescently labeled myelin coverage in the Corpus callosum of Plp1^TM117FL/FL;CNP-mEGFP control mice (1) and Plp1^TM117KO/KO;CNP-mEGFP mice with high (2) or low (3) intensity output. (G) Quantification of fluorescently labeled myelin coverage in the Corpus callosum of Plp1^TM117KO/KO;CNP-mEGFP male mice (red) and their Plp1^TM117FL/FL;CNP-mEGFP control littermates (black) (n = 3 to 4 per group; unpaired t test). (H) Transmission electron microscopy analysis of myelin in the Corpus callosum of Plp1^TM117KO male mice (5–9) and their Plp1^TM117FL control littermates (1–4) (n = 3 to 4 per group). Lines correspond to mean values and error bars represent ±SEM. *P < 0.05. CB: cerebellum, cbf: cerebellum related fiber tracts, CC: Corpus callosum, cm: cranial nerves, CTXsp: cortical subplate, eps: extrapyramidal fiber system, HPF: hippocampal formation, HY: hypothalamus, lfbs: lateral forebrain bundle system, MB: midbrain, mfbs: medial forebrain bundle system, MY: medulla, P: pons, PAL: pallidum, scwm: supracallosal cerebral white matter, STR: striatum, TAM: tamoxifen, TH: thalamus.

Our previous data from Olig2^TM117HET mice suggested that haploinsufficiency of Tmem117 across oligodendrocytes leads to male-specific growth retardation comparable to that observed in homozygous Olig2^TM117KO mice (SI Appendix, Fig. S2B). Therefore, we first assessed whether downregulation of Tmem117 in mature oligodendrocytes, through deletion of a single allele, affects myelin content across the adult brain. Male Tmem117^fl/+; Plp1-creER^T(tg/+);CNP-mEGFP^(tg/tg) (Plp1^TM117KO/+;CNP-mEGFP) mice and their Tmem117^fl/+; Plp1-creER^T(+/+);CNP-mEGFP^(tg/tg) (Plp1^TM117FL/+;CNP-mEGFP) littermate controls were injected i.p. with tamoxifen at P60-P65 to induce recombination of the Tmem117 locus in mature oligodendrocytes. Four weeks later the fixed brains were collected, rendered transparent by SHIELD clearing and imaged by light-sheet microscopy. Data were subsequently registered to the Allen Brain Atlas to enable quantitative assessment of fluorescent myelin signal across brain regions. This analysis revealed substantial interindividual variability among Plp1^TM117KO/+;CNP-mEGFP mice. While two male animals displayed fluorescence intensities comparable to controls (high intensity), one mouse exhibited a pronounced reduction in signal across the entire brain (low intensity; Fig. 3 B and C and Movies S2–S4). A similar pattern was observed in female Plp1^TM117KO/+;CNP-mEGFP mice, with two animals showing marked reductions in fluorescence and the remaining two resembling control levels (SI Appendix, Fig. S5 A–C and Movies S5–S7).

To determine whether this variability could be attributed to the presence of a remaining functional allele, we next assessed myelin content in homozygous Plp1^TM117KO mice using confocal microscopy on brain sections. Male Tmem117^fl/fl; Plp1-creER^T(tg/+);CNP-mEGFP^(tg/tg) (Plp1^TM117KO;CNP-mEGFP) mice and their Tmem117^fl/fl; Plp1-creER^T(+/+);CNP-mEGFP^(tg/tg) (Plp1^TM117FL;CNP-mEGFP) littermate controls were treated with tamoxifen at P60–P65, and brains were collected 4 wk later. Coronal sections (25 μm) were imaged, and representative regions (Cortex, Striatum, Thalamus, Hypothalamus, and Corpus callosum) were selected for quantitative analysis. Consistent with previous observations, Plp1^TM117KO;CNP-mEGFP mice displayed pronounced interindividual variability, with 3 out of 5 males (Fig. 3 D and E) and 1 out of 5 females (SI Appendix, Fig. S5 D and E) showing reduced fluorescence intensity across all regions examined, indicative of decreased myelin content. The effect was most prominent in the highly myelinated Corpus callosum. Higher magnification imaging revealed well-aligned myelinated fibers in the Corpus callosum of control mice, whereas Plp1^TM117KO;CNP-mEGFP mice exhibited disrupted and misaligned signal in both males (Fig. 3F) and females (SI Appendix, Fig. S5F). Notably, white matter from Plp1^TM117KO;CNP-mEGFP mice contained areas devoid of fluorescence, consistent with focal myelin loss (Fig. 3 F and G and SI Appendix, Fig. S5 F and G), that were more prominent in the “low intensity” samples (Fig. 3F3 and SI Appendix, Fig. S5F3).

To determine whether these alterations reflect myelin decompaction, we performed transmission electron microscopy (TEM) on Corpus callosum samples from an independent cohort of male mice (Fig. 3H). These analyses were consistent with the variability observed using fluorescent imaging: 2 out of 4 Plp1^TM117KO mice exhibited clear demyelination (Fig. 3 H5 and H6), one showed severe myelin decompaction and swellings (Fig. 3H7), and one displayed milder myelin sheath decompaction (Fig. 3 H8 and H9).

Given the widespread myelin defects, we next assessed whether Plp1^TM117KO mice exhibit motor related behavioral phenotypes. Motor coordination was evaluated using the rotarod test, and general activity was assessed using the open field test (OFT). Four weeks after tamoxifen administration, both male and female Plp1^TM117KO mice showed mild impairments in rotarod performance (SI Appendix, Fig. S5 H and I), while no significant differences were observed in locomotor activity or anxiety-related behavior in the OFT (SI Appendix, Fig. S5 J–M).

Considering that in the Plp1^TM117KO mouse model Tmem117 is knocked-out only in mature oligodendrocytes at the moment that tamoxifen is administered, we next assessed if the myelin defects we observe are long lasting or can be resolved later on by newly formed oligodendrocytes. Plp1^TM117KO;CNP-mEGFP mice of both sexes and their Plp1^TM117FL;CNP-mEGFP littermate controls were treated with tamoxifen at P60–P65, and brains were collected 8, 12, and 24 wk later (SI Appendix, Fig. S6A). Coronal sections (25 μm) were imaged, and representative regions (Cortex, Striatum, Thalamus, Hypothalamus, and Corpus callosum) were selected for quantitative analysis. In the case of Plp1^TM117KO male mice, both fluorescent intensity per brain region (SI Appendix, Fig. S6 B and C) and myelin coverage assessed in higher-resolution images (SI Appendix, Fig. S6 D and E) were comparable to Plp1^TM117FL controls. Suggesting that the defects detected at the 4-wk timepoint are only transient and restored later on by the formation of new myelin. In contrary, female Plp1^TM117KO mice show strong decrease in fluorescent intensity at the 8-wk timepoint (SI Appendix, Fig. S6F) that is not observed at the later timepoints (SI Appendix, Fig. S6 G and H). While closer analysis of the myelin coverage in higher-resolution images reveals areas devoid of fluorescence across all timepoints (SI Appendix, Fig. S6 I and J), suggesting that focal demyelination is not fully restored even 4 mo after tamoxifen injection.

To further explore the molecular consequences of Tmem117 loss that could contribute to the myelin defects observed, we collected the Corpus callosum of Plp1^TM117KO male mice and their Plp1^TM117FL littermate controls 2 wk after induction of recombination by tamoxifen injection (Fig. 4A). Quantitative Real-Time PCR (qRT-PCR) verified the efficient recombination of the Tmem117 locus (Fig. 4B) and showed an increase in spliced XBP1 (sXBP1; Fig. 4C) indicative of endoplasmic reticulum (ER)-stress induction. Proteomic analysis identified 483 differentially expressed proteins (410 upregulated, 73 downregulated; Fig. 4D). STRING analysis of the 483 differentially expressed proteins revealed that the two most affected pathways in regard to the KEGG pathway database were protein processing in the ER and regulation of actin cytoskeleton (Fig. 4E). A closer look at the networks formed by the differentially abundant proteins, focusing only on high-confidence interactions, pointed toward hubs related to respiratory electron transport, mitochondrial translation, vesicle tethering, autophagy, butanoate metabolism, cell adhesion/extracellular matrix, spliceosome/interferon signaling, and amyloid formation (Fig. 4F).

Fig. 4.

Depletion of Tmem117 from mature oligodendrocytes triggers ER-stress and a wide range of molecular adaptations in the Corpus callosum. (A) Schematic representation of the experimental timeline. (B and C) Real-time PCR results of samples deriving from the Corpus callosum of male Plp1^TMEM117KO mice (red) and Plp1^TMEM117FL control littermates (black) verifying the recombination of the Tmem117 locus (B) and revealing increased levels of spliced Xbp1 (C) 2 wk after TAM injection (n = 7 per group; unpaired t test). (D) Volcano plot depicting the differentially abundant proteins in the Corpus callosum of male Plp1^TMEM117KO mice compared to Plp1^TMEM117FL control littermates (n = 4 samples per group; 5,517 proteins in total; 483 with P < 0.05; 90 with P < 0.01). (E) STRING KEGG pathway enrichment analysis for all the differentially abundant proteins in the Plp1^TMEM117KO Corpus callosum that showed a P < 0.05. (F) STRING functional protein association network analysis with k-means clustering performed for all the differentially abundant proteins in the Plp1^TMEM117KO Corpus callosum that showed a P < 0.05. Lines correspond to mean values and error bars represent ±SEM. *P < 0.05, ****P < 0.0001. IF: interferon, IP: inositol phosphate, TAM: tamoxifen.

Taken together, these results demonstrate that Tmem117 is essential for maintaining oligodendrocyte homeostasis and myelin integrity in both sexes and its loss induces ER stress and disrupts a wide array of cellular pathways, likely contributing to oligodendrocyte instability and demyelination.

Tmem117 Decreases Intracellular Calcium by Modulating NCX1 Activity.

The broad cellular disruptions observed, alongside the restricted expression pattern of Tmem117, prompted us to ask whether Tmem117 supports homeostasis via a conserved molecular mechanism. Therefore, we decided to look into the specific features of Tmem117-enriched cells. From an evolutionary perspective, Tmem117 appears for the first time in vertebrates, but alignment of distant members based on the PFMA motif reveals expression of a TMEM117 protein domain also in cnidaria. To identify the transcriptomic signatures of Tmem117-enriched cells across evolution we performed differential expression analysis of Tmem117-enriched cells versus cells showing minimal to no expression of Tmem117 in three datasets: cells of the human brain white matter (12), mouse cells from 20 different organs (10) and cells of the sea anemone (Nematostella vectensis; v1g11324-enriched cells) (13). The results from M. musculus and N. vectensis were transformed into human orthologs and were overlapped with the results from H. sapiens. As expected, there was a very small overlap between all three organisms containing only 17 transcripts (Fig. 5A). From those 14 ribosomal proteins were commonly downregulated in the Tmem117-enriched cells of all organisms, eno1 was contraregulated (upregulated in M. musculus and downregulated in N. vectensis and H. sapiens) and only two transcripts were commonly upregulated: Tmem117 and Slc8a1.

Fig. 5.

Tmem117 decreases intracellular calcium by modulating NCX1 activity. (A) Venn diagram depicting the overlap of the differentially expressed genes in Tmem117-enriched cells compared to cells that express low to no Tmem117 across three evolutionary distinct organisms (bold: common upregulated, red: contraregulated, underlined: common downregulated). (B) Schematic representation of the experimental outline for panels C–G. (C) Quantification of intracellular calcium in GT1-7 cells expressing Tmem117 (blue) versus mock transfected (gray) or nontransfected (black) cells (n = 48 to 66 wells per group; one-way ANOVA; P values correspond to Tukey’s multiple comparisons test results]. (D) Quantification of intracellular calcium in MIN6B1 cells expressing Tmem117 (blue) versus mock transfected (gray) or nontransfected (black) cells (n = 6 to 9 wells per group; two-way ANOVA; P values correspond to Tukey’s multiple comparisons test results). (E–G) Ratiometric quantification of intracellular calcium in HEK293T cells coexpressing TMEM117-GFP and NCX1. Correlation of the GFP intensity (used as a proxy for TMEM117 expression levels) with intracellular calcium without (E) or with (F) the NCX1 inhibitor SEA0400. Representative images (G) with cells showing higher expression of TMEM117-GFP highlighted by the white arrows (n = 68 to 108 cells per group; Pearson’s r correlation analysis). (H) Western blot quantification of NCX1 and representative blot of HEK293T cells coexpressing GFP and NCX1 (black) or TMEM117-GFP and NCX1 (green) (n = 4 per group; unpaired t test). (I and J) Proximity ligation assay quantification (I) and representative images (J) of HEK293T cells coexpressing GFP and NCX1 (black) or TMEM117-GFP and NCX1 (green) (n = 6 per group; unpaired t test). (K) Schematic representation of the experimental outline for panels L–N. (L) Real-time PCR results of samples deriving from the Corpus callosum of male Plp1^TMEM117KO mice (red) and Plp1^TMEM117FL control littermates (black) verifying the recombination of the Tmem117 locus (n = 10 to 14 per group; unpaired t test). (M) Western blot quantification of Tmem117 and representative blot of samples from the Corpus callosum of male Plp1^TMEM117KO mice (red) and Plp1^TMEM117FL control littermates (black) (n = 7 to 15 per group; unpaired t test). (N) Western blot quantification of NCX1 and representative blot of samples from the Corpus callosum of male Plp1^TMEM117KO mice (red) and Plp1^TMEM117FL control littermates (black) (n = 9 to 15 per group; unpaired t test). Lines correspond to mean values and error bars represent ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. LG: low glucose, PLA: proximity ligation assay, TAM: tamoxifen.

Slc8a1 encodes for NCX1, a sodium-calcium exchanger vital for calcium homeostasis. We had observed in previous studies that Tmem117 depletion led to a strong increase in intracellular calcium in AVP neurons (7). Therefore, we decided to investigate in vitro if overexpression of Tmem117 in NCX1 expressing cell lines would alter intracellular calcium (Fig. 5B). Cells of the mouse hypothalamic neuronal cell line GT1-7 transfected with a plasmid encoding for the mouse Tmem117 CDS and loaded with the fluorescent calcium indicator Fluo-4 showed lower levels of intracellular calcium upon treatment with KCl compared to nontransfected cells or cells transfected with a mock plasmid not containing the Tmem117 sequence (Fig. 5C). We then performed similar experiments in the mouse pancreatic beta cell line MIN6B1 to get insight not only into the intracellular calcium levels but also into their effect on the secretory capacity of the cells. Again, similarly to GT1-7 experiments, the cells that were transfected with the Tmem117 plasmid showed lower intracellular calcium levels upon KCl treatment (Fig. 5D). In line with the lower intracellular calcium levels, the insulin secretion of Tmem117 overexpressing cells was decreased (SI Appendix, Fig. S7A). Interestingly the total insulin content of the Tmem117 overexpressing cells was also decreased (SI Appendix, Fig. S7B), an effect that could possibly be explained by a decreased translational activity suggested by our differential expression analysis (Fig. 5A). Western blot analysis of the same samples, apart from verifying the overexpression of Tmem117 (SI Appendix, Fig. S7 C and D), also revealed a negative correlation between the levels of Tmem117 and the amount of insulin secreted (SI Appendix, Fig. S7E).

To investigate whether this effect on intracellular calcium is mediated through NCX1 we cotransfected HEK293T cells with a plasmid expressing the human TMEM117 tagged with GFP in the C-terminus and a plasmid expressing the human NCX1. The NCX1 plasmid was also carrying a sequence providing resistance over the antibiotic zeocin. Therefore, the cells that survived in a zeocin containing medium and had GFP fluorescence were expressing both proteins of interest. Forty-eight hours after transfection cells were treated with medium containing the ratiometric calcium indicator Fura red alone or in combination with the NCX1 inhibitor SEA0400 for 1 h. Then cells were washed, fixed, and analyzed for intracellular calcium levels using confocal microscopy. As shown in Fig. 5E, a negative correlation between the levels of TMEM117-GFP expression and the concentration of intracellular calcium was observed. Interestingly, this correlation was not present in the cells that were treated with the NCX1 inhibitor (Fig. 5F), proving that the lower intracellular calcium observed in Tmem117 overexpressing cells is due to NCX1 activity. Representative images can be found in Fig. 5G, with cells showing higher expression of TMEM117-GFP highlighted with white arrows.

To check whether this higher NCX1 activity in TMEM117 overexpressing cells is due to higher levels of NCX1 expression we quantified NCX1 levels with immunofluorescence and western blot. Quantification of the NCX1 immunofluorescent analysis revealed a positive correlation between TMEM117-GFP fluorescent intensity and NCX1 fluorescent intensity (SI Appendix, Fig. S7 F and G), suggesting that presence of TMEM117 in higher levels leads to higher levels of NCX1 protein. This suggestion was further proved by western blot analysis that revealed a profound enrichment of NCX1 protein in TMEM117-GFP transfected cells compared to GFP only transfected control cells (Fig. 5H). Furthermore, to investigate if this stabilization of NCX1 by TMEM117 is through a direct interaction we utilized a proximity ligation assay (PLA). As shown in Fig. 5 I and J, the PLA assay revealed a strong signal in TMEM117-GFP expressing cells that was not present in GFP only transfected control cells.

Last, to evaluate the relevance of the Tmem117–NCX1 mechanism in vivo we isolated RNA and protein from the Corpus callosum of Plp1^TM117KO male and female mice and their Plp1^TM117FL littermate controls 4 wk after induction of recombination by tamoxifen treatment (Fig. 5K). Quantification by real time PCR revealed efficient recombination of the Tmem117 locus in both male (Fig. 5L) and female (SI Appendix, Fig. S7I) Plp1^TM117KO samples. While protein quantification by western blot verified the decrease in Tmem117 protein levels (Fig. 5M and SI Appendix, Fig. S7J) and a concurrent downregulation of NCX1 protein levels (Fig. 5N and SI Appendix, Fig. S7K) in both sexes. Interestingly, the protein levels of NCX3 in the same samples appeared comparable between Plp1^TM117KO mice and their Plp1^TM117FL controls (SI Appendix, Fig. S7 L and M).

These results identify Tmem117 as a molecular stabilizer of NCX1, suggesting a unifying mechanism by which Tmem117 maintains calcium homeostasis and supports cell viability in specialized cell types such as oligodendrocytes and neuroendocrine cells.

Discussion

Tmem117 was initially identified in a genetic screen of 36 recombinant BXD mouse strains that aimed at uncovering hypothalamic regulators of the CRR. A clinical quantitative trait locus (cQTL) was mapped to a region on chromosome 15, and among all genes within this locus, only two transcripts—Irak4 and Tmem117—showed a strong correlation with glucagon secretion during IIH (8). In our previous work, we next identified an expression quantitative trait locus (eQTL) for Tmem117 mapping to the same genomic region and we further demonstrated its functional relevance in AVP-producing neuroendocrine cells (7). Here, by mining publicly available RNAseq datasets, we expand on these findings, identifying an enrichment of Tmem117 in cells of the oligodendrocyte lineage and establishing its essential role in maintaining myelin homeostasis.

Closer inspection of single-cell RNAseq data revealed that although Tmem117 is particularly enriched in oligodendrocytes, it is also expressed in distinct subpopulations of neurons and hypothalamic astrocytes (Fig. 1 A and C). Beyond the CNS, analysis of the Tabula Muris dataset revealed a similarly selective expression profile in peripheral tissues, including subsets of large intestine epithelial cells, cardiomyocytes, and kidney collecting duct cells. This cell-type specificity prompted us to perform a differential expression analysis comparing Tmem117-enriched cells to nonenriched populations. Applying an evolutionary filter to this analysis led to the identification of Slc8a1, which encodes the sodium-calcium exchanger NCX1, as the only transcript consistently coenriched across all Tmem117-enriched cells. For these cross-species comparisons, we mapped M. musculus and N. vectensis transcripts to their H. sapiens orthologs. Consequently, while Slc8a1 emerged as the main candidate, its paralogs Slc8a2 (NCX2) and Slc8a3 (NCX3) are also orthologs of the N. vectensis transcript v1g239709, which was enriched in cells expressing v1g11324 (the gene encoding the TMEM117 protein domain). Although we focus here on in vitro validation of the TMEM117–NCX1 interaction, our data raise the possibility that TMEM117 also modulates NCX2 and NCX3 function. Our western blot data showcasing downregulation of NCX1 and not NCX3 in the Corpus callosum of Plp1^TM117KO mice point toward a specific contribution to NCX1 stabilization, but given the known importance of NCX3 in mature oligodendrocytes (14), its potential involvement—alongside NCX1—in the observed phenotypes becomes a priority for future studies. Our in vitro findings show that TMEM117 stabilizes NCX1 and modulates intracellular calcium dynamics, pointing to a regulatory mechanism with broad physiological relevance. Calcium homeostasis is fundamental to cellular function, and NCX activity plays a pivotal role in numerous pathological contexts (15). Notably, a recent study reported that Tmem117 knockdown is protective against cardiac hypertrophy (16)—a condition in which NCX1 is upregulated. Our data suggest a mechanistic basis for this observation: TMEM117-dependent stabilization of NCX1. In oligodendrocytes, NCX1 and NCX3 are differentially regulated during lineage progression—NCX1 is predominant in OPCs and downregulated with maturation, whereas NCX3 follows the opposite pattern (14). The enrichment of Tmem117 in both OPCs and mature oligodendrocytes (Fig. 1A) supports the hypothesis that it may serve as a stage-specific modulator of calcium signaling via these exchangers.

Apart from molecular interactions, our study highlights a broader physiological principle: oligodendrocyte homeostasis is critical—particularly in males—for CRR and systemic metabolic regulation. We demonstrate that Tmem117 deletion throughout the oligodendrocyte lineage impairs CRR and causes myelin abnormalities in male mice. Strikingly, inducible deletion of Tmem117 in mature oligodendrocytes during adulthood recapitulates these effects and leads to long-term alterations in metabolic profiles. This suggests that even transient disruptions in mature oligodendrocyte function—despite the capacity for OPC-mediated remyelination—can result in enduring physiological consequences. CRR has traditionally been considered a strictly neuronal process. Glucose-sensing neurons in key brain regions detect fluctuations in blood glucose and trigger neuroendocrine responses to restore homeostasis (17). Our results suggest that nonneuronal cells, specifically oligodendrocytes, also play a critical role—likely by influencing axonal conduction and signal fidelity within this circuitry. Furthermore, our findings related to weight gain, glucose tolerance, and hepatic lipid deposition demonstrate that oligodendrocyte dysfunction alone can impact whole-body metabolism. To date, most studies have adopted a bottom–up perspective, examining how systemic metabolic imbalances affect CNS cell populations (18). Our data introduce a complementary, top–down model in which oligodendrocyte dysfunction and disrupted myelin integrity act as upstream drivers of systemic metabolic imbalance. Importantly, these effects were sexually dimorphic. We observed them exclusively in male mice, while female mice exhibited either no phenotype or, intriguingly, enhanced CRR during proestrus (Fig. 3G). This points toward a role for sex hormones in modulating central glucose sensing and neuroglial interactions. Previous studies have reported sex differences in oligodendrocyte biology and myelin dynamics (19–21), and sexual dimorphism in systemic metabolism is well documented (22). Our data suggest that depletion of Tmem117 from mature oligodendrocytes leads to NCX1 downregulation and myelin defects in both sexes. If anything, the effect appears to persist longer in female mice. However, the impaired CRR and long-term metabolic imbalances are observed exclusively in males, indicating a male-specific metabolic vulnerability to disrupted oligodendrocyte homeostasis. Whether these sex-specific outcomes arise from differences in the myelination of neural circuits controlling glucose homeostasis, intrinsic properties of the oligodendrocyte lineage, or sexually dimorphic regulation of Tmem117 itself remains an open question for future investigation.

The importance of oligodendrocytes in providing metabolic support to axons has long been established (5). However, when considering whole-body metabolism, most studies have focused on how metabolic imbalances affect myelin integrity and drive the progression of demyelinating diseases (23, 24). Only recently have a few preclinical studies begun to examine how disruption of oligodendrocyte homeostasis influences metabolic health (25–28). Our data support this emerging view, showing that even a transient disruption can lead to prolonged metabolic effects in male mice. Clinically, patients with several demyelinating diseases, particularly multiple sclerosis (MS) and chronic inflammatory demyelinating polyneuropathy, show a higher prevalence for insulin resistance and diabetes (29, 30). However, these associations are generally attributed to chronic inflammation, immune dysregulation, treatment effects, and shared cardiometabolic risk factors, rather than myelin loss itself. Although it is impossible to directly isolate the contribution of myelin loss per se in clinical settings, our findings suggest that myelin loss may represent an additional factor contributing to the increased prevalence of metabolic disease, potentially in a sexually dimorphic manner.

That being stated, several important questions remain unresolved. Our study does not establish whether alterations in Tmem117 contribute to acquired forms of counterregulatory failure, such as those observed in recurrent hypoglycemia, hypoglycemia-associated autonomic failure, or diabetes. Likewise, although our findings demonstrate that disruption of oligodendrocyte homeostasis can impair CRR and systemic metabolic regulation in an experimental model, they do not establish that myelin abnormalities are a causal driver of defective counterregulation in humans. Future studies will therefore be required to determine whether oligodendrocyte dysfunction contributes to clinically relevant metabolic disorders and to identify the specific neural circuits and mechanisms through which myelin integrity influences counterregulatory responses. Nevertheless, our findings provide direct evidence that perturbation of oligodendrocyte homeostasis is sufficient to disrupt CRR and metabolic health in male mice, highlighting a previously underappreciated role for oligodendrocytes in the regulation of systemic energy balance.

Beyond Tmem117’s role in oligodendrocyte homeostasis and the accompanied systemic consequences, several additional questions arise from our findings. Our whole brain light-sheet microscopy data supported by high-resolution confocal imaging and electron microscopy analysis of the Corpus callosum suggests that our Plp1^TMEM117KO mouse line may represent a model for inducible demyelination. Induction of ER stress and increased reactive oxygen species are among the key factors leading to oligodendrocyte disfunction and subsequent demyelination in MS (31, 32). Up to date the mouse models available for MS research are partially capturing some aspects of disease pathophysiology (33), with the main focus either on the inflammatory aspect (autoimmune encephalomyelitis, virus-induced demyelination) or on the demyelination aspect (toxin-induced demyelination). This incomplete resemblance has been proposed as a possible explanation for the low translational success of existing models (34), prompting for better preclinical models that combine these two main aspects while resembling the “CNS first” pathology in which dysfunction of oligodendroglial lineage cells leads to demyelination and subsequent immune response. In this context, the Plp1^TMEM117KO mouse line may represent a promising candidate, with the additional advantage that recurrent demyelinating events can be triggered through repetitive tamoxifen administration. However, more extensive characterization of the inflammatory consequences and the remyelination capacity is required to support such a claim. In parallel, we are also exploring tools to directly assess how Tmem117 influences NCX1 and NCX3 in oligodendrocytes in vivo—an effort that could yield valuable insights for targeting demyelinating diseases. Finally, given Tmem117’s restricted expression in tissues such as the heart, kidney, and colon, we hope that our mechanistic insights into NCX1 regulation will inspire further investigation into its role in other organ systems and disease contexts.

In conclusion, our study identifies Tmem117 as a modulator of NCX1 activity with highly cell-specific expression and establishes oligodendrocyte homeostasis as a key regulator of systemic metabolic health. These findings open the door to further mechanistic dissection and suggest therapeutic avenues for diseases involving myelin dysfunction and calcium signaling dysregulation.

Materials and Methods

A combination of in vivo, ex vivo, and in vitro approaches was used to investigate the role of Tmem117 in oligodendrocytes and metabolic regulation. Conditional mouse models, metabolic phenotyping, vagal nerve recordings, and behavioral assays were combined with histological, ultrastructural, transcriptomic, and proteomic analyses. Myelin organization and brain-wide alterations were assessed using tissue clearing coupled to light-sheet imaging and automated registration to the Allen Brain Atlas, confocal microscopy, and electron microscopy. In parallel, cultured neuronal, pancreatic β-cell, and HEK293T cell models were used to examine the cellular and molecular effects of Tmem117 manipulation on calcium signaling, insulin secretion, and NCX1-related pathways. Detailed descriptions of experimental procedures, bioinformatic analyses, and statistical methods are provided in the SI Appendix.

Declaration of Generative AI And AI-Assisted Technologies in the Writing Process.

During the preparation of this work the authors used Microsoft Copilot and ChatGPT exclusively for text polishing as well as language and grammar corrections. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Supplementary Material

Appendix 01 (PDF)

Movie S1.

Depletion of Tmem117 from all oligodendrocyte lineage cells leads to growth retardation in male mice. Representative video depicting the growth retardation phenotype of Olig2^TM117KO male mice in comparison with Olig2^TM117KO female mice and Olig2TM117FL male and female control littermates.

Movie S2.

3D reconstruction of myelin signal in the male mouse brain. Representative video depicting the intensity and distribution of the fluorescent myelin signal in the brain of a Plp1^TM117FL/+;CNP-mEGFP control male mouse.

Movie S3.

3D reconstruction of myelin signal in the male Plp1TM117HET mouse brain. Representative video depicting the intensity and distribution of the fluorescent myelin signal in the brain of a Plp1^TM117KO/+;CNP-mEGFP male mouse that displayed high signal intensity.

Movie S4.

3D reconstruction of myelin signal in the male Plp1TM117HET mouse brain. Representative video depicting the intensity and distribution of the fluorescent myelin signal in the brain of a Plp1^TM117KO/+;CNP-mEGFP male mouse that displayed low signal intensity.

Movie S5.

3D reconstruction of myelin signal in the female mouse brain. Representative video depicting the intensity and distribution of the fluorescent myelin signal in the brain of a Plp1^TM117FL/+;CNP-mEGFP control female mouse.

Movie S6.

3D reconstruction of myelin signal in the female Plp1^TM117HET mouse brain. Representative video depicting the intensity and distribution of the fluorescent myelin signal in the brain of a Plp1^TM117KO/+;CNP-mEGFP female mouse that displayed high signal intensity.

Movie S7.

3D reconstruction of myelin signal in the female Plp1^TM117HET mouse brain. Representative video depicting the intensity and distribution of the fluorescent myelin signal in the brain of a Plp1TM117KO/+;CNP-mEGFP female mouse that displayed low signal intensity.

Data, Materials, and Software Availability

The original codes used for data analysis are available at https://doi.org/10.5281/zenodo.21475221 (35). All raw MS data together with raw output tables are available via the Proteomexchange data repository (www.proteomexchange.org) with the accession PXD064620 (36). All the imaging data used for the quantifications included in the manuscript are available in figshare: https://doi.org/10.6084/m9.figshare.33006602 (37); https://doi.org/10.6084/m9.figshare.33014234 (38); https://doi.org/10.6084/m9.figshare.33014678 (39); https://doi.org/10.6084/m9.figshare.33016136 (40); https://doi.org/10.6084/m9.figshare.33016166 (41); https://doi.org/10.6084/m9.figshare.33016247 (42); https://doi.org/10.6084/m9.figshare.33016259 (43); https://doi.org/10.6084/m9.figshare.33016265 (44); https://doi.org/10.6084/m9.figshare.33016271 (45); https://doi.org/10.6084/m9.figshare.33016301 (46); https://doi.org/10.6084/m9.figshare.33016319 (47); https://doi.org/10.6084/m9.figshare.33016325 (48); https://doi.org/10.6084/m9.figshare.33016331 (49); https://doi.org/10.6084/m9.figshare.33016337 (50); https://doi.org/10.6084/m9.figshare.33016346 (51); https://doi.org/10.6084/m9.figshare.33016388 (52); https://doi.org/10.6084/m9.figshare.33016400 (53); https://doi.org/10.6084/m9.figshare.33016409 (54); https://doi.org/10.6084/m9.figshare.33016418 (55); https://doi.org/10.6084/m9.figshare.33016433 (56); https://doi.org/10.6084/m9.figshare.33049979 (57); and https://doi.org/10.6084/m9.figshare.33050003 (58).