Expression and subcellular localization of mitochondrial docking protein, syntaphilin, in oligodendrocytes and CNS myelin sheath

Oligodendrocytes produce lipid-rich myelin sheaths that provide metabolic support to the underlying axon and facilitate saltatory conduction. Oligodendrocyte mitochondria supply the bulk of energy and carbon-chain backbones required for lipid synthesis. The sparsity of mitochondria in the myelin sheath suggests that tight regulation of mitochondrial trafficking is crucial for their efficient distribution in the cell. In particular, retention of mitochondria at axoglial junctions would support local lipid synthesis and membrane remodeling during myelination. How mitochondrial docking in oligodendrocytes is regulated is not known. Our findings indicate that syntaphilin (SNPH), a mitochondrial docking protein that has been characterized in neurons, is expressed by oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in vitro and present in the myelin sheath in vivo. We have previously reported that bath application of netrin-1 promotes the elaboration of myelin basic protein-positive membranes, and that localized presentation of a netrin-1 coated microbead results in rapid accumulation of mitochondria at the site of oligodendrocyte-bead adhesion. Here we show that netrin-1 increases the redistribution of SNPH to oligodendrocyte processes during the expansion of myelin basic protein-positive membranes and that SNPH clusters at the oligodendrocyte plasma membrane at sites of adhesion with netrin-1-coated beads where mitochondria are retained. These findings suggest roles for SNPH in oligodendrocytes regulating netrin-1-mediated mitochondrial docking and myelin membrane expansion.

structural basis for saltatory conduction. Myelination is a highly dynamic process that requires the addition of new plasma membrane at the growing edge of the myelin sheath (Snaidero et al., 2014). A single oligodendrocyte can produce a myelin membrane with a surface area of 20 Â 10 5 μm 2 (Pfeiffer et al., 1993). During axon ensheathment, cytoplasm is extruded from the majority of the myelin sheath and restricted to non-compacted channels that connect the cell body to the cytoplasmic channel at the extending edge (Snaidero et al., 2014). Mitochondria are found within the cytoplasmic paranodal loops that form specialized junctions with the axolemma flanking the node of Ranvier Rinholm et al., 2016). Mitochondria are a key source of energy and carbon-chain precursors required for lipid synthesis (Schulz, 1991); thus, the proper distribution of mitochondria in the myelin sheath is likely crucial for normal myelination. In particular, the retention of mitochondria within paranodal loops positions them in close proximity to regions of membrane remodeling where local lipid synthesis is critical. In neurons, intracellular signaling regulates mitochondrial localization, for instance, at focal sites of NGF signaling (Chada & Hollenbeck, 2004).
Recently, we reported that mitochondria in oligodendrocytes are retained at sites of contact with locally enriched netrin-1 . Netrin-1, an extracellular protein expressed by mature neurons and oligodendrocytes, stimulates expansion of the myelin membrane and is enriched at CNS paranodal axo-glial junctions (Jarjour et al., 2008;Manitt et al., 2001;Rajasekharan et al., 2009). Further, local extracellular enrichment of netrin-1 is sufficient to recruit paranodal proteins DCC and neurofascin 155 (NF155) in oligodendrocytes , and the absence of expression of netrin-1 or its receptor, deleted in colorectal cancer (DCC), results in severe disorganization of the paranodal junction in vivo (Bull et al., 2014;Jarjour et al., 2008). Netrin-1 promotes mitochondrial fusion and function in oligodendrocytes via a mechanism dependent on src family kinase activation and ROCK inhibition , however, the mechanisms underlying netrin-1-induced mitochondrial docking in oligodendrocytes are not known.
In neurons, the retention of mitochondria at metabolically demanding sites is critical for normal function. To meet the energy demands of nerve conduction, axonal firing increases the size of mitochondrial stationary sites within the axon at the node of Ranvier and paranode (Ohno et al., 2011). Disruption of axoglial junctions by mutating the axonal adhesion protein Caspr results in the abnormal localization of axonal mitochondria at paranodes, demonstrating that myelination is a key modulator of mitochondrial transport (Sun et al., 2009). In neurons, the mitochondrial docking protein syntaphilin (SNPH), which has been considered to be axonspecific, constrains mitochondrial movements by anchoring the mitochondrial outer membrane to the microtubule cytoskeleton (Kang et al., 2008). SNPH-dependent mitochondrial docking is regulated by its interaction with the motor proteins KIF5 and dynein (Chen et al., 2009;Chen & Sheng, 2013). The coupling of KIF5 and SNPH inactivates the KIF5 ATPase and halts mitochondrial movement in a calcium-dependent manner (Chen & Sheng, 2013).
Synaptic activity increases the retention of mitochondria at synapses, and loss of SNPH function abolishes activity-dependent immobilization of mitochondria in hippocampal neurons. Further, an increase in presynaptic calcium concentration, that follows a reduction of mitochondrial calcium buffering, provokes an increase in synaptic vesicle release and enhances short-term facilitation (Kang et al., 2008). Mitochondrial trafficking and calcium metabolism thus appear to be of major importance in both axonal conduction and synaptic activation.
Mitochondrial dysfunction is implicated in the progression of multiple sclerosis (MS), a neurodegenerative disease characterized by axon demyelination in the CNS (Witte et al., 2009). Chronically demyelinated MS lesions contain $3.2-fold more SNPH compared to normal appearing white matter, which correlates with increased mitochondrial mass in demyelinated axons (Mahad et al., 2009).
Destruction of the protective myelin sheath disrupts saltatory conduction and increases the insertion of Na + channels into the axonal membrane (Craner et al., 2004). The resultant pathological influx of Na + ions strains the Na + /K + ATPases required for Na + expulsion.
Consequently, mitochondria are retained at demyelinated segments along an axon to compensate for the large amounts of ATP consumed.
Studies in mouse models provide evidence for a biphasic role for SNPH in MS, whereby SNPH is neuroprotective in acute, earlyphase MS (Joshi et al., 2015;Ohno et al., 2011) but detrimental to axon survival in chronic, late-phase MS (Joshi et al., 2015). Loss of SNPH in the cuprizone or experimental autoimmune encephalomyelitis (EAE) mouse models of acute MS inhibits formation of mitochondrial stationary sites in demyelinated axons, leading to exacerbated axon degeneration (Joshi et al., 2015;Ohno et al., 2011). Strikingly, deletion of SNPH in the Shiverer mouse model of chronic MS yields positive effects on axon survival, mitochondrial health, and lifespan (Joshi et al., 2015). SNPH-deficiency restores mitochondrial respiratory chain activity and reduces ROSrelated oxidative stress in demyelinated axons. As demyelination progresses and energy demand increases, SNPH in degenerating axons may interfere with trafficking of damaged mitochondria to the cell body for mitophagy, ultimately leading to increased ROS production by dysfunctional mitochondria at demyelinated sites (Joshi et al., 2015).
Here we investigated the expression, subcellular distribution, and function of SNPH in oligodendrocytes. Our findings indicate that SNPH is expressed by OPCs and oligodendrocytes. SNPH exhibits a punctate distribution in the oligodendrocyte soma, myelin-like membrane in vitro, and paranodal loops of the myelin sheath in vivo. We show that application of netrin-1 promotes the redistribution of SNPH to oligodendrocyte processes during the elaboration of myelinlike membranes and that SNPH aggregates in oligodendrocytes at sites of adhesion with netrin-1-coated microbeads. These findings identify a potential role for SNPH in the regulation of netrin-1-dependent mitochondrial docking in oligodendrocytes that may affect myelin membrane expansion.

| Animals
Sprague-Dawley rat pups were obtained from Charles River Canada (Montreal, Quebec, Canada). C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All procedures were performed in accordance with the Canadian Council on Animal Care guidelines for the use of animals in research.

| Quantitative real-time PCR (qRT-PCR)
RNA was collected at each time point using the Qiagen RNeasy Mini Kit, according to the manufacturer's protocol. Briefly, cells were lysed in RLT lysis buffer and scraped from the plate bottom using a spatula. Lysate was placed in a Qiashredder column (Qiagen, MD, USA), and spun at 15,000g for 2 min. Lysate was then mixed with 70% ethanol in RNase-free H 2 O (Qiagen, MD, USA) at a ratio of 1:1, transferred to an RNAeasy spin column and centrifuged at 15,000g for 25 s. The eluate was discarded, and the column washed with RW1 buffer and centrifuged at 15,000g for 25 s. Eluate was again discarded and the column washed twice in succession with RPE buffer. Finally, RNA was eluted in RNase-free H 2 O. Total cDNA was produced by RT-PCR from 150 ng of RNA using the RT2 First Strand Kit (Qiagen, MD, USA) according to the manufacturer's protocol. For gene expression analyses, qPCR was carried out using these RT-PCR products. Each reaction was carried out in technical triplicate, using four biological replicates (n = 4) for each time point. Amplification was carried out using the following protocol in a CFX Connect thermocycler: 95 C for 2 min, followed by 40 cycles of 95 C for 5 s, then 60 C for 30 s. Analysis was carried out using the CFX Connect software (Bio-Rad Laboratories, CA, USA) using the delta-delta Ct method, with a beta-actin (actb) template as a reference.

| Western blotting
Oligodendrocytes, and E18 rat cortical neurons cultured as described (Goldman et al., 2013), were homogenized in ice-cold RIPA buffer (10 mM phosphate buffer pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitors (2 μg/mL aprotinin, 5 μg/mL leupeptin, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF). Tissue lysates were prepared from freshly isolated adult mouse cortex, cerebellum and spinal cord. Isolated tissues were homogenized on ice with RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein per sample were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in TBS containing 0.1% Tween (TBST) for 1 h at rt. Primary antibodies were diluted in 5% milk in TBST and incubated with membranes overnight at 4 C. After three TBST washes, membranes were probed with horseradish peroxidase-conjugated secondary antibodies (5% milk in TBST) for 1 h at rt. Blots were developed using Immobilon Western Chemiluminescent HRP Substrate

| Immunocytochemistry and immunohistochemistry
Rat OPCs and oligodendrocytes grown in culture were fixed in 2% paraformaldehyde (PFA) + 10% sucrose for immunocytochemistry. Fixed cells and slides with myelinated nerve fibers were blocked with 3% bovine serum albumin (BSA), 3% heatinactivated horse serum (HIHS), 0.3% Triton-X 100 in PBS for 1 h at rt. Primary antibodies were diluted in the same blocking solution and were incubated with the samples overnight at 4 C on a gentle shaker. Cells were then washed 3 Â 10 min with PBS. This was followed immediately by a 1-h, rt incubation with secondary antibodies diluted in 3% BSA, 3% HIHS in PBS. Finally, cells were washed 3 Â 10 min in PBS and briefly washed with doubledistilled water (ddH 2 0) before mounting with Fluoro-Gel mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA).
For antigen retrieval, when applied before blocking, slides with tissue sections were immersed in boiling 0.1 M citrate buffer, pH 6.0, for 20 min. Fluorescent images were taken using a Leica TCS SP8 confocal microscope, Zeiss Axio Observer.Z1 or Zeiss Axiovert S100 TV epifluorescent microscope. 3D image reconstruction was carried out using the Leica Application Suite X (LAS X) software. Signal intensity measurements were done using FIJI image analysis software and quantification of the overlap between signals used Manders' coefficient calculation with the JACoP plugin. Measurement of fluorescent signal intensity in OLs used MBP immunolabeling to identify the cell body and processes of mature OLs and define regions of interest (ROIs) that were outlined for quantification.

| Antibodies
The following antibodies were used in this study: mouse monoclonal

| Structured illumination microscopy (SIM)
Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4 (GE Healthcare, IL, USA) equipped with a 60Â/1.42 NA PlanApo oil immersion lens (Olympus), 405, 488, 568 and 642 nm solid state lasers and sCMOS cameras (pco.edge). Image stacks of 5-6 μm with 0.125 μm thick z-sections and 15 images per optical slice (3 angles and 5 phases) were acquired using immersion oil with a refractive index of 1.518. Images were reconstructed using Wiener filter settings of 0.003 and optical transfer functions (OTFs) measured specifically for each channel with SoftWoRx 6.1.3 (GE Healthcare, IL, USA) to obtain superresolution images with a 2-fold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide (GE Healthcare, IL, USA) and SoftWoRx 6.5.2 (GE Healthcare, IL, USA).

| Stochastic optical reconstruction microscopy (STORM)
For STORM super resolution imaging, mice were perfused with 4% PFA, post-fixed in 30% sucrose, and fixed in OCT. Thirtymicrometer thick sections of the optic nerve were stored at

| Bead adhesion assay
Briefly, uniform polystyrene microbeads (7.3 μm diameter, nonfluorescent and "Dragon Green" fluorescent beads; Bangs Laboratories, IN, USA) were washed three times in sterile PBS and coated with recombinant netrin-1 protein (10 μg/mL) overnight followed by three washes in sterile PBS to remove non-adherent protein.
Microbeads were pelleted by centrifugation (7 min at 6500 rpm) and resuspended in warmed oligodendrocyte defined culture media.
Cells were incubated with resuspended microbeads for 2 h (37 C in a humidified 5% CO 2 incubator). Following three rigorous PBS washes to remove non-adherent microbeads, cells were fixed with 4% PFA or lysed for western blot analysis. For western blotting, equal volumes of bead isolate per condition were loaded onto an SDS-page gel.

| Statistical analysis
Statistical analyses were performed using GraphPad Prism software.
All data are presented as the mean ± standard error of the mean. Snph mRNA expression time-point datasets were tested using a one-way ANOVA with a Tukey post-hoc test. Comparisons of small datasets (n < 10 per group) were done using a Mann-Whitney nonparametric test. Comparisons of larger datasets were done using a Student's t-test if variances were not significantly different between groups (nonsignificant Fisher test), or a Mann-Whitney nonparametric test.
A p-value <0.05 was considered significant.

| Syntaphilin is expressed in oligodendrocytes and CNS myelin
Snph mRNA is expressed in human and rat brain tissue homogenates (Lao et al., 2000). Although SNPH is expressed in rodent neurons and human multiple sclerosis (MS) lesions (Joshi et al., 2015;Kang et al., 2008;Lao et al., 2000;Mahad et al., 2009;Ohno et al., 2011), the expression of SNPH by oligodendrocytes had not been reported.
We initially consulted the Allen Brain Atlas to visualize the distribution of cells expressing Snph mRNA in white matter tracts of the adult mouse brain (Figure 1a These results indicate that Snph mRNA expression peaks in immature OLs, while SNPH protein is readily detected throughout OPC and oligodendrocyte differentiation and maturation. The mechanisms that regulate the amounts of Snph mRNA and protein in oligodendrocytes remain to be determined.

| Subcellular localization of syntaphilin in oligodendrocytes
The subcellular distribution of SNPH was examined in rat oligodendrocytes in cell culture throughout differentiation. Cells were colabeled using antibodies against SNPH and MBP, and phalloidin to label F-actin. Initially, OPCs exhibit a bipolar morphology. As OPCs differentiate into oligodendrocytes, they extend multiple processes F I G U R E 2 Expression profile of SNPH mRNA and protein during OPC differentiation in vitro. Representative epifluorescence microscopy images of rat OPCs (a-c) and oligodendrocytes (d-h) at different stages of maturation that were collected for quantitative real-time PCR (qRT-PCR) analysis. (i) qRT-PCR detected relatively low levels of SNPH mRNA expression in OPCs (OPC-10h) with significantly increased expression in oligodendrocytes (1-10d). The ΔΔCt method was used to calculate mRNA levels which were normalized to β-actin (actb) expression. Graph represents four biological replicates. (j) Representative western blot of SNPH protein in cell homogenates of oligodendrocytes as they mature in vitro. The histograms present quantification of western blot immunoreactive band optical density from seven different cell cultures as biological replicates. One-way ANOVA, Tukey test (*p < 0.001). Error bars indicate SEM. Scale bar = 100 μm.
that become progressively more branched throughout maturation.
Concurrent with extension of the cytoskeleton, oligodendrocytes produce a lipid-rich plasma membrane sheet that forms the myelin sheath in vivo. SNPH was detected in the soma of OPCs (Figure 3a,b), and in the soma, branching processes, and cytoplasmic channels that tra-

| SNPH is associated with mitochondria in mature oligodendrocytes
To examine whether SNPH interacts with mitochondria, potentially acting as a docking protein in oligodendrocytes as it does in neurons (Kang et al., 2008), we used SIM and STORM super-resolution microscopy ( Figure 5). Mature oligodendrocytes were cultured for 7 DIV and immunolabeled for the outer mitochondrial membrane protein TOM20, for SNPH, and for F-actin using phalloidin. Using SIM, we observed punctate clusters of SNPH in the soma and processes in close association with the mitochondrial marker TOM20 (Figure 5a

| Potential role of syntaphilin in netrin-1-induced mitochondrial docking and myelin-like membrane sheet expansion in oligodendrocytes
To estimate the proportion of mitochondria potentially anchored by SNPH during oligodendrocyte maturation, we quantified the overlap between SNPH and TOM20 immunoreactivity. Our findings indicate an increase in the proportion of TOM20 signal overlapping with SNPH when comparing 1 DIV and 8 DIV oligodendrocytes (Figure 6a), suggesting more sites of SNPH anchorage associated with mitochondria in mature cells in vitro. The proportion of SNPH overlapping TOM20 did not significantly change during maturation (Figure 6b), suggesting that a similar proportion of the total SNPH in the cell is engaged in mitochondrial docking.
We previously reported that netrin-1 promotes process branching and the formation of MBP-positive myelin-like membranes (Rajasekharan et al., 2009), and that mitochondria rapidly aggregate in oligodendrocytes at sites of adhesion with netrin-1 coated microbeads . To investigate possible roles for SNPH in netrin-1-induced recruitment of mitochondria, we bath applied 200 ng/mL netrin-1 protein to 8 DIV F I G U R E 3 SNPH protein distribution during OPC differentiation in vitro. Representative confocal images of SNPH protein in rat OPCs (a, b) and oligodendrocytes (c-e). Aggregates of SNPH protein were detected in OPC soma, with a more dispersed distribution in the cell processes in oligodendrocytes (OLs). SNPH is readily detected localized to the extending MBP-positive membrane sheet in 5d and 10d OLs. Scale bar = 20 μm. mature oligodendrocytes. Following immunolabeling, we detected SNPH in MBP-positive myelin-like membrane sheets in control cells and cells exposed to netrin-1 for 2 h in vitro (Figure 6d and supplementary Video S2). Following this treatment with netrin-1, no significant change in the amount of SNPH protein was detected by western blot analyses of oligodendrocyte whole cell homogenates (Figure 6c).
In contrast, quantification of immunocytochemical fluorescence revealed a significant increase of SNPH intensity in netrin-1-treated oligodendrocytes (Figure 6e). To determine if netrin-1 induces a redistribution of SNPH within oligodendrocytes, we quantified SNPH immunoreactivity in cell bodies and cell processes. Significant increases in SNPH immunoreactivity were only detected in the cell processes (Figure 6f,g), suggesting that netrin-1 redistributes SNPH to the processes, coincident with promoting myelin-like membrane expansion (Rajasekharan et al., 2009(Rajasekharan et al., , 2010. No significant difference in the intensity of the mitochondrial marker TOM20 was found between control cells and oligodendrocytes treated with netrin-1 for 2 h (Figure 6h-j).
Due to the interaction between SNPH and mitochondria in oligodendrocytes ( Figure 5), and the redistribution of SNPH following application of netrin-1 (Figure 6), we investigated whether SNPH is involved in netrin-1-mediated mitochondrial docking. Mature rat oligodendrocytes at 7 DIV were incubated with netrin-1-coated microbeads for 2 h (Figure 7a). Western blot analyses revealed significantly more SNPH protein associated with netrin-1-coated microbeads compared to control beads (Figure 7b). Previously, we demonstrated that the outer mitochondrial protein, TOM20, is also significantly enriched with netrin-1-coated microbeads compared to controls . Using SIM super-resolution microscopy, punctate clusters of plasmamembrane-associated SNPH were detected at sites of netrin-1-coated microbead adhesion to oligodendrocytes (Figure 7c,d).
Overall, these results provide evidence that netrin-1 is sufficient to induce SNPH redistribution within oligodendrocyte myelin-like membrane sheets, potentially promoting local mitochondrial docking.

| DISCUSSION
Here, we demonstrate that Snph mRNA is expressed by oligodendrocytes throughout their development. SNPH protein was detected within OPC cell bodies and in oligodendrocytes, localized within the cell body and MBP-positive myelin-like membrane sheets.
In neurons, the release of neurotransmitters from synaptic vesicles into the synaptic cleft is dependent on the formation of a SNAP-Receptor (SNARE) complex, composed of proteins such as vesicle-associated synaptobrevin associating with plasma membranebound SNAP-25 and syntaxin-1 (Sollner & Rothman, 1994;Sudhof, 1995). SNPH competes with SNAP-25 for binding to syntaxin-1, thereby preventing the formation of the SNARE complex and inhibiting synaptic vesicle exocytosis (Das et al., 2003;Lao et al., 2000). Many of the genes encoding the main components of the SNARE machinery mediating vesicle release in neurons, including SNAP25, syntaxins and VAMP proteins, are expressed by oligodendrocytes (Lam et al., 2022;Marques et al., 2016) (http://linnarssonlab.org/ oligodendrocytes/), and these proteins may regulate the fusion of transport vesicles with the plasma membrane. Among them, VAMP3 and VAMP7 control SNARE-mediated exocytosis of vesicles transporting myelin proteolipid protein (PLP) to the myelin membrane in oligodendrocytes co-cultured with cortical neurons (Feldmann et al., 2011). the MBP-positive myelin-like membrane (Figure 6g). Phosphorylation of SNPH by cAMP-dependent protein kinase (PKA) is predicted to decrease its binding to syntaxin-1, thereby acting as an "off" switch for SNPH-mediated inhibition of exocytosis in neurons (Boczan et al., 2004), but the detailed regulation of SNPH function has yet to be described in neurons and oligodendrocytes. In the future, it will be crucial to identify what SNARE-complex components interact with SNPH in oligodendrocytes, and what post-translational modifications are involved in regulating its activity.
To date, SNPH is the only known mitochondrial docking protein in neurons, and it has also been identified as a cargo of mitochondrial derived vesicles (Kang et al., 2008;Lin et al., 2017). To investigate the interaction between SNPH and mitochondria in oligodendrocytes, we With oligodendrocyte maturation mitochondria may alter their length and morphology, complicating the interpretation of the correspondence between SNPH and TOM20 immunoreactivity. Additional studies will be required to determine the precise relationship between the distribution of SNPH and docked mitochondria at different stages of oligodendroglial maturation.
The presence of SNPH within non-compact paranodal loops and the compact myelin sheath in vivo (Figure 1g,h) suggests that SNPH may function within these regions to regulate mitochondrial trafficking and vesicle release. Previously, we demonstrated that mitochondria in oligodendrocytes are recruited to, and remain stationary at, local sources of netrin-1  but the mechanisms regulating netrin-1-dependent mitochondrial docking in oligodendrocytes are not known. Using super-resolution microscopy, we detected clusters of SNPH at sites of oligodendrocyte adhesion with netrin-1-coated microbeads (Figure 7c,d). Netrin-1 and its receptor DCC are enriched at CNS paranodes (Bull et al., 2014;Jarjour et al., 2008), where they may contribute to SNPH-mediated mitochondrial docking within the cytoplasm of the oligodendroglial paranodal loops (Meyer & Rinholm, 2021;Rinholm et al., 2016). Future studies examining Snph loss-of-function are required to identify the role of SNPH in the expansion of the F I G U R E 5 SNPH is closely associated with mitochondria in oligodendrocytes. (a-e) SIM images of mature rat oligodendrocytes cultured for 7 days in vitro. Cells were immunolabeled with antibodies against SNPH (red), outer mitochondrial membrane protein TOM20 (green), and F-Actin (blue). Panel (b) illustrates magnification of the boxed region in panel A showing clusters of TOM20 labeling in the oligodendrocyte cell body. A TOM20 containing process extends away from the cell body. Clusters of SNPH protein are sparsely distributed in the soma and cell processes (arrows). (c) SNPH appears to be located at sites of mitochondrial fission and (d, e) appears closely associated with mitochondria and the Actin cytoskeleton. (f-h) Immunolabeling for SNPH (green) and TOM20 (red) distributed along a process of a mature oligodendrocytes cultured for 7 days in vitro. STORM imaging localizes individual blinking fluorophores and represents the position of each fluorophore as a colored sphere. The $20 nm lateral and $50 nm axial resolution provided by STORM imaging reveals close interactions between SNPH and TOM20 labeling the outer mitochondrial membrane. Scale bars: (a) = 10 μm, (b, c, e, g) = 1 μm, (d, f) = 5 μm, (h) = 200 nm.
oligodendrocyte myelin membrane and cytoskeleton in the presence or absence of exogenous netrin-1.
As an extracellular guidance cue, netrin-1 directs the migration of commissural neurons to the floorplate in the developing spinal cord (Bin et al., 2015;Kennedy et al., 1994;Kennedy et al., 2006;Lai Wing Sun et al., 2011). Previous findings indicate that the orientation of axonal growth cones depends on netrin-1-dependant activation of a transmembrane influx of Ca 2+ , and Ca 2+ release from intracellular stores (Hong et al., 2000;Low et al., 2012;Tang & Kalil, 2005). Calcium can inhibit mitochondrial trafficking by binding Miro, a Rho GTPase connecting mitochondria to the motor protein KIF5 (Cai & Sheng, 2009;Wang & Schwarz, 2009). Miro binding to calcium causes the release of KIF5, which allows inhibition of KIF5 motor function by SNPH (Chen & Sheng, 2013). We speculate that netrin-1-induced local increases in intracellular calcium may result in Ca 2+ -dependent docking of mitochondria at the neuronal growth cone, a site with high energetic requirements and activity-dependent mitochondrial aggregation (Morris & Hollenbeck, 1993). If netrin-1 induces an increase of intracellular calcium in OLs, as it does in neurons (Hong et al., 2000), we would predict that netrin-1-coated microbeads may induce a local intracellular increase of calcium at sites of adhesion with oligodendrocytes, thus triggering Ca 2+ -dependent mitochondrial docking and recruitment of SNPH. Future loss of function experiments will examine the effect of silencing Snph expression on netrin-1-induced mitochondrial docking in oligodendrocytes.

SNPH-mediated mitochondrial immobilization is implicated in reg-
ulating the progression of axon degeneration in mouse models of MS (Joshi et al., 2015;Ohno et al., 2011). The amount of SNPH is highly elevated in human chronic MS lesions (Mahad et al., 2009) and the functional significance of this finding has been primarily associated with SNPH in neurons. Our study is the first to characterize SNPH expression in oligodendrocytes. We show that SNPH is expressed by differentiating OPCs and localizes along the branching cytoplasmic channels of the compacted myelin-like membranes of mature oligodendrocytes in vitro. The localization of SNPH within cytoplasmic paranodal loops and along myelinated internodes in vivo supports a function for SNPH in mature CNS myelin. Our findings show that bath application of netrin-1 recruits SNPH to oligodendroglial processes, and that subcellular enrichment of netrin-1 presented as a coated microbead is sufficient to locally recruit SNPH, where it may mediate mitochondrial docking and promote myelin membrane expansion in oligodendrocytes by regulating vesicular release at the growing edge of the compacted membrane.

ACKNOWLEDGMENTS
SIM imaging for this manuscript was performed at the McGill University Life Sciences Cell Imaging and Analysis Network Facility (CIAN). This work was supported by Multiple Sclerosis Society of Canada operating grants held by T.E.K. (#3009) and R.K. (#2989). Diane S. Nakamura was supported by a Multiple Sclerosis Society of Canada F I G U R E 6 Netrin-1 increases SNPH localization to the myelin-like membrane sheet in oligodendrocytes. (a) Proportion of TOM20 overlapping with SNPH immunolabeling during oligodendroctye differentiation in vitro visualized using confocal microscopy. (b) Proportion of SNPH overlapping with TOM20 immunolabeling during oligodendroctye differentiation in vitro. (c) Western blot analysis of SNPH in whole cell homogenates of mature oligodendroctyes comparing control verses 2 h netrin-1 (200 ng/mL). (d) Confocal images of 8DIV oligodendrocytes immunolabeled for TOM20 (green), SNPH (red) and MBP (white). Scale bars = 10 μm. (e-g) Quantification of SNPH immunofluorescence signal intensity (raw integrated density) in control cells and in oligodendrocytes exposed to 200 ng/mL netrin-1 for 2 h, in the whole cell, cell soma, or in cell processes excluding the soma. (h-j) Quantification of TOM20 signal intensity (raw integrated density) in control cells and in oligodendrocytes exposed to 200 ng/mL netrin-1 for 2 h, in the whole cell, cell soma or cell processes. Error bars indicate SEM.
F I G U R E 7 SNPH aggregates within oligodendrocytes at a local source of extracellular netrin-1. (a) Netrin-1-coated or uncoated control microbeads were incubated with mature rat oligodendrocytes in vitro for 2 h. Microbeads were collected by scraping, and beadassociated proteins were isolated via a series of vigorous washes, vortex-shearing and centrifugation. (b) Western blot analyses of beadassociated protein showed an increase in SNPH protein at netrin-1 beads. (c, d) Structured illumination microscopy (SIM) images of a netrin-1-coated microbead (blue) adhered to an oligodendrocyte through interactions with the cell membrane. Immunostaining for TOM20 and SNPH revealed an accumulation of SNPH at the cell membrane at the surface of netrin-1-coated beads. Student's t-test (**p < 0.01). Error bars indicate SEM. Scale bar = 5 μm.
studentship. Jean-David M. Gothié was supported by Roche and Multiple Sclerosis Society of Canada post-doctoral fellowships. We thank members of the Kennedy lab, Daryan Chitsaz, Teddy Fisher, Nonthué Uccelli, Kira Feighan, and Laura Neagu-Lund for assistance with cells cultured for these studies.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.