Primary Schwann cell cultures were prepared from neonatal rat sciatic nerves according to Kim and Maurel (2009) with few alterations. Pregnant wild-type (WT) Sprague–Dawley rats were obtained from Janvier Labs (France). Sort1^–/– Sprague–Dawley rats were bred at Aarhus University (Gonçalves et al., 2020). In brief, sciatic nerves were dissected from P1-P3 Sprague–Dawley rat pups and placed in ice-cold Leibovitz’s L-15 medium (Gibco, Cat. #11415). Following dissection, the harvested nerves were digested with 0.25% trypsin (Gibco, Cat. #25200-072) and 0.1% collagenase I (Sigma, Cat. #C9722) for 30 min at 37°C and dissociated by trituration in DMEM (Sigma, Cat. #D0819) containing 10% FBS (Thermo Fisher, Cat. #10270) before being plated on poly-L lysine-coated culture dishes in DMEM supplemented with 10% FBS and Primocin™ (InvivoGen, Cat. #ant-pm-2). Contaminating fibroblasts were removed by 48 h incubation with the anti-metabolic agent Cytosine-ß-arabino furanoside hydrochloride (Ara-C; 10 μM final concentration, Sigma, Cat. #C1768), which kills mitotic cells. The cultures were washed twice with HBSS (Gibco, Cat. #14170-088) and the Schwann cells expanded in the growth medium [DMEM containing 10% FBS, recombinant human neuregulin-1-β1 EGF domain (10 ng/ml, R&D Systems, Cat. #396-HB-050) and forskolin (2.5 μM, Sigma, Cat. #F-6886)].

Sciatic nerve crush injury was performed in both 12-weeks-old WT mice (Janvier Labs, France) and Sort1^–/– mice (Jansen et al., 2007). Mixed groups with both males and females were used throughout the experiments.

The animals were anesthetized with isoflurane (IsoFlo vet, Abbot). Eye ointment was applied to protect the eyes from drying during the procedure and a subcutaneous injection of buprenorphine (0.3 mg/ml; Temgesic, RB Pharmaceuticals, Cat. #521634) and ampicillin (0.25 mg/ml; Ampicillin PCD, PharmaCoDane, Cat. #175689) was administered before surgery, to minimize post-surgery pain and risk of infection. The thigh and legs were shaved and disinfected before application of lidocaine (10 mg/ml; AstraZeneca, Cat. #PS02671) and exposure of the sciatic nerve at the mid-thigh level by separation of the biceps femoris and the gluteus superficialis. The left sciatic nerve was crushed with a non-serrated clamp for 30 s (Varejo et al., 2004; Ronchi et al., 2010). A sham surgery was performed on the contralateral side, where the sciatic nerve was exposed and the skin sutured immediately after. Care was taken to minimize damage to the musculature to guarantee ideal conditions for functional recovery. Post-surgery analgesic (0.006 mg/ml buprenorphine) was supplied in the drinking water for a minimum of 2 days. The mice were housed with littermates and cared for in a pathogen-free environment, in a 12 h light/dark cycle and with water and chow ad libitum. All experiments were approved by the Danish Animal Experiments Inspectorate under the Ministry of Justice (permission no. 2017-15-0201-01192) and carried out according to the European Council directive, and institutional and national guidelines.

Walking tract analysis (Mendes et al., 2015) was performed to access locomotor functional recovery before (0) and on days 3, 7, 9, 11, 18, 25, and 60 after injury (n = 6 mice per genotype). The mice were allowed to walk through a homemade catwalk consisting of a 50 cm × 5.5 cm corridor with a transparent Plexiglas floor to a box on the side of the catwalk containing bedding from their cages. Green light was sent horizontally through the Plexiglas floor and ambient red light was used to enhance contrast to the green light reflected down when the mouse placed a paw on the floor. The paw placement during walking was accurately determined from a video recorded with a camera below. A liner was placed on top of the Plexiglas floor. This way, the paw prints and gait could be analyzed in ImageJ by setting the scale accordingly to obtain print measurements.

The obtained footprints were then measured in ImageJ to calculate the sciatic functional index (SFI) using the empirical equation adapted for mice by Inserra et al. (1998): SFI = 118.9 × [(ETS-CTS)/CTS] − 51.2 × [(EPL-CPL)/CPL] − 7.5, where ETS represents operated experimental toe spread (distance between the first and fifth toes), CTS stands for control toe spread, EPL for operated experimental print length, and CPL for control print length. Footmarks made at the beginning of the trial were excluded and three analyzable walks were evaluated from each run for individual step parameter calculation. The pre-injured SFI values (time point = 0) were used as control for comparison. The SFI scores that we processed ranged from 0 to −130, with 0 representing normal or completely recovered nerve function and −100 or more, a non-functional nerve; thus, the mice who dragged their paws were arbitrarily assigned a value of −100.

The stride length and the step length (from the center of the right paws to the center of the left paws and vice versa) were measured in ImageJ from 3 consecutive steps. The step width and the angle of the paw to the mouse midline were additionally measured from the 3 consecutive steps. The mice who dragged their paws were arbitrarily assigned a value of 90°.

A grip strength test was performed after the walking tract analysis to evaluate the hind limb muscle strength as an indicator of motor regeneration and neuromuscular function before (0) and on days 3, 9, 11, 18, 25, 31, 38, 45, and 60 after injury. Combined hind limb grip strength was measured as tension force using the commercial grip strength meter from Bioseb (Chaville, France), which digitally displays the maximum force applied as the peak tension (in Newton) when the grasp is released. The mice were subjected to 5 consecutive trials and the maximum value was taken as an index of the hind limb strength.

Motor (sciatic) and sensory (sural) nerve conduction velocities (MNCV, SNCV, respectively) were performed 60 days after crush injury according to Oh et al. (2010) using a Viking Quest apparatus (Natus Neurology Incorporated, United States) (n = 6 mice per group). NCV was measured from the crush-injured side and from the contralateral side, which was subjected to sham surgery. Briefly, for the sural nerve, recording electrodes were placed in the dorsal part of the foot, with supramaximal stimulation at the ankle. Sural sensory NCV (m/s) was calculated by dividing the distance between the recording and stimulating electrodes (mm) by the onset latency (ms) of the sensory nerve action potential after supramaximal antidromic stimulation. Sciatic-tibial motor NCV was recorded by placing electrodes dorsally in the foot and orthodromically stimulating first at the ankle, then at the sciatic notch. The distance between the two sites of stimulation (mm) was then divided by the difference between the two onset latencies (ankle distance and notch distance, ms) to calculate the final sciatic-tibial motor NCV (m/s).

The operated mice (n = 6 per genotype) were euthanized 25 days after injury with intraperitoneal injections of pentobarbital (5 mg/ml). The mice were then transcardially perfused with ice-cold Tyrodes solution followed by fixation in ice-cold 5% glutaraldehyde in 300 mOsm phosphate buffer containing 0.1 M sucrose. The samples were postfixed in 1% osmium tetroxide for 1 h at 4°C and washed in the phosphate buffer. The samples were dehydrated in 50% ethanol and stained with 2% uranyl acetate in 50% ethanol. The samples were then dehydrated in a series of ascending concentrations of ethanol and 100% acetone. A three-step infiltration in a mixture of acetone-embedding medium (1:3, 1:1, 3:1) was performed before embedding (48 h at 60°C) in the Epoxy Embedding Medium Kit (Sigma-Aldrich, Sweden AB). The blocks were initially trimmed and sectioned using a Leica UC7 ultra microtome (Leica Microsystems GmbH, Vienna, Austria). Ultrathin sections (60 nm thickness) were collected onto formvar-coated copper slot grids, and counter-stained with uranyl acetate and lead citrate. Images were taken using a 100 kV transmission electron microscope (TEM, JEM 1230, JEOL Ltd., Tokyo, Japan). For each nerve, a panorama picture was created (x1,200) for orientation and quantification of myelinated fibers. For C-fiber quantification, 30 pictures were randomly taken (each third window; x15,000), and fibers were counted and normalized against the area to obtain fiber density.

Sciatic nerves dissected 5 days after injury were fixed overnight with 4% PFA and cryoprotected in 25% (w/v) sucrose in DPBS. The tissue was embedded in OCT compound mounting medium (VWR, Cat. #361603E) and snap-frozen on dry ice using isopentane before sectioning with a Leica CM1900 cryostat. About 10 μm longitudinal sections were thawed at RT for 10 min and antigen retrieval was performed with Target Retrieval Solution (Dako, Cat. #S1699) for 30 min at 80°C. The sections were rinsed once in TBS (50 mM tris base and 150 mM NaCl, pH 7.4) and incubated in TBS containing 5% donkey serum and 0.3% Triton-X-100 to block the unspecific binding of antibodies. The sections were incubated with primary antibodies (Table 1) diluted in TBS containing 5% donkey serum overnight in a humidified chamber at 4°C. The following day, the sections were incubated for 4 h with secondary antibodies (Table 1) diluted in TBS containing 5% donkey serum and Hoechst 33258 was used for nuclear stain. The slides were mounted with Dako fluorescence mounting medium (Dako, Cat. #S3023), sealed with nail polish, and stored at 4°C.

Images were acquired with a Zeiss Axio Imager 2 microscope (Carl Zeiss, Germany) equipped with a Hamamatsu digital camera (ORCA-flash4.0 digital camera, model C11440-22CU, Hamamatsu Photonics Deutschland GmbH, Germany) and a Plan-Apochromat 20x/0.8 lens or the LSM 780 confocal microscope (Carl Zeiss) equipped with a 63x water-immersion objective or a 20x/0.8 M27 objective (Carl Zeiss). Subsequent image analysis was performed with ImageJ.

The Schwann cells were fixed for 10 min in 4% formaldehyde and permeabilized with DPBS containing 0.1% Triton-X-100 (PanReac Applichem, Cat. #A4975) 3 times for 10 min. Unspecific binding of antibodies was blocked by subsequent incubation in DPBS containing 5% donkey serum and 1% BSA (Sigma, Cat. #A8806). The cells were then incubated with primary antibodies (Table 1) diluted in DPBS and containing 1% BSA overnight at 4°C. The samples were left for 1 h at RT before being washed 3 times for 10 min in DPBS and then incubated for 4 h with the secondary antibodies (Table 1), diluted in 1% BSA. Hoechst 33258 was used for nuclear staining (1:10.000, Sigma). The sections were then mounted with Dako Fluorescent mounting medium (Dako, Cat. #S3023) and sealed with nail polish.

Sort1^–/– and WT primary Schwann cells were lysed in ice-cold lysis buffer [10 mM Tris–HCl and 1 mM disodium EDTA, pH 8.0 (Sigma-Aldrich, Cat. #93283)] containing 1% IGEPAL (Sigma-Aldrich, Cat. #I8896), cOmplete Mini (Roche, Cat. # 4719956001), and PhosStop (Roche, 4906845001), incubated for 30 min on ice and frozen overnight at −20°C.

Naïve mice (day 0) or crush-injured mice were euthanized by decapitation after isoflurane gas-induced anesthesia at days 1, 7, or 25 post-injury (n = 3 animals per time point/group). The sciatic nerves were lysed in TNE buffer supplemented with protease (cOmplete Mini, Roche) and phosphatase inhibitors (PhosStop, Roche), after which a 10.000 g centrifugation for 10 min at 4°C was performed. The supernatant was stored at −20°C until use. A vacuum concentrator was used to increase protein concentration in the lysates, when necessary. Total protein concentration for both cell and tissue lysates was determined with the Bicinchoninic acid assay (Sigma, Cat. #BCA1-1KT). About 50 or 100 μg total protein was mixed with NuPAGE LDS sample buffer (Life Technologies, Cat. #NP0008) and a minimum of 10 mM dithioerythritol (DTE, AppliChem). The protein samples were separated on 4–12% Bis-Tris protein gels (Life technologies) in MOPS SDS running buffer (Thermo Fisher, Cat. #NP0001) containing NuPAGE Antioxidant (Invitrogen, Cat. #NP0005) and electroblotted with nitrocellulose iBlot Gel Transfer Stacks (Invitrogen, Cat. #IB301001) using the iBlot DryBlotting System (Invitrogen) according to manufactures guidelines. The membranes were subsequently blocked in TST buffer [50 mM Tris base, 500 mM NaCl, and 0.05% Tween-20 (PanReac Applichem, Cat. #A4974)] containing 2% skimmed milk powder and 2% Tween-20 and subsequently incubated with primary antibodies (Table 1), diluted in blocking buffer overnight at 4°C. The following day, the membranes were washed 3 times for 5 min in washing buffer (2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, and 140 mM NaCl) containing 0.2% skimmed milk powder and 0.05% Tween-20 and incubated for 1 h at RT with HRP conjugated secondary antibodies (Table 1). The membranes were then washed 3 times for 5 min and developed with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Cat. #RPN2236). Fuji film LAS4000 was used for visualization and densitometry.

The viability of Schwann cells was assessed by seeding Schwann cells on poly-L lysine-coated coverslips (250.000 cells/ml) in DMEM containing 10% FBS, at 37°C and 5% CO₂. The following day, the medium was replaced with or without NT-3 (50 ng/ml, Sigma, Cat. #SRP3128). The viability assay was performed 24 h after stimulation, according to the manufacture’s protocol (Live/Death Viability/Cytotoxicity Kit, Molecular Probes, Cat. #L3224). Calcein-positive (live) cells and ethidium homodimer-1 (dead) cells were counted and the percentage of live cells was calculated (n = 4 for each genotype).

The proliferation rate was assessed (n = 3 for each genotype) in parallel with the viability assay. The Schwann cells were fixed with 4% formaldehyde (Sigma, Cat. #158127) in Dulbecco’s phosphate-buffered saline (DPBS, Sigma, Cat. #D8537) 48 h after stimulation, and immunostained with an antibody against Ki-67. The Ki-67 positive cells and total cells were counted using ImageJ and the percentage of Ki-67 positive cells was calculated.

The scratch wound healing assay was used to examine the migration of Sort1^–/– Schwann cells (n = 3 independent experiments). Sort1^–/– or WT Schwann cells were seeded in an IncuCyte^® 96 well ImageLock plate (Satorius, Cat. #4379) at 31,000 cells/cm³ (10,000 cells/well) in Schwann cell growth media. The plate was incubated at RT for 20 min followed by incubation at 37°C. When the cells formed a confluent monolayer, the media was changed to starvation media (DMEM containing 2% B27 and 1% heat-inactivated FBS). The cells were starved for 18 h, after which a 700–800 μm scratch was induced using the 96-pin mechanical WoundMaker™ (Satorius, Cat. #4493). The media was replaced with either DMEM containing 2% B27, 1% FBS, 2.5 μM forskolin, and 10 ng/ml neuregulin-1 (negative control), DMEM containing 2% B27, 10% FBS, 2.5 μM forskolin, and 10 ng/ml neuregulin-1 (positive control), or DMEM containing 2% B27, 1% FBS, 2.5 μM forskolin, 10 ng/ml neuregulin-1, and 50 ng/ml of NT-3. The plates were then immediately placed in the IncuCyte Zoom Live-Cell Imaging System from Essen Bioscience and pictures were taken with the 10x objective every 3 h for 48 h. A cell mask was created and adjusted to fit the cell type after which the IncuCyte™ Scratch Wound Cell Migration Software Module (Essen BioScience) was used to calculate the Relative Wound Density (the Relative Wound Density is a measurement of the cell density in the wound area relative to the cell density in the non-wound area).

Dorsal root ganglia (DRG) were dissected from neonatal WT rats on postnatal days 1–3 and dissociated with 0.25% trypsin for 30 min at 37°C before being seeded onto poly-L lysine and laminin (Sigma, Cat. #L2020)-coated coverslips. The DRG neurons were purified and maintained in the presence of NGF (100 ng/ml, Promega, Cat. #G514A) for 2 weeks with cycles of maintenance medium (MEM) (Sigma, Cat. #M2279) containing 10% FBS, Primocin (0.1 mg/ml), 0.4% glucose, and GlutaMAX (2 mM, Fisher Scientific, 35050-061), and anti-mitotic medium [DMEM containing Primocin (0.1 mg/ml), pyruvate (1 mM), rat transferrin (10 mg/L, Sigma, Cat. #SRP6513), progesterone (20 nM, Sigma, Cat. #P8783), putrescine (100 μM, Sigma, Cat. #P5780), insulin (5 mg/L, Sigma, Cat. #I6634) supplemented with FdU (10 μM, Sigma, Cat. #F0503), and uridine (10 μM, Sigma, Cat. #U3750)]. Purified Sort1^–/– or WT Schwann cells were seeded onto the DRG neurons (100.000 cells/well) and allowed to proliferate for 1 week in a maintenance medium before the addition of ascorbic acid (50 μg/ml, Sigma, Cat. #A5960) to induce myelin formation, in the presence or absence of 50 ng/ml NT-3. The medium was changed every 2–3 days. The Schwann cell-DRG neuron co-cultures were fixed for 10 min with 4% formaldehyde in DPBS, 11 days after myelin induction. The cells were permeabilized and unspecific binding of antibodies was blocked with 0.3% Triton-X-100 and 10% FBS in DPBS for 1 h. Subsequently, the cells were incubated with mouse anti-Myelin Basic Protein (1:50, Sigma, Cat. #MAB381) and rabbit anti-neurofilament M (1:200, Millipore, Cat. #AB1987) overnight in a humified box at 4°C. The following day, the Schwann cells were washed 3 times for 10 min in DPBS before 4 h incubation at RT with Alexa Fluor 488 donkey anti-mouse IgG (1:300, Abcam, Cat. #AB150105) and Alexa Fluor 568 donkey anti-rabbit IgG (1:300, Abcam, Cat. #AB175470), diluted in blocking buffer, with Hoechst 33258 (1:10.000, Sigma) as a nuclear stain. The cells were washed and mounted with Fluorescent mounting medium (Dako), sealed with nail polish, and stored at 4°C until image acquisition with the LSM 780 laser scanning confocal microscope (Carl Zeiss) equipped with a Plan-Apochromat 20x/0.8 lens objective (Carl Zeiss). The myelin segments were counted in ImageJ (n = 3 for each genotype, 6 images were acquired per coverslip for each experiment, and myelin segments per image averaged).

Statistical comparison of data was accomplished using one-way or two-way ANOVA or multiple analysis and Bonferroni’s or Bonferroni Holm’s multiple comparisons post hoc test, with Graph Pad Prism software. Quantitative data are reported as mean ± SEM. Statistical significance was established for *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The sciatic nerve crush model was chosen to investigate the role of sortilin on nerve regeneration. The crush injury model with the intact basal lamina, as opposed to a transection injury where the basal lamina is disrupted, provides a system to test if sortilin enhances or inhibits peripheral nerve regeneration without variables imposed by transection injuries [reviewed in Griffin et al. (2010)]. We have previously described the impact of sortilin on neuropathic pain (Richner et al., 2019), and herein, we focus on functional and morphometric analysis of the injured nerve. The overall functional recovery was evaluated over 60 days by the sciatic functional index (SFI), which is a metric tool to evaluate sciatic nerve function (Inserra et al., 1998). We found that at day 7 post-injury, there was a maximum reduction for WT mice, with a steady recovery over the next few weeks until reaching baseline level from day 25. Although Sort1^–/– mice displayed a similar overall pattern, the SFI values were significantly reduced on days 25 and 60, relative to both WT and pre-injury baseline (Figure 1A). Next, the recovery was further examined by measuring the dynamic gait parameters (Mendes et al., 2015). Parameters such as stride and step length did not reveal any major altered patterns or endpoints between the genotypes (Figures 1B–D). However, the step width was significantly higher at the endpoint in WT as compared to the Sort1^–/– mice (Figure 1E). The paw angle is the angle between the paw axis and the midline of the mouse body during movement and it can be affected by abnormalities of the muscles controlling the paw or the innervating nerves. We correspondingly observed increased paw angle 3 days after injury, which corroborates loss of motor and sensory nerve muscle innervation, but with no significant differences detected between the two genotypes (Figure 1F). Muscle strength was evaluated by the grip strength test at pre-injury baseline (day 0) and at different time intervals up to 60 days after nerve crush, with a recovery pattern for Sort1^–/– mice indistinguishable from that of WT mice (Figure 1G).

Due to the reduced SFI recovery in Sort1^–/– mice (Figure 1A), we finally assessed the nerve electrophysiological properties by measuring the motor and sensory nerve conduction velocities (MNCV and SNCV, respectively) at the terminal endpoint day 60. As expected, the injured WT mice displayed a significant reduction in both SNCV and MNCV as compared with pre-injury (Figure 1H). The corresponding parameters for Sort1^–/– mice were identical to those of WT mice at both naïve or injury conditions (Figure 1H).

Histological examination using electron microscopy analysis of the sciatic nerve transverse sections demonstrated overall normal histology of the sciatic nerve in naïve Sort1^–/– mice (Figure 2). The endoneurium was occupied by myelinated fibers with different caliber sizes (Figure 2A) and unmyelinated axons (C-fibers) that appeared in large groups entailed by Schwann cell cytoplasm, forming Remak bundles (Figure 2B). No difference in myelinated or C-fiber densities was observed between Sort1^–/– and WT sciatic nerve in naïve conditions (Figures 2A,B), per previous studies (Jansen et al., 2007; Vaegter et al., 2011). As expected, pathology was prominent in the injured tissue of WT mice 25 days after injury. Accordingly, axonal degeneration of myelinated axons and removal or degradation of myelin by Schwann cells was evident. These parameters were indistinguishable between the genotypes (Figures 2A,B), as were the levels of total fat droplets (Figure 2C), indicating that the Sort1^–/– status did not affect lipid degeneration or reutilization after nerve crush. The densities of myelinated axons (Figure 2A) and C-fibers (Figure 2B), 25 days after injury were also identical between Sort1^–/– and WT mice.

For both genotypes, we observed that C-fibers appeared in lower average numbers in the Remak bundles on the injured side as compared to the contralateral sham sciatic nerve (Figure 2B, bottom chart), with extensive Schwann cell cytoplasmic branches spreading throughout the endoneurium. Nonetheless, a prominent feature of the Sort1^–/– mice entailed a more than twofold increase in the number of C-fibers only partially enclosed by Schwann cell cytoplasm with areas where the C-fiber was naked and not engulfed by the basement membrane (Figures 3A,B), demonstrating a potentially important role for sortilin in the Remak bundle formation following injury.

The sortilin expression was confirmed by immunohistochemistry in Schwann cells of naïve WT murine sciatic nerves co-localizing with the Schwann cell marker S100β (Figure 4A). Furthermore, the sciatic crush injury resulted in considerably increased sortilin immunoreactivity in Schwann cells at 5 days post-injury (Figure 4B). These in vivo observations prompted us to investigate the potential involvement of sortilin on Schwann cell neurotrophin signaling using primary cell cultures. Immunofluorescence (Figure 5A) and WB analysis (Figure 5B) confirmed sortilin expression in cultured WT Schwann cells with no detectable sortilin in Sort1^–/– Schwann cells. Quantification of viability and proliferation, as addressed by the live and death assay (Figure 5C) and the Ki67-positive cell percentages (Figure 5D), respectively, were indistinguishable between WT and Sort1^–/– Schwann cells, indicating that Sort1 expression is not essential for Schwann cell proliferation or viability.

We have previously demonstrated the involvement of sortilin in neurotrophin signaling in dorsal root ganglia neurons (Vaegter et al., 2011). To further examine such a role in Schwann cells, primary WT and Sort1^–/– cultures were stimulated with either BDNF or NT-3. Using WB analysis, we found that NT-3 stimulation significantly increased the pAKT, pMAPK/ERK, pRSK, and pCREB protein levels in WT Schwann cells (Figure 6A). In contrast, BDNF did not activate these pathways, confirming previous reports that Schwann cells do not express the full-length catalytically active form of the BDNF neurotrophin receptor TrkB (Funakoshi et al., 1993). Interestingly, relative to WT, NT-3 stimulation of Sort1^–/– Schwann cells significantly reduced the pMAPK/ERK response by 47 ± 10%, while the downstream mediators, pRSK and pCREB, were significantly reduced by 45 ± 15 and 35 ± 9%, respectively (Figure 6A). As NT-3 has been reported to facilitate Schwann cell migration (Yamauchi et al., 2003, 2005a), we compared WT and Sort1^–/– Schwann cell migration in the scratch wound healing assay but observed similar migration rates between the two groups of primary Schwann cells (Figure 6B). NT-3 is furthermore described to inhibit myelination (Chan et al., 2001) and we compared the performance of WT and Sort1^–/– Schwann cells in their ability to myelinate segments of WT dorsal root ganglia neurons in an in vitro co-culture system. While we obtained a similar degree of myelination for both genotypes in the absence of NT-3, we were also unable to confirm an inhibitory effect of NT-3 on myelination in either genotype but rather observed a (non-significant) trend toward a stimulatory effect (Figure 6C).

In conclusion, sortilin ablation in cultured primary Schwann cells reduced the activation of NT-3 stimulated intracellular signaling pathways. Surprisingly, we did not observe any NT-3-dependent cellular responses in either genotype on in vitro characteristics of proliferation, survival, migration, or myelination.