Contact for reagents and resource sharing

Requests for further information, resources, and reagents should be directed to and will be fulfilled by Kelly Monk (monk@ohsu.edu).

Experimental model and subject details

All animal experiments were performed in compliance with institutional ethical regulations for animal testing and research at Washington University, Oregon Health and Science University (OHSU), and University of California San Francisco (UCSF). Experiments were approved by the Animal Care and Use Committee of Washington University School of Medicine (St. Louis, MO), the Institutional Animal Care and Use Committee of OHSU (Portland, OR), and the Institutional Animal Care and Use Program of UCSF (San Francisco, CA).

The Fbxw7 conditional-ready mice (Fbxw7fl/fl)11 were obtained from Jackson laboratories (Stock #: 017563) on a pure C57BL/6 background. Fbxw7fl/fl mice were mated to DhhCre(+) mice10 that had also been maintained on a pure C57BL/6 background (>7 generations) to generate DhhCre(+);Fbxw7fl/+ (Het) mice. Fbxw7 Hets were backcrossed to Fbxw7fl/fl animals to obtain DhhCre(+);Fbxw7fl/fl (cKO) mice. For all cKO experiments, we used DhhCre(−);Fbxw7fl/+ or DhhCre(−);Fbxw7fl/fl littermates as controls. For the double mutant experiments with Fbxw7 and mTOR, we obtained mTOR conditional-ready (mTORfl/fl) mice43 from Jackson laboratories (Stock #: 0110009), also on a pure on C57BL/6 background. The mTORfl/fl mice were crossed with DhhCre(+);Fbxw7fl/fl animals to generate DhhCre(+);Fbxw7fl/+;mTORfl/+ animals. Finally, to obtain double mutants, we crossed DhhCre(+);Fbxw7fl/+;mTORfl/+ to DhhCre(−);Fbxw7fl/+;mTORfl/+ animals and analyzed DhhCre(+);Fbxw7fl/+;mTORfl/fl animals (“HetΔmTOR” for brevity). In all cases, mice of both sexes were analyzed, in equal ratios whenever possible. In all cases mutants were compared with littermate sibling controls.

All mouse lines were genotyped as previously described10,11,43.

Transmission electron microscopy (TEM)

TEM was performed on mouse sciatic nerves at P3, P21, P42 and ≥6 months44. Nerves were immersion-fixed in modified Karnovsky’s fixative (4% PFA, 2% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.4) at least overnight at 4 °C. Samples were then washed with 0.1 M sodium cacodylate to remove fixative, and then post-fixed for 1 h in 2% osmium tetroxide in 0.1 M sodium cacodylate. Nerves were then dehydrated with increasing concentrations of ethanol followed by propylene oxide (PO). Samples were then infiltrated for 1–2 h in 2:1 PO:EPON, and then overnight in 1:1 PO:EPON with gentle agitation at room temperature. Samples were then transferred to 100% EPON while residual PO was allowed to fully evaporate (>4 h). For all time points, four non-overlapping images at ×1000 magnification were quantified, and all mouse TEM data is expressed per 1000 μm2 area. Control genotypes were DhhCre(−);Fbxw7fl/+ and DhhCre(−);Fbxw7fl/fl. For P3: N = 4 controls, N = 6 DhhCre(+);Fbxw7fl/+ (Hets), and N = 5 DhhCre(+);Fbxw7fl/fl (cKO). At P21: N = 4 controls, N = 4 Hets, and N = 4 cKO. For P42 samples: N = 4 controls, N = 3 Hets, and N = 4 cKO. At 6 months of age: N = 5 controls, N = 4 Hets, and N = 5 cKO. For the double mutant analyses, control genotypes used were DhhCre(−);Fbxw7fl/+;mTOR+/+ DhhCre(−);Fbxw7fl/+;mTORfl/fl, DhhCre(−);Fbxw7fl/+;mTORfl/+, DhhCre(−);Fbxw7fl/fl;mTORfl/fl, and DhhCre(−);Fbxw7+/+;mTORfl/fl. Fbxw7 “Hets” were DhhCre(+);Fbxw7fl/+;mTOR+/+ littermate siblings, and “HetΔmTOR” animals were DhhCre(+);Fbxw7fl/+;mTORfl/fl siblings. At P3: N = 4 controls, N = 6 Hets, N = 5 HetΔmTOR, and N = 1 DhhCre(+);Fbxw7fl/fl;mTORfl/fl (cKOΔmTOR). For P21: N = 6 controls, N = 4 Hets, and N = 3 HetΔmTOR.

Serial block-face scanning electron microscopy (SBF-SEM)

SBF-SEM was performed on a DhhCre(+);Fbxw7fl/fl nerve at P180 (N = 1; multiple regions). Nerves were fixed in 4% PFA overnight at 4 °C. Nerves were then processed for SBF-SEM by the Multiscale Microscopy Core at Oregon Health & Science University. Images were collected with 10 nm lateral resolution and 50 nm slice thickness using an FEI Teneo VolumeScope Microscope. Nerves were sectioned and imaged overnight to obtain a depth of ~50 μm. Eleven different regions of interest were imaged and analyzed across two technical experiments. Sections were annotated using FIJI, the movie was composed using Microscopy Image Browser software, and data compilation was performed using Amira. The movie shown is a representative example.

Behavioral studies

Behavior testing was performed using mice of both sexes from 5 to 6 months of age (N = 11 controls, N = 10 cKO). The experimenter was blind to the genotypes of the mice during all data acquisition. All behavior data was analyzed using a t-test with Welch’s correction to determine statistical significance.

Gross motor function (Rotarod)

An accelerating Rotarod (Ugo Basile, Comerio, Italy) as used to assess motor coordination45. Mice received two training sessions separated by 1 h. The first training session consisted of two trials of 120 s spent walking on the Rotarod at a fixed spped of 4 r.p.m. The second training session consisted of one trial of 120 s at 4 r.p.m. Latency to fall as the Rotarod accelerated from 4 to 40 r.p.m. over 5 min was assessed. Five consecutive experimental trials were performed with a 5 min rest interval between trials.

Locomotor activity (open field)

Prior to testing, mice were habituated to the test room in their cages for 1 h. Locomotor activity in an open field16 was then assessed by recording photo beam breaks in a 42 (length) × 42 (width) × 30 (height) cm chamber for 60 min using a VersaMax Animal Activity Monitoring System (AccuScan Instruments). We then calculated the total distance traveled and the horizontal activity (beam breaks) over the entire chamber.

Movement initiation

To assess movement initiation, we recorded the time it took each mouse to exit an 18 × 18 cm square (all four paws outside the square) marked on a flat horizontal surface.

Complex motor function (pole test)

We used the pole test to evaluate performance of a complex motor task that requires skilled forelimb use, strength, and balance15. Mice were placed on a vertical metal pole that is 49 cm in height and 0.9 cm in diameter with the head of the mouse oriented upward. The time required for the mouse to turn around such that the mouse’s head is oriented downward and the hind limbs are straddling the pole was recorded. In addition, the time required for the mouse to climb down to the base of the pole was recorded.

Cold sensitivity (acetone evaporation test)

Cold sensitivity of the hind paws was measured by applying a drop of acetone to the plantar surface of the hind paw. Five separate applications of acetone were applied to each hind paw. For each application the mouse was observed for 5 min. The percentage of applications for which the mouse responded (shaking, licking, or elevating the hind paw) to acetone application was recorded for each mouse17.

Gait analysis

We used the Noldus CatWalk XT system to quantify multiple locomotor and gait parameters including: run speed, stride length, paw print area (mm2), maximum contact area (mm2), and maximum contact mean intensity (arbitrary units [a.u.]). Briefly, the mouse voluntarily traverses a meter-long glass plate and its footprints are captured by a video camera. CatWalk XT quantifies parameters related to print dimensions and gait dynamics.

Mechanical sensitivity (von Frey)

The innocuous mechanical thresholds of both hind paws were assessed with the von Frey test. Mice were placed in plastic behavior boxes with open bottoms on a wire mesh. Varying diameter von Frey monofilaments (Stoelting, Chicago, IL) were pressed against the plantar surface of the hind paw until the filament bent. The force applied to the hind paw is dependent on the diameter of the filament. The up/down method described by Chaplan was used to determine the withdrawal threshold46.

Heat sensitivity (Hargreaves)

Heat sensitivity was evaluated by using a paw thermal stimulation system in which a source of radiant heat (active intensity = 15) was applied to the plantar surface of the hind paw and the paw withdrawal latency was measured47. We performed three trials on each paw. The withdrawal latencies obtained in each of the six trials were averaged to obtain the withdrawal latency for each mouse.

Myelinating Schwann cell cultures

Dissociated SC/dorsal root ganglia (DRG) sensory neuron co-cultures were prepared as previously described48,49,50. Briefly, DRGs were isolated from individual embryonic day 13.5 (E13.5) DhhCre(−);Fbxw7fl/fl (control) or DhhCre(+);Fbxw7fl/fl (cKO) mouse littermate embryos. DRGs were washed with L15 medium and then incubated in 0.25% trypsin at 37 °C for 30 min. Trypsin was removed, and DRGs were washed with L15 + 10% FBS and centrifuged gently at 1000 r.p.m. for 10 min. The medium was replaced with DRG medium (high glucose MEM with 10% FBS and 100 ng/mL NGF), and DRGs were triturated with a fire-polished Pasteur pipette until homogenous. The suspension was plated at 150,000 cells in the center of a collagen-coated 25 mm coverslip. Cultures were maintained in DRG medium for 5–6 days, after which 50 µg/mL ascorbic acid was added to the medium to induce myelination. Cultures were fixed in 4% PFA for 15 min after ten days in media containing ascorbic acid. Cultures were blocked and permeabilized with 20% normal goat serum (NGS) and 0.2% Triton X-100 in PBS for 1 h at room temperature, and then incubated overnight at 4 °C with the following antibodies in 20% NGS: mouse anti-neurofilament medium chain (1:200; Millipore), rat monoclonal anti-MBP (1:100; Millipore), and rabbit anti-Krox20 (1:500; provided by Dies Meijer). Cultures were then incubated with Alexa Fluor AffiniPure goat anti-mouse 488 (1:1000), AffiniPure goat anti-rat 594 (1:1000), and goat anti-rabbit 647 (1:500) for 1 h at room temperature, counterstained with DAPI, and dried before mounting in Prolong Gold Mountant (Invitrogen). Cultures were imaged as z-stacks using a ×40 oil 1.3NA objective on a Zeiss AxioImager with ApoTome. Entire cultures were imaged and analyzed by an experimenter blinded to genotype, and counts represent the number of multipolar MBP(+)/Krox20(+) SCs per culture. Data represent two technical replicate cultures from each of three independent mouse embryos per genotype.

Western blot analyses

To assess mTOR protein levels in the sciatic nerve, we dissected nerves from the sciatic notch to just proximal to the trifurcation. These nerve segments were flash-frozen in liquid nitrogen, cut into small pieces with microdissection scissors, and homogenized in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Na 2 EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na 3 VO 4 , 1 μg/ml leupeptin) with phosphatase inhibitor mixtures 1 and 2 (Invitrogen, ThermoFisher Scientific, Waltham, MA). Equal protein amounts (15 μg) were loaded and analyzed by SDS-PAGE and western blot. Antibodies used were anti-mTOR (1:1000; Cell Signaling Technologies, Danvers, MA) and anti-α-tubulin (1:1000; Abcam, Cambridge, MA). Western blot images were quantified using FIJI. All bands were normalized to background and mTOR bands were compared with α-tubulin levels.

RNA isolation and reverse transcription

Total RNA was extracted from flash-frozen P21 mouse sciatic nerves (N = 3 DhhCre(−);Fbxw7fl/fl littermate controls and N = 3 DhhCre(+);Fbxw7fl/fl animals), using a standard TRIzol extraction protocol (Life Technologies, ThermoFisher Scientific, Waltham, MA). Briefly, TRIzol was added to the frozen tissue samples, which were then allowed to thaw at room temperature for 10 min. During this incubation time, and while still in TRIzol, nerves were cut into much smaller pieces using microdissection scissors. Samples were homogenized via disruption with a plastic-tipped electric homogenizer, followed by passage through a syringe and 22.5 g needle at least ten times, and then a 27 g needle at least ten more times until no lumps of tissue were observed. Once the nerves had been homogenized, we proceeded as usual with the standard TRIzol RNA extraction procedure as per manufacturer instructions.

Total RNA (500 ng) was then reverse transcribed in 20 μl using the Superscript III First Strand cDNA Synthesis Kit (Invitrogen, ThermoFisher Scientific, Waltham, MA) using random hexamers, as per manufacturer instructions. All cDNA products were diluted 1:5 prior to use in qPCR reactions.

Quantitative reverse transcription PCR

To assay mRNA expression levels of mTOR and members of the mTOR signaling pathway, we used the RT2 Profiler PCR Array for Mouse mTOR Signaling (Qiagen, PAMM-098ZA, Valencia, CA). A complete gene list can be found on the manufacturer’s website. All assays were performed on a ViiA7 Real-Time PCR system (Applied Biosystems, ThermoFisher Scientific, Waltham, MA), in a total volume of 10 μl using 2X SsoFast Evagreen Supermix (BioRad, Hercules, CA) and 50 ng of cDNA per reaction. Standard qPCR settings were used: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s (sec) then 60 °C for 30 s, followed by melt curve analysis. As suggested by the RT2 profiler manual, we adjusted the ramp rate to 1 °C/s. All controls including housekeeping genes, positive controls for amplification, and controls for genomic DNA contamination were included as standards in the array.

qPCR data were analyzed using Microsoft Excel. Relative expression was calculated using the ΔΔCt method51. Genomic contamination was negligible in all samples. To control for input variations, ΔCt was calculated by comparing the Ct of each gene of interest (GOI) to the average Ct of the five housekeeping genes (Actb, B2m, Gapdh, Gusb, and Hsp90ab1) for that sample. ΔΔCt was then calculated relative to expression compared with that seen in the littermate control. Average relative expression (RQ), or fold change (2−ΔΔCt), over controls is shown in Fig. S3. All error bars depict RQmax and RQmin, which represent the maximum and minimum limits of possible RQ values based on the standard error of the ΔCt values. The gray line at y = 1 represents the controls.

RNA sequencing data mining

To analyze the expression of Fbxw7, mTOR, and other Fbxw7 targets, we mined previously reported RNA sequencing data24 from wild-type sciatic nerves at P3 and P21. We generated a list of candidates through a series of basic literature searches focusing primarily on targets of Fbxw7 that might have a role in SCs (c-Jun, Notch, Myc, Ccne1 (cyclinE), PGC1, SREBP1, and GATA1). This list is not intended to represent an exhaustive list of Fbxw7 targets. Using Excel, we searched the raw data for the gene names of Fbxw7, mTOR, and our candidate targets. We then recorded the raw FPKM value for each animal at each time point (N = 4 for P3 and N = 3 for P21) and transferred that information into Graphpad PRISM for analysis. We then averaged the FPKM values for each time point, calculated the standard deviation (S.D.; error bars shown), and generated the graph shown in Fig. 6a using PRISM 8 for MacOS.

Immunofluorescence

Sciatic nerves were isolated and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. After washing with PBS and 30% sucrose, nerves were embedded in OCT and frozen at −80 °C. Cryo-sections were acquired in cross-section orientation at 15 μm thickness. Slices were brought to room temperature and then incubated with blocking solution (2% bovine serum albumin, 2% normal goat serum, 0.2% Triton in 1× PBS). The following primary antibodies were diluted in blocking solution and incubated for 2 h at room temperature: Anti-S100 beta [EP1576Y] (Alexa Fluor 488—Abcam 1:200) and Anti-c-Jun (60A8, Cell Signaling 1:200). Samples were then washed three times (5 min each) in 1× PBS, and slides were incubated with secondary species-specific Invitrogen antibodies for 1 h or mounted using Vectashield with DAPI (Vector Labs) to label nuclei. Fluorescent images were obtained with a Zeiss AxioImager microscope. CZI files were analyzed using FIJI. All data were quantified blindly. N = 3 per genotype. c-Jun data are shown in Fig. 6.

Quantification and statistical analysis

All data are reported as mean + standard deviation (S.D.). Statistically significant differences were determined using one-way ANOVA for all experiments with more than two groups but only one dependent variable. Similarly, two-way ANOVA was used for experiments with multiple groups and two dependent variables. All experiments with only two groups and one dependent variable were compared using an unpaired t-test with Welch’s correction, which assumes unequal variance. Figure legends specify which test was used for specific experiments. In all cases, * = p < 0.05; ** = p < 0.01; *** = p < 0.001; and **** = p < 0.0001; NS = not significant. In all cases, asterisks immediately above a bar indicate the significance of that sample relative to the control sample. If any other comparisons, such as DhhCre(+);Fbxw7fl/+ (Het) to DhhCre(+);Fbxw7fl/fl (cKO), were significant, this is indicated with a bar spanning above the two samples being compared with the appropriate asterisks. If not indicated otherwise, the comparison was not significant. In most cases, DhhCre(+);Fbxw7fl/+ samples were not statistically distinguishable from DhhCre(+);Fbxw7fl/fl.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.