Fig 1.
P0-S63del protein is degraded via ERAD.
(A) Immunofluorescence staining on inducible HEK293 cells expressing P0-wt, P0-S63C or P0-S63del variants after 17 hrs of induction with 100 ng/ml tetracycline. Anti-HA antibody recognizes P0 (green), whereas anti-Calnexin (CNX) antibody (red) stains the ER; cells nuclei are visualized with DAPI (blue). White arrowhead points at aggregate-like intracellular structures formed specifically by P0-S63C variant. Scale bar, 10μm. (B) EndoH/PNGaseF assays on lysates from induced HEK293 cells, followed by Western blot against the HA-tagged P0s. (C) Steady state expression of P0 variants in cells either mock-treated or induced for 17 and 48 hr with 100 ng/ml tetracycline. One of two independent blots is shown. Quantification relative to WT is shown below the panel. Samples normalized for cell number. (D) Pulse-chase experiments on HEK293 cells induced for 17 hr. Cells were pulsed with [35S]-methionine/cysteine for 10 min and chased for 10 min, 120 min, 180 min or 180 min with PS341. P0 proteins were immunoprecipitated with anti-HA antibody and separated in SDS-PAGE under reducing conditions. Arrowheads indicate two bands that specifically co-immunoprecipitate with the P0-S63del variant. (E) Quantification of (D). Symbols represent the values obtained in the different replicates, whereas columns indicate the mean. Samples normalized for cell number; n = 1–3 replicates per condition. (F) Co-immunoprecipitation of HA-tagged P0s followed by Western blot against either BiP, detected with the anti-KDEL antibody, or CNX; induced HEK293 cells were treated for 3 hr with/without the proteasome inhibitor PS341 before lysis. (TCE, total cell extract).
Fig 2.
Morphology of Der2SCKO and S63del//Der2SCKO sciatic nerves at P28.
(A) Transverse semithin sections from WT, Der2SCKO, S63del and S63del//Der2SCKO P28 sciatic nerves. Arrowheads indicate fibers with similar diameter for myelin thickness comparison. n = 5–6 mice/genotype. Scale bar, 10μm; 100x magnification. (B-C) Morphometric g-ratio (axon diameter/fiber diameter) analysis on P28 sciatic nerve sections. Scatter-plots of g-ratio distribution with trend lines are shown. ~1500–1700 fibers/genotype; n = 3 nerves per genotype. (D) Graph representing the frequency of myelinated axons (as percentage of all myelinated axons) per axon size interval (μm) at P28.
Fig 3.
Depletion of Derlin-2 leads to P0-S63del accumulation and ER-stress activation.
(A) Western blot analysis for Derlin-2 on HEK293 cells transfected with scramble (SCR) or Der2 siRNA. One representative blot of two is shown. (B) Pulse chase experiment on P0-S63del expressing HEK293 cells induced for 18 hr with 100 ng/ml tetracycline after Der2 silencing. Cells were pulsed with [35S]-methionine/cysteine for 10 min and chased for 10 min and 150 min. One representative gel of two is shown. (C) Quantification of (B). (D) Western blot for the ER stress marker HERP on HEK293 cells expressing the HA-tagged P0-S63del protein, after Derlin-2 silencing. One representative blot of two is shown.
Fig 4.
Measurement of ER stress/UPR levels in mutant sciatic nerves at P28.
(A-C) Western blot analysis for the ER stress/UPR markers BiP, GRP94 and P-eIF2α on P28 sciatic nerve lysates; β-Tubulin was used as loading control. One representative blot of four is shown. (D-F) Protein levels of BiP, GRP94 and P-eIF2α as measured by densitometric analysis. (G-I) qRT-PCR analysis on P28 sciatic nerve extracts for BiP, CHOP and spliced Xbp1; WT animals were used as the reference group. n = 4–5 RT from independent pools of three nerves per genotype. Error bars, SEM; *P < 0,05, **P < 0,01, ***P < 0,001 by unpaired, 2-tails, Student’s t test.
Fig 5.
Morphology of Der2SCKO and S63del//Der2SCKO adult sciatic nerves.
(A) Transverse semithin sections from WT, Der2SCKO, S63del and S63del//Der2SCKO sciatic nerves at 12 mo. Arrowheads indicate demyelinating/remyelinating fibers. n = 4–5 mice/genotype. Scale bar, 10μm; 100x magnification. (B) Morphometric g-ratio analysis performed on sciatic nerve semithin sections at 6 mo. Scatter-plot of g-ratio distribution with trend lines is reported; ~800–1200 fibers/genotype. n = 3–4 nerves per genotype. (C) Number of demyelinated fibers in 6 and 12 mo sciatic nerves (expressed as a percentage of total myelinated fibers per sciatic nerve field). 8–10 non-overlapping fields per nerve were analyzed from n = 3–5 nerves per genotype. Error bars, SEM; * and § P < 0,05, ** and §§ P < 0,01 by Student’s t test. (D) Transverse semithin sections of 6 mo Der2SCKO and S63del//Der2SCKO motor fascicles of sciatic nerves in which large onion bulbs and naked axons are visible (arrowheads). Scale bar, 10μm.
Fig 6.
Ultrastructural and neurophysiological analyses in adult Der2SCKO and S63del//Der2SCKO nerves.
(A) EM images from WT, Der2SCKO, S63del and S63del//Der2SCKO sciatic nerves at 6 mo. Black arrowheads show axons of similar caliber for myelin thickness comparison. Yellow arrowhead shows a Schwann cell with ongoing demyelination. Scale bar, 5μm (B) Mean g-ratio quantification (WT 0.63± 0.004; Der2SCKO 0.65±0.003; S63del 0.70±0.004; S63del//Der2SCKO 0.74±0.005); n = 50–70 fibers per nerve, from three nerves per genotype. Error bars, SEM; **P < 0,01, ***P < 0,001 by one-way ANOVA with Tukey’s post hoc test. (C-C’) Ultrastructural analysis of myelin in 6 mo WT, Der2SCKO, S63del and S63del//Der2SCKO shows proper myelin compaction with normal periodicity in Der2SCKO as compared to WT (C’). (D) Measurement of nerve conduction velocities (NCV; m/s), (E) F wave latencies (FWL; ms) and (F) compound muscle action potential (CMAP) amplitudes (mV) on 6 mo sciatic nerves. In (E), only detectable FWLs are reported in the graph; 30% of FWLs in Der2SCKO and 7% in S63del//Der2SCKO nerves, versus 0% in WT and S63del controls, were totally absent. n = 8–12 nerves per genotype. Error bars, SEM; *P < 0,05, **P < 0,01 by one-way ANOVA with Tukey’s post hoc test.
Fig 7.
Morphology of Der2SCKO and S63del//Der2SCKO adult quadriceps and saphenous nerves.
(A) Transverse semithin sections of saphenous and quadriceps nerves from WT, Der2SCKO, S63del and S63del//Der2SCKO at 12 mo. Red arrowheads indicate onion bulbs. n = 4–5 nerves/genotype. Scale bar, 10μm; 100x magnification. (B) Image of an onion bulb by EM analysis on 12 mo S63del//Der2SCKO quadriceps nerves. Scale bar, 2μm; ax, axon. (C) Number of onion bulbs in 12 mo quadriceps nerve (expressed as a percentage of total myelinated fibers in the whole nerve); n = 3–4 nerves/genotype. Error bars, SEM; *P < 0,05, **P < 0,01 by Student’s t test. (D-G) EM analysis on 12 mo quadriceps and saphenous nerves. In (D), a normal fiber from WT quadriceps nerve is shown as control. Panel (E) shows a demyelinating fiber from S63del//Der2SCKO quadriceps nerves in which the cell surrounding the axon is full of myelin debris (md). In (F) and (G), signs of axonal degeneration detected in S63del//Der2SCKO quadriceps and saphenous nerves, respectively. (D-E) scale bar, 2μm; (F-G), scale bar, 1μm.
Fig 8.
Measurement of ER stress/UPR levels in adult sciatic nerves.
(A-C) Western blot analysis for the ER stress/UPR markers BiP, GRP94 and P-eIF2α on 6 mo sciatic nerve lysates; β-Tubulin was used as loading control. One representative blot of three is shown. (D-E-F) Protein levels of BiP, GRP94 and P-eIF2α as measured by densitometric analysis. (G-H-I) qRT-PCR analysis for BiP, CHOP and spliced Xbp1 on 6 mo sciatic nerve extracts; the WT was used as the reference group. n = 5–6 independent RT per genotype. Error bars, SEM; *P<0,05, **P<0,01, ***P<0,001 by unpaired, 2-tails, Student’s t test.
Fig 9.
GlcNAc treatments ameliorate myelination in S63del DRG explants.
(A) Myelinating DRG explants were dissected from E13.5 WT and S63del littermate embryos. Treatment with 10 mM GlcNAc was performed for 2-weeks, in parallel to the induction of myelination with ascorbic acid. Myelinated internodes were detected with antibodies against myelin basic protein (MBP; red). Neurofilament (NF)-M (green) staining marks the axons; nuclei are visualized with Hoechst (blue). Scale bar, 100μm. (B-C) Graphs showing the number of MBP+ internodes/mm2 (B) and the internodal length (C), respectively. 5–12 DRG explants/condition/dissection; n = 4 independent dissections. Error bars, SEM. *P <0,05 by unpaired, 2 tails, Student’s t test. (D) Western blot analysis for P0 on lysates from DRG explants treated with 10 mM GlcNAc as in (A). One representative blot of three is shown. (E) Protein levels of P0 as measured by densitometric analysis. (F-G) qRT-PCR analysis for CHOP and Sel1L on extracts form DRG explants treated with 10 mM GlcNAc for 3 weeks. n = 3 independent embryos per genotype, 7–8 DRG/embryo/condition. Error bars, SEM; *P<0,05, **P<0,01, ***P<0,001 by unpaired, 2-tails, Student’s t test.