Journal: Nature Communications

Article Title: AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A

doi: 10.1038/s41467-021-22593-3

Figure Lengend Snippet: a Representative image of Western blot showing PMP22 and MPZ protein levels (upper panel) and total protein as loading control (lower panel) in rat sciatic nerve lysates from WT ctr.sh, CMT1A ctr.sh, CMT1A sh1 or CMT1A sh2 three months after injection. b Graph shows the average level of PMP22 / MPZ ratio in sciatic nerve lysates normalized to total protein loaded as loading control ( n = 7 animals per group). Statistical test shows one-way ANOVA followed by Tukey’s post hoc, two-sided. ** p = 0.0027 between WT ctr.sh and CMT1A ctr.sh, ** p = 0.0056 between CMT1A ctr.sh and CMT1A sh1, ** p = 0.008 between CMT1A ctr.sh and CMT1A sh2; ns, not significant; arb. units, arbitrary unit. All error bars show SEM. Source data are provided as a Source Data file.

Article Snippet: The efficiency of shA, shB, sh1 and sh2 to silence human PMP22 expression was done by transfecting HEK293 cells with pCMV3-hPMP22-Flag (HG14519-CF, Sinobiological) and pAAV vectors expressing shRNAs as described previously.

Techniques: Western Blot, Injection

Journal: Nature Communications

Article Title: AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A

doi: 10.1038/s41467-021-22593-3

Figure Lengend Snippet: a Representative images of Western blot showing MPZ protein levels (upper panel) and total protein as loading control (lower panel) in sciatic nerve lysates from WT ctr.sh, CMT1A ctr.sh, CMT1A sh1 or CMT1A sh2 three months after injection. b Graph shows mean MPZ level in sciatic nerve lysates normalized to total protein level loaded as loading control ( n = 7 animals per group). Statistical test shows one-way ANOVA followed by Tukey’s post hoc, two-sided. * p = 0.0122 between WT ctr.sh and CMT1A ctr.sh, ** p = 0.0028 between CMT1A ctr.sh and CMT1A sh1, ** p = 0.0022 between CMT1A ctr.sh and CMT1A sh2; ns, not significant; arb. units, arbitrary unit. All error bars show SEM. Source data are provided as a Source Data file.

Article Snippet: The efficiency of shA, shB, sh1 and sh2 to silence human PMP22 expression was done by transfecting HEK293 cells with pCMV3-hPMP22-Flag (HG14519-CF, Sinobiological) and pAAV vectors expressing shRNAs as described previously.

Techniques: Western Blot, Injection

Journal: Nature Communications

Article Title: AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A

doi: 10.1038/s41467-021-22593-3

Figure Lengend Snippet: a, b Representative images of CARS imaging on WT ctr.sh, CMT1A ctr.sh, CMT1A sh1 and CMT1A sh2 rat sciatic nerves twelve months post-injection ( n = 6 animals for WT ctr.sh, n = 8 animals for CMT1A ctr.sh, n = 5 animals for CMT1A sh1 and n = 5 animals for CMT1A sh2). a CMT1A ctr.sh nerve shows typical myelin sheath defects such as thin myelin sheath or demyelination (white arrows), focal hypermyelination (blue arrows) and myelin degeneration with myelin ovoids (stars). These defects are less abundant in CMT1A sh2 rat sciatic nerves. The insert represents a zoom of a node of Ranvier (arrowheads). Scale bars: 10 µm and 2 µm for the insert. b Nodes of Ranvier were labeled with arrowheads. Nodes of Ranvier are more abundant in CMT1A ctr.sh sciatic nerves compared to WT ctr.sh nerves indicating shorter internodes. CMT1A sh1 and CMT1A sh2 rat sciatic nerves showed less nodes than CMT1A ctr.sh nerves. Scale bars: 50 µm. c Graph showing the mean number of nodes of Ranvier on the total number of myelinated fibers per field ( n = 6 animals for WT ctr.sh, n = 8 animals for CMT1A ctr.sh, n = 5 animals for CMT1A sh1 and n = 5 animals for CMT1A sh2). Statistical test shows one-way ANOVA followed by Tukey’s post hoc, two-sided. **** p < 0.0001 between WT ctr.sh and CMT1A ctr.sh, * p = 0.0271 between CMT1A ctr.sh and CMT1A sh1, ** p = 0.0043 between CMT1A ctr.sh and CMT1A sh2. d Representative images of electron microscopy semi thin sections on WT ctr.sh, CMT1A ctr.sh, CMT1A sh1 and CMT1A sh2 rat sciatic nerves twelve months post-injection. CMT1A ctr.sh nerve shows typical myelinated fiber defects such as large demyelinated axons (orange stars), large hypomyelinated axons (green arrowheads) and small hypermyelinated axons (blue arrows). These defects are less abundant in CMT1A sh1 and CMT1A sh2 rat sciatic nerve. Scale bars: 10 µm. Graphs showing e the mean number of myelinated fibers per area unit (µm 2 ), f the mean percentage of axon caliber distribution per axon diameter and g g-ratio relative to axon diameter. ( n = 6 animals for WT ctr.sh, n = 8 animals for CMT1A ctr.sh, n = 5 animals for CMT1A and n = 6 animals for CMT1A sh2). Statistical tests show one-way ANOVA followed by Tukey’s post hoc, two-sided ( e ), **** p < 0.0001 between WT ctr.sh and CMT1A ctr.sh, ** p = 0.0082 between CMT1A ctr.sh and CMT1A sh1, ** p = 0.002 between CMT1A ctr.sh and CMT1A sh2) or two-way ANOVA followed by Dunnett’s post hoc, two-sided ( f ), * p < 0.05, *** p < 0.001, **** p < 0.0001 (all p- values of two-sided multiple comparison tests are available in the Source Data File); ns, not significant). All error bars represent SEM. Source data are provided as a Source Data file.

Article Snippet: The efficiency of shA, shB, sh1 and sh2 to silence human PMP22 expression was done by transfecting HEK293 cells with pCMV3-hPMP22-Flag (HG14519-CF, Sinobiological) and pAAV vectors expressing shRNAs as described previously.

Techniques: Imaging, Injection, Labeling, Electron Microscopy

Journal: Nature Communications

Article Title: AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A

doi: 10.1038/s41467-021-22593-3

Figure Lengend Snippet: Graphs showing a NCV (meter/second), b Rotarod test (second), c grip test (Newton) one to twelve months after injection and d Randall Selitto test (gram) six and twelve months after injection in WT ctr.sh, CMT1A ctr.sh, CMT1A sh1 and CMT1A sh2 animals ( n = 7 animals per group). Statistical analysis shows two-way ANOVA followed by Tukey’s post hoc, two-sided, ( a , b and c ) comparing all groups paired two by two (** p < 0.01, *** p < 0.001, **** p < 0.0001, all p -values of two-sided multiple com p arison tests are available in Table ) or one-way ANOVA followed by Tukey’s post hoc, two-sided, for six and twelve months post-injection in d . At six months post-injection, **** p < 0.0001 between WT ctr.sh and CMT1A ctr.sh, between CMT1A ctr.sh and CMT1A sh1, between CMT1A ctr.sh and CMT1A sh2; at twelve months post-injection, *** p = 0.0008 between WT ctr.sh and CMT1A ctr.sh, ** p = 0.0033 between CMT1A ctr.sh and CMT1A sh1, ** p = 0.0091 between CMT1A ctr.sh and CMT1A sh2; ns, not significant. Results are expressed as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: The efficiency of shA, shB, sh1 and sh2 to silence human PMP22 expression was done by transfecting HEK293 cells with pCMV3-hPMP22-Flag (HG14519-CF, Sinobiological) and pAAV vectors expressing shRNAs as described previously.

Techniques: Injection, Randall–Selitto Test

Journal: Nature Communications

Article Title: AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A

doi: 10.1038/s41467-021-22593-3

Figure Lengend Snippet: a Principal component analyses (PCA) of all nine transcriptional biomarkers in forepaw skin biopsies on twelve-month-old animals. Note little to no overlap between WT ctr.sh (gray, n = 7) and CMT1A ctr.sh (red, n = 8), whereas the treated groups, CMT1A sh1 (blue, n = 7) and CMT1A sh2 (green, n = 7), show more overlap with the WT ctr.sh group than with the CMT1A ctr.sh group. The mean of each group is given as a center point including the confidence interval (95%) given as an ellipse. b Correlation matrix from all animals (total n = 28 with n = 7 per group) including the expression levels of the skin biomarkers (green labels) and the four functional phenotypic analyses (purple labels): GS, grip strength; NCV, nerve conduction velocity; ROD, Rotarod; RST, Randall-Selitto test). Shown is data from a two-sided Pearson’s correlation analyses with graphical representation of the correlation coefficients, from red (−1) to blue (+1) (indicated by circle size and color), and the respective p- values (asterisks indicate p < 0.05, all the exact p -values are available in the Source Data File). c Principal component analyses (PCA) of the three best biomarkers ( Nrg1-I , Gria1 , Cda ; see correlation matrix in b ) in forepaw skin biopsies on twelve-month-old animals (same analysis as in a ). Source data are provided as a Source Data file.

Article Snippet: The efficiency of shA, shB, sh1 and sh2 to silence human PMP22 expression was done by transfecting HEK293 cells with pCMV3-hPMP22-Flag (HG14519-CF, Sinobiological) and pAAV vectors expressing shRNAs as described previously.

Techniques: Expressing, Functional Assay, Randall–Selitto Test