The perijunctional zone is a molecularly distinct muscle subdomain altered in Duchenne muscular dystrophy

Figures

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  Fig. 1. Defining the perijunctional zone. a Representative immunofluorescence labeling of known PJZ proteins at the NMJ using antibodies against AnkG, AnkR, Nav1.4, β1 spectrin (β1 Sp), and Dystrophin (Dys) (green) from at least 3 independent experiments. AChRs are labeled using α-Bungarotoxin (red). The black and white lines in the images were used for the fluorescence intensity line scans in b. Scale bar, 20 µm. b Fluorescence intensity line scans corresponding to the lines shown in (a). Intensity measurements were normalized to the maximum value in each channel. c Normalized peak-to-peak distance (normalized width) for AChR fluorescence intensity and PJZ proteins. Unpaired two-tailed t-test. DF = 4 for each comparison. N = 3 mice each, with n = 45 NMJs measured for AnkR, β1 Sp, and Nav1.4, n = 63 NMJs measured for AnkG, and n = 103 NMJs measured for Dys. p-values are shown; AnkG, t = 3.745; AnkR, t = 9.214; Nav1.4, t = 9.979; β1 Spectrin, t = 6.009; Dys, t = 6.009. All experiments were performed in 2–3-month-old mice.

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  Fig. 2. NF-TurboID biotinylates PJZ proteins. a The NF-TurboID transgene, consisting of a fusion between rat Neurofascin-155 and the biotin ligase TurboID, is restricted to striated muscle tissue by the human skeletal actin (HSA) promoter. The transgene also includes a hemagglutinin (HA) tag. Created in BioRender. Rasband, M. (2026) https://BioRender.com/wts3puw. b NF-TurboID is restricted to the PJZ through binding to AnkG and AnkR. Following the injection of biotin, the TurboID ligase biotinylates proteins within ~10 nm of the ligase. Biotinylated proteins are isolated through streptavidin (strep) capture. c Mass spectrometry is used to identify and rank captured proteins. d Immunoblot for rat Neurofascin (rNfasc; top) and HA (bottom) using rat brain membrane (RBM) homogenate, WT STM (Ctrl), and NF-TurboID STM homogenates. Arrowheads indicate the location of rat Nfasc155. Experiments were repeated at least 3 times. e Representative immunolabeling of Ctrl and NF-TurboID STM using antibodies against rat Neurofascin (green) and AnkG (red). AChRs (red) were detected using α-BTX. Scale bar, 25 µm. Experiments were repeated at least 3 times. f Streptavidin blot of Ctrl STM and NF-TurboID STM…

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  Fig. 3. Identification of candidate PJZ proteins. a and b Comparison of the average normalized peptide spectral mass (PSM) count for proteins identified in WT and NF-TurboID mice after biotinylation. The diagonal dashed line is a best-fit estimate for the endogenously biotinylated carboxylases. The dashed horizontal line in (b) indicates an arbitrary cutoff for candidate protein inclusion (PSM = 18.62 corresponding to the average value for Nav1.4). a and b are the same data but shown with different scales. c Ranking of biotinylated proteins by Log2-fold change of NF-TurboID/WT. The horizontal dashed line indicates Log2-fold change = 2. d Volcano plot showing the Log2-fold change against the statistical significance (-Log10(p-value)) between proteins identified in WT and NF-TurboID samples. p-values were calculated using an unpaired two-tailed t-test, and the horizontal dashed line indicates a p-value = 0.05; the vertical dashed line indicates Log2-fold change = 2. N = 3 groups of 3 mice per genotype; STM muscles from 3 mice were pooled for each experiment. e Candidate PJZ proteins ranked by their average Log2-fold change between WT and NF-TurboID mice. The horizontal dashed line…

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  Fig. 4. Validation of candidate PJZ proteins. a and b Immunofluorescence labeling for the candidate PJZ proteins Plin4, Speg, Synm, Atp2a1, and Cmya5 (green) compared to AChR clustering (indicated by α-Bungarotoxin, a, red) or AnkG (b, red). Immunostaining was performed in a minimum of 3 mice with at least 10 NMJs observed for each mouse. The line in each image was used to generate the fluorescence intensity line scans to the right of the images. Intensity measurements are normalized to the maximum intensity value for each channel. Scale bars, 25 µm. All experiments were performed in 2–3-month-old mice.

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  Fig. 5. Proteomics of the dystrophic PJZ. a Streptavidin labeling (green) of Ctrl and NF-TurboID NMJs after biotin injection; AChRs (red) were labeled using α-Bungarotoxin. The lines in the NF-TurboID panels indicate where fluorescence intensity line scans were performed. Intensity measurements were normalized to the maximum value in each channel. Scale bar, 20 µm. Experiments were repeated at least three times. b Streptavidin blot of Ctrl STM and NF-TurboID STM homogenates following biotin injection. Experiments were repeated at least two times. c Volcano plot showing the Log2-fold change against the statistical significance (−Log10(p-value)) between proteins identified in mdx;NF-TurboID and NF-TurboID samples. p-values were calculated using an unpaired two-tailed t-test, and the horizontal dashed line indicates a p-value = 0.05; the vertical dashed line indicates Log2-fold change = 1.2. N = 3 mice per genotype. Red dots are representative known NMJ or PJZ proteins, blue dots are introduced NF-TurboID. Selected proteins are indicated by their gene name in black. DF = 4. d Log2-fold change of PJZ proteins comparing mdx;NF-TurboID and NF-TurboID mice. Red bars indicate known NMJ or PJZ…

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  Fig. 6. Plin4 KO mice have normal NMJs. a Immunofluorescence labeling for Plin4 (green) and AChR (red, α-Bungarotoxin) in WT, Plin4 het, and Plin4 KO mice. Experiments were repeated at least three times. Fluorescence intensity line scans corresponding to the lines in each image are shown to the right. The fluorescence intensity was normalized to the maximum value in each channel. Scale bar, 20 µm. b Analysis of NMJ morphology comparing WT and Plin4 KO mice. Error bars = ±SEM. Unpaired two-tailed t-test. All measurements, DF = 4. N = 3 mice per genotype with n = 69 NMJs for Plin4 KO and 57 NMJs for WT mice. No statistically significant difference was measured for any comparison: AChR area, t = 2.277, p = 0.085; endplate area, t = 1.794, p = 0.147; fragments, t = 0.01276, p = 0.990; compactness, t = 0.2223, p = 0.835; and endplate diameter, t = 2.091, p = 0.105. c Immunofluorescence labeling of PJZ proteins (AnkG, AnkR, β1 Sp, Nav1.4, and Dys), Plin4, and AchR (red, α-Bungarotoxin) in Plin4 KO muscle (note: there is no Plin4 co-labeling with the Dys since both are rabbit polyclonal antibodies). Scale bar, 20 µm. Fluorescence intensity line scans corresponding to the lines in each image are…

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  Fig. 7. Plin4 KO mice have normal NMJ and skeletal muscle function. a CMAP recordings of NMJ function from the FDB muscle of WT and Plin4 KO mice. Nerves were stimulated at 20 Hz, 40 Hz, and 60 Hz. Values plotted as percent of the first CMAP value. Error bars = ±SEM; unpaired two-tailed t-test at each stimulus number. Values were not significantly different at any stimulus or frequency. At 20 Hz, DF = 27, N = 15 WT and N = 14 Plin4 KO mice; 40 Hz, DF = 27, N = 15 WT and N = 14 Plin4 KO mice; 60 Hz, DF = 23, N = 13 WT and N = 12 Plin4 KO mice. b and c Latency to fall (b) or slip (c) on the Rotarod Unpaired two-tailed t-test; statistically significant p-values (p < 0.05) are shown above trial numbers. DF = 11. Error bars = ±SEM. N = 7 WT mice and 6 Plin4 KO mice. d Wire hang analysis for skeletal muscle strength. WT and Plin4 KO mice latency to fall from a wire mesh was measured over 10 trials. An unpaired t-test was used to compare genotypes at each trial. No statistically significant difference was observed. DF = 10 or 11. Error bars = ±SEM. N = 7 WT mice and 6 Plin4 KO mice. e An extended running wheel analysis of muscle fatigue. The distance traveled by WT and Plin4 KO animals was…

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  Fig. 8. Plin1 is expressed in muscle. a Immunoblot of Plin1 in muscle homogenates from WT, mdx, and Plin4 KO STM. Plin1 (top), α tubulin (bottom); the α tubulin blot is the same as that shown in Fig. 5c. N = 3 mice per genotype. b Quantification of Plin1 expression in STM normalized to α-tubulin. One-Way ANOVA, Tukey’s multiple comparisons. No significant differences were measured for any comparisons. WT vs mdx, q = 0.5963. WT vs Plin4 KO, q = 0.9810. mdx vs Plin4 KO, q = 0.3848. DF = 6. N = 3 mice per genotype. Data are presented as mean values +/− SEM. c Immunofluorescence labeling of Plin1 (green) at WT, mdx, and Plin4 NMJs; AChRs (red, α-Bungarotoxin). Scale bar, 20 µm. d Fluorescence labeling of lipid droplets using Oil Red O (ORO, green) and AChRs (α-Bungarotoxin, red) in WT, mdx, and Plin4 KO NMJs. Experiments were repeated at least 3 times. Scale bar, 20 µm. e Quantification of lipid droplet (LD) density in muscle and at the NMJ. One-Way ANOVA with Tukey’s multiple comparison test. All p-values > 0.05. Muscle, F = 1.890. NMJ, F = 0.2124. DF = 8. N = 3 mice per genotype, with n = 25 measurements per genotype and in muscle or at the NMJ. Data are presented as mean values +/− SEM.…