i.c.v. delivery of AAV9-synapsin-promoted caveolin-1 attenuates neuromuscular deficits and neuromuscular degeneration in hSOD1G93A mice

Figures

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  Figure 1. i.c.v. AAV9-SynCav1 delivery increases Cav-1 protein expression in the motor cortex and the spinal cord in hSOD1G93A mice (A) Schematic of experimental design and timeline (BioRender, https://BioRender.com/t168yx7). (B–D) Representative immunoblots on motor cortex (B), cervical (C), and lumbar cord (D), respectively. (E–G) Quantification of (B)–(D) for both sexes. Cav-1 was normalized to GAPDH. Each point represents one animal. Animal numbers for analysis: WT (male n = 6; female n = 5), hSOD1G93A + CTRL (male n = 6 [6 saline]; female n = 6 [5 saline, 1 AAV9-SynRFP]), and hSOD1G93A + SynCav1 (male n = 6; female n = 6). (H) Representative images of ventral horn MN transduction in hSOD1G93A control and hSOD1G93ASynCav1 visualized with ChAT (red), NeuN (blue), and Cav-1 (green) co-staining. Yellow arrows denote examples of Cav-1-negative MNs; white arrows denote examples of Cav-1-positive MNs. Scale bar: 100 μm. (I) Quantification of Cav-1+ MNs in cervical and lumbar ventral horn for both sexes. Data are mean (SD). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test (E–G) or unpaired t test (I). Significance was assumed when p < 0.05. ∗p <…

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  Figure 2. SynCav1-treated hSOD1G93A male mice show maintained RW motor performance at 18 weeks of age and a trend toward GS at 22 weeks of age (A and B) Left to right: average voluntary RW velocity (m/min) per 2 h over 36 h, total voluntary RW distance over 36 h, and GS test in male (A) and female (B) hSOD1G93A mice treated with control or SynCav1. Data are presented as mean (SD). For RW: hSOD1G93A + CTRL (male n = 15 [11 saline, 4 AAV9-SynRFP]; female n = 13 [10 saline, 3 AAV9-SynRFP]) and hSOD1G93A + SynCav1 (male n = 14; female n = 13). For GS: hSOD1G93A + CTRL (male n = 9 [7 saline, 2 AAV9-SynRFP]; female n = 11 [8 saline, 3 AAV9-SynRFP]) and hSOD1G93A + SynCav1 (male n = 9; female n = 10). Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test for RW velocity and unpaired t test for total wheel distance and GS. NS indicates no significance (p > 0.05), and significance was assumed when p < 0.05. ∗∗p < 0.01.

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  Figure 3. SynCav1 attenuates muscle electrophysiological abnormalities in late-stage symptomatic hSOD1G93A mice at 22 weeks of age (A) Annotated representative pictures of MEPs for WT, SOD1-CTRL, and SOD1-SynCav1. MEP onset is denoted by 1, peak amplitude is denoted by 2, and end of response is denoted by 3. (B) Quantification of MEP latency and amplitude in the triceps muscles. (C) Representative resting EMG traces from the gastrocnemius muscle. (D) Integration of resting EMG amplitudes. Data are presented as mean (SD). For MEPs: WT (male n = 5; female n = 6), hSOD1G93A + CTRL (male n = 8 [8 saline]; female n = 9 [6 saline, 3 AAV9-SynRFP]), and hSOD1G93A + SynCav1 (male n = 10; female n = 9). For EMG: WT (male n = 5; female n = 7), hSOD1G93A + CTRL (male n = 8 [8 saline]; female n = 8 [7 saline, 1 AAV9-SynRFP]), and hSOD1G93A + SynCav1 (male n = 8; female n = 9). Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s comparisons test for MEP latency and EMG amplitude, followed by Welch’s t test for MEP amplitude (male) and unpaired t test (female). Significance was assumed when ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005.

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  Figure 4. SynCav1 partially mitigates MN degeneration in cervical and lumbar ventral horns in late-stage symptomatic SOD1G93A mice at 22 weeks of age (A) Representative images of MNs in the ventral horn of the cervical spinal cord identified by ChAT (red), NeuN (blue), and Cav-1 (green) immunofluorescence. Scale bar: 100 μm. (B and C) Quantification of MN number and total ChAT+ area (μm2) in the cervical (B) and lumbar (C) regions for both sexes. (D and E) Quantification of Cav-1 intensity in MNs along with correlation analysis of Cav-1 fluorescence and MN area in cervical (D) and lumbar (E) regions for both sexes. Each point represents an MN for (D) and (E). Data are presented as mean (SD). Animal numbers for cervical histological analysis: WT (male n = 5; female n = 5), hSOD1G93A + CTRL (male n = 7 [7 saline]; female n = 6 [6 saline]), and hSOD1G93A + SynCav1 (male n = 8; female n = 7). For lumbar histological analysis: WT (male n = 6; female n = 6), hSOD1G93A + CTRL (male n = 7 [6 saline, 1 AAV9-SynRFP]; female n = 7 [5 saline, 2 AAV9-SynRFP]), and hSOD1G93A + SynCav1 (male n = 7; female n = 7). Statistical analysis was performed using one-way ANOVA with Fisher’s LSD multiple…

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  Figure 5. i.c.v. AAV9-SynCav1 delivery produces peripheral neuromuscular target engagement in diaphragm muscles. hSOD1G93A mice received i.c.v. AAV9-SynCav1 (WPRE), and diaphragm tissues were harvested for RT-qPCR and agarose gel validation (A) Representative agarose gels showing SynCav1 and synaptophysin transcripts in diaphragm tissue. Spinal cord tissue from validated SynCav1-treated mice was used as positive controls. (B) RT-qPCR quantification of SynCav1 expression normalized to PGK1. Animal numbers for analysis: hSOD1G93A + CTRL (male n = 5 [4 saline, 1 AAV9-SynRFP]; female n = 5 [4 saline, 1 AAV9-SynRFP]) and hSOD1G93A + SynCav1 (male n = 5; female n = 5). Data are presented as mean (SD). Statistical analyses were performed using Mann-Whitney tests. ∗∗p < 0.01.

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  Figure 6. SynCav1 preserves NMJ innervation in the diaphragm of hSOD1G93A mice at 22 weeks of age (A) Representative images of diaphragm NMJ with α-bungarotoxin (green) and synaptophysin (red). Scale bar: 20 μm. (B) Quantification of NMJ occupancy, defined as the percentage of the postsynaptic α-bungarotoxin area overlapping with presynaptic synaptophysin, was analyzed. Animal numbers for analysis: WT (male n = 5; female n = 5), hSOD1G93A + CTRL (male n = 5 [4 saline, 1 AAV9-SynRFP]; female n = 5 [3 saline, 2 AAV9-SynRFP]), and hSOD1G93A + SynCav1 (male n = 5; female n = 5). Data are presented as mean (SD). Statistical analysis was performed using one-way ANOVA with Fisher’s LSD multiple comparisons test. Significance was assumed when ∗p < 0.05 and ∗∗∗p < 0.005.