Neuron-targeted caveolin-1 improves neuromuscular function and extends survival in SOD1G93A mice

Abstract

Interventions that preserve motor neurons or restore functional motor neuroplasticity may extend longevity in amyotrophic lateral sclerosis (ALS). Delivery of neurotrophins may potentially revive degenerating motor neurons, yet this approach is dependent on the proper subcellular localization of neurotrophin receptor (NTR) to plasmalemmal signaling microdomains, termed membrane/lipid rafts (MLRs). We previously showed that overexpression of synapsin-driven caveolin-1 (Cav-1) (SynCav1) increases MLR localization of NTR [e.g., receptor tyrosine kinase B (TrkB)], promotes hippocampal synaptic and neuroplasticity, and significantly improves learning and memory in aged mice. The present study crossed a SynCav1 transgene-positive (SynCav1⁺) mouse with the mutant human superoxide dismutase glycine to alanine point mutation at amino acid 93 (hSOD1^G93A) mouse model of ALS. When compared with hSOD1^G93A, hSOD1^G93A/SynCav1⁺ mice exhibited greater body weight and longer survival as well as better motor function. Microscopic analyses of hSOD1^G93A/SynCav1⁺ spinal cords revealed preserved spinal cord α-motor neurons and preserved mitochondrial morphology. Moreover, hSOD1^G93A/SynCav1⁺ spinal cords contained more MLRs (cholera toxin subunit B positive) and MLR-associated TrkB and Cav-1 protein expression. These findings demonstrate that SynCav1 delays disease progression in a mouse model of ALS, potentially by preserving or restoring NTR expression and localization to MLRs.-Sawada, A., Wang, S., Jian, M., Leem, J., Wackerbarth, J., Egawa, J., Schilling, J. M., Platoshyn, O., Zemljic-Harpf, A., Roth, D. M., Patel, H. H., Patel, P. M., Marsala, M., Head, B. P. Neuron-targeted caveolin-1 improves neuromuscular function and extends survival in SOD1^G93A mice.

Keywords: MLR; TrkB; mitochondria; motor neurons; neuroplasticity.

Figure 1

SOD1^G93A/SynCav1⁺ mice exhibit preserved body weight and greater longevity. A) Body weight curves for Tg-neg, SynCav1⁺, SOD1^G93A, and SOD1^G93A/SynCav1⁺ (n = 20/group). Data are expressed as means ± sem. **P < 0.01, ***P < 0.001 vs. SOD1^G93A. Two-way ANOVA with Tukey’s multiple comparisons post hoc test with significance set at P < 0.05. B) Kaplan-Meier survival curve of male SOD1^G93A (n = 19) and SOD1^G93A/SynCav1⁺ (n = 17) mice. Mean survival (mean ± sem): SOD1^G93A/SynCav1⁺, 180.3 ± 4.5 d; SOD1^G93A, 155.5 ± 4.4 d; P = 0.0004, unpaired Student’s t test. Median survival: SOD1^G93A/SynCav1⁺, 182 d; SOD1^G93A, 153 d; P = 0.0006, unpaired Student’s t test (log-rank Mantel-Cox test).

Figure 2

SOD1^G93A/SynCav1⁺ mice exhibit greater MEPs in upper and lower limbs. To identify conductivity of descending motor axons, MEPs were elicited by electrical stimulation of motor cortex and extrapyramidal system and responses recorded in upper trapezius or lower gastrocnemius muscles. A) Amplitude of MEP from upper limb. B) Amplitude (mV) of MEP from lower limb (gastrocnemius muscle). C) Latency (ms) of MEP from lower limb. D) Resting EMG (mVs) from lower limb. Two-way ANOVA with Tukey’s multiple comparison post hoc test. All data are expressed as means ± sem. (n = 9–10/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. SOD1^G93A.

Figure 3

SOD1^G93A/SynCav1⁺ mice perform better on voluntary running wheel (RW) task. The 12-wk (A–C) and 16-wk (D–F) mean RW velocity (m/s) and total distance (m) for 36 h. Data are expressed as means ± sem. (n = 9–10/group). One-way ANOVA with Tukey’s multiple comparisons post hoc test with significance set at P < 0.05. **P < 0.01, ****P < 0.0001.

Figure 4

SOD1^G93A/SynCav1⁺ mice display more ChAT-positive α-motor neurons in spinal ventral horn column at 12 wk. A) Immunofluorescence confocal microscopy for ChAT-positive neurons in cervical (C3), thoracic (T1), and lumbar (L4) spinal cord at 12 wk. Scale bars, 50 μm. B–D) Quantitation of total number of α-motor neurons in C3 (B), T1 (C), and L4 (D) ventral horn segments from the spinal cord. Data are expressed as means ± sem. (n = 4/group). N.s., not significant. One-way ANOVA with Tukey’s multiple comparisons post hoc test with significance set at P < 0.05. **P < 0.01.

Figure 5

SOD1^G93A/SynCav1⁺ mice have increased MLR and MLR-localized TrkB in spinal cord tissue. A) Immunoblot of TrkB, Cav-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in spinal cord homogenates from Tg-neg, SOD1^G93A, and SOD1^G93A/SynCav1⁺ at 12 wk. B) quantitation of data in A. C) Spinal cord tissue subjected to sucrose density fractionation followed by immunoblot of TrkB, CT-B, and Cav-1. D) quantitation of data in C. All fractions (4–12) were generated from equal protein loading of 0.5 mg/ml. Red line indicates buoyant fractions 4 and 5. Data represent arbitrary units (A.U.) means ± sem. (n = 4/group). One-way ANOVA with Tukey’s multiple comparisons post hoc test with significance set at P < 0.05. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 6

SOD1^G93A/SynCav1⁺ mice exhibit preserved mitochondrial morphology L4 ventral horn motor neurons. A) Electron micrographs of motor neuron mitochondria cross sections of spinal cords from lumbar L4 ventral horn of wild-type (Tg-neg; first panel), hSOD1^G93A (second panel), SynCav1⁺ (third panel), and hSOD1^G93A/SynCav1⁺ (fourth panel) mice. Scale bars, 1 µm. B) Quantitation of average cristae area density, mitochondrial perimeter, and area. Data represent means ± sem. (n = 3/group). One-way ANOVA with Tukey’s multiple comparisons post hoc test with significance set at P < 0.05. ^#P < 0.05.