Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice

Abstract

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1^G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1^G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1^G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

Keywords: amyotrophic lateral sclerosis; caveolin-1; gene therapy; hSOD1G93A; membrane/lipid raft (MLRs); motor neuron; neuromuscular junction.

Figure 1

Subpial AAV9-SynRFP delivery to the lumbar spinal cord (L1) resulted in RFP transgene expression across entire lumbar cords. Spinal cords were collected 30 days post-AAV9-SynRFP. (A) Horizontal section of spinal cords showed strong RFP expression around lumbar cords. (B) Coronal sections of spinal cord were stained with motor neuron marker ChAT. Representative images showed strong RFP expression in the lumbar segment including ChAT positive lower motor neuron at the ventral horn area, while cervical and thoracic exhibit weak RFP signal predominantly in white matter.

Figure 2

AAV9-SynCav1 subpial delivery significantly delays disease onset, preserves BW and extends survival in hSOD1^G93A mice. (A-B) Body weight (BW), (C-D) Average disease onset timepoint (defined by peak BW) and (E-F) Kaplan-Meier survival curve for male and female mice. Groups: hSOD1^G93A naïve (no surgery), hSOD1^G93A + control and hSOD1G93A + SynCav1 mice. BW was expressed as mean ± SEM, Two-way ANOVA with Šídák's multiple comparisons test was used for A and B. One-way ANOVA with Dunnett's multiple comparison test was used for C and D (all compared to hSOD1^G93A + control group). Kaplan-Meier Curve was expressed as median days (d) survival and analyzed using a Mantel-Cox Log-rank test (***p < 0.005, ****p < 0.0001).

Figure 3

Decreased neuronal Cav-1 expression in the lumbar spinal cord MN from end-stage hSOD1^G93A mice. (A-B) Lumbar spinal cords from end-stage hSOD1^G93A mice (21-23 wk) were subjected to IB assays to measure Cav-1, ChAT (motor neuron marker), and mutant hSOD protein (B8H10). While Cav-1 expression was significantly decreased in the end-stage naïve hSOD1^G93A mice, subpial delivery of AAV9-SynCav1 resulted a 6-8 folds increase of Cav-1 protein expression, with no significant change in expression of hSOD1G93A. (C) Representative IB of lumbar spinal cord homogenates showed decreased MLR-localized Cav-1 (i.e., buoyant fractions 4-5) in naïve hSOD ^G93A mice compared to WT and hSOD ^G93A + SynCav1. (D) IF microscopy was performed to measure Cav-1 and NeuN-positive MN in lumbar sections of 10 wk (early symptomatic) and 23 wk (end-stage) hSOD ^G93A mice. (E) Higher magnification of ventral horn area revealed polarized Cav-1 expression on large NeuN-positive MN cell bodies at 10 wk (red arrowheads in left panels). At 23 wk, both Cav-1 expression on NeuN-positive MN and the size of NeuN-positive MN cell bodies were decreased in the ventral horn region (arrowheads in right panels) from hSOD ^G93A mice. Data are presented as mean ± SEM. Data were analyzed using One-way ANOVA with Fisher's LSD multiple comparisons post hoc test. (n = 3-6; *p < 0.05). Scale bar, 50 μm.

Figure 4

AAV9-SynCav1 treated hSOD1^G93A mice perform better on voluntary running wheel (RW) motor test. The 16 wk male and female total distance (km) (A, B) and mean RW velocity (m/s) (C, D) measured for 36 h. Data are expressed as means ± SEM. (n = 10-19/group). Student's t-test was used for A and B. 2-way ANOVA with Fisher's LSD multiple comparison test was used for C and D (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001).

Figure 5

SynCav1 prevented lumbar ventral horn motor neuron loss in 19 wk hSOD1^G93A mice. (A) Motor neuron (MN) was identified by the MN marker choline acetyl transferase (ChAT) in the lumbar ventral horn. Scale bar, 20 μm. (B-E) Number and average cell body area (μm²) of MNs per animal. A minimal of 3 images was used to calculate the average value. (F-G) Cav-1 expression was measured in individual ChAT+ somas and normalized to its area. Data are presented as mean ± SEM. Student's t-test (n = 3-8 mice/group; *p < 0.05, **p < 0.01, ****p < 0.001). (H-I) Simple linear regression with Pearson's r correlation analysis was performed comparing MN soma area with Cav-1 expression.

Figure 6

SynCav1 delivery preserved NMJ morphology in the gastrocnemius muscle. (A) Representative images of neuromuscular junction (NMJ) identified by bungarotoxin-alpha (Btx-α, post-synaptic) and synaptophysin (pre-synaptic vesicles). (B, C) NMJ occupancy is defined by pre-synaptic area overlaid with post-synaptic area (%). Student's t-test was performed for B and C (n = 3-5 mice/group; *p < 0.05, ****p < 0.0001).