AAV9‐synapsin‐caveolin‐1 (AAV9‐SynCav1) gene delivery preserves motor neuron and neuromuscular junction morphology, motor function and body weight, and extends survival in hSOD1G93A mice
Authors: Wang Shanshan , Ichinomiya Taiga , Head Brian 
Published: May 2022

IntroductionAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from loss of upper and lower motor neurons in the brain and spinal cord. Due to the unknown etiology of sporadic ALS (SALS), interventions that are neuroprotective are urgently needed. Evidence shows that caveolin‐1 (Cav‐1), a membrane/lipid raft (MLR) scaffolding and neuroprotective protein, is decreased in degenerating neurons along with Cav‐1‐associated pro‐survival signaling proteins. We have previously shown that synapsin‐promoted Cav‐1 (SynCav1) augments neuroplasticity, preserves synaptic ultrastructure and hippocampal learning and memory in a mouse model of Alzheimer’s disease. The current study investigated whether spinal cord delivery of adeno‐associated virus serotype 9 (AAV9)‐SynCav1 could also be neuroprotective and extend survival in a mouse (hSOD1G93A) model of ALS.MethodsAAV9‐SynCav1 (10 ul; 1.9 x 1013 genomic copies (g.c.)/ml) subpial lumber spinal cord delivery was performed at 8 weeks (wk) of age. Weekly body weight (BW) measurements were recorded and survival was determined using Kaplan‐Meier curve. Running wheel (RW) tests were recorded at 8 (pre‐surgery baseline) and 16 wk (symptomatic age). Immunoblot (IB) and immunofluorescence microscopy (IF) assays for choline acetyltransferase (ChAT) and Cav‐1 were performed at 19 wk (before end‐stage age). To measure neuromuscular junctions (NMJs) in tibialis muscle, IF for bungarotoxin‐a (Btx, marker of post‐synaptic nicotinic acetylcholine receptors) and synaptoporin (Synphy, pre‐synaptic vesicle marker) was performed. Values were expressed as mean ± S.E.M while Kaplan‐Meier (Log‐rank (Mantel‐Cox) test) was expressed as median days (d) survival. Statistical significance was defined as p < 0.05.ResultsMale (Figure 1) and female (Figure 2) hSOD1G93A mice that received AAV9‐SynCav1 exhibited significantly longer survival compared to non‐treated hSOD1G93A mice (male: 176 d for SynCav1‐hSOD1G93A vs 158 d for non‐treated hSOD1G93A, p=0.004; female: 180 d for SynCav1‐hSOD1G93A vs 164 d for non‐treated hSOD1G93A, p<0.001). IF revealed that AAV9‐SynCav1 significantly increased motor neuron cell body area (Figure 3A, B), total ChAT‐positive motor neurons within the ventral horn (Figure 3C), increased Cav‐1 expression per motor neuron (Figure 3D), which resulted in a direct correlation between motor neuron area and Cav‐1 expression (R2 = 0.66) in hSOD1G93A mice at 19 wk. Further IF of anterior tibial muscle showed that preserved NMJ morphology (Btx) and NMJ occupancy of Synphy expression in AAV9‐SynCav1‐hSOD1G93A (Figure 4).ConclusionAAV9‐SynCav1 gene therapy significantly prolongs survival, preserves motor neurons and motor neuron morphology in the ventral horn of the spinal cord, and preservers NMJs in the tibialis muscle of hSOD1G93A mice. We are currently testing if alternative routes of AAV9‐SynCav1 delivery (e.g., intrathecal, intraventricular, systemic) can recapitulate the neuroprotective effects achieved using subpial delivery.