Chemogenetic Activation of Hypoglossal Motoneurons in Neuromuscular Disease

Abstract

The tongue is essential for breathing, swallowing, mastication, speech, coughing, and suckling. Many conditions and neuromuscular diseases are associated with lingual dysfunction, making it a priority to develop treatments which can improve tongue motor function. Our laboratory established that intralingual delivery of adeno‐associated virus serotype 9 (AAV9) encoding a particular gene can drive expression of that gene in hypoglossal motoneurons (XII MNs). This method can also drive expression of designer receptors exclusively activated by designer drugs (DREADDs) in XII MNs (Fleury‐Curado et al, 2021). Here we tested if intralingual delivery of AAV9‐hSyn‐hM3(Gq)‐mCherry could drive functional DREADD expression in XII MNs in a mouse model exhibiting tongue neuromuscular pathology. Specifically, we studied a mouse model of Pompe disease (Gaa‐/‐). The prevalence of Pompe disease is estimated to be 1 per 8,700 live births and is associated with macroglossia, dysphagia, dysarthria, sleep disordered breathing, and XII MN pathology. Intralingual injections of AAV9‐hSyn‐hM3(Gq)‐mCherry (2.4 x 1011 vg) or saline (sham) were administered to the base of tongue in anesthetized Pompe mice (AAV9‐DREADD n=11, saline n=4) or wild type (C57BL//6J mice (AAV9‐DREADD, n=5) at 4‐6 weeks of age. Lingual EMG activity was measured during spontaneous breathing under isoflurane anesthesia 12 weeks later. Tonic and phasic inspiratory EMG responses to intraperitoneal injection of saline (i.e., vehicle) or the DREADD ligand JHU37160‐dihydrochloride (J60) were assessed. Saline produced no significant changes in lingual EMG measures. All Pompe mice studied responded to J60 with an increase in tonic lingual EMG activity (time to response, TTR = 219±68 seconds; 1034±350% baseline at 30 min). Only 1 of 5 mice showed discernable increase in tonic EMG output after J60. The DREADD ligand produced an increase in phasic inspiratory bursting in all Pompe mice (TTR = 197±78 seconds; 506±248% baseline at 30 min). The increase in phasic bursting also occurred in all WT mice (TTR = 161±22 seconds; 512±151% baseline at 30 min). The DREADD ligand also caused a decrease in respiratory rate (bursts per minute) that was more pronounced in Pompe mice (79±5% baseline at 30 min post‐J60) compared to WT mice (91±12% baseline at 30 min post‐J60. Histological evaluation of the medulla confirmed expression of the mCherry fluorophore in XII MNs in all animals that received intralingual injection of AAV9‐hSyn‐hM3(Gq)‐mCherry. We conclude that intralingual delivery of an AAV9 vector encoding a DREADD can drive functional expression of DREADDs in XII MNs in a mouse model of Pompe disease that displays lingual myofiber atrophy and XII MN pathology. Thus, the ongoing neuromuscular pathology did not prevent the efficacy of chemogenetic tongue muscle activation. Indeed, the DREADD ligand caused a robust increase in both tonic and phasic lingual EMG activity in Pompe mice. The tonic EMG response appears to be more consistent in Pompe vs WT mice.