Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-triplet repeat expansion in the mutant huntingtin gene (mHTT) (1). The pathology develops when more than 35 CAG repeats are carried in the HTT gene. A hallmark of HD is progressive neurodegeneration and the formation of intracellular mHTT inclusions (2). The striatum and corticostriatal pathway are predominantly affected (3-5). The events leading to neuronal pathology were believed to be entirely cell-autonomous. Emerging data shows that mHTT can propagate from one neuron to a functional, connected one, in vitro and in vivo (6-8). Healthy human neurons receiving mHTT have been shown to develop pathological neurodegenerative-like changes (6). Interestingly, mHTT inclusions have been found in neuronal tissues and peripheral ones as skeletal muscle (9-11). Moreover, HD skeletal muscle shows functional dysfunction already in presymptomatic HD carriers and undergoes severe wasting in HD patients (12-14). Taking this information together, we hypothesized that the constant crosstalk between neurons and muscles given by the neuromuscular endplate junctions creates a possible route for mHTT to escape the nervous system towards the periphery, where it could play a role in disease onset and / or progression. Therefore, we established a human-derived neuromuscular in vitro system to investigate whether mHTT trans-neuromuscular spreading can trigger muscle dysfunction in HD.
Using a novel human induced pluripotent stem cells (hiPSCs)-derived neuromuscular co-culture method, we collected evidence supporting the hypothesis of mutant HTT fragment Exon1 with 72Q (HTTEx1Q72) trans-neuromuscular spreading. Indeed, we established two in vitro HD models to study this new potential disease pathway. We showed that HTTEx1Q72 can propagate from neuron-to-myotubes via functional NMJs. We observed mHTT aggregation, the hallmark of HD, in the myotubes (puncta staining indicates the presence of HTT aggregates but information about the ultrastructure or composition of these structures are not given). We also observed that trans-neuromuscular spreading of HTTEx1Q72 causes structural and functional pathological alterations in myotubes at the level of the mitochondria, myotubes contraction and nuclear aggregate accumulation. Moreover, our data suggest the active involvement of the NMJs in this process. Indeed, upon increased neuronal depolarization, we observed an increase in the number of puncta in the myotubes. This data suggests that synaptic activity is involved in the trans-neuromuscular spreading of mHTT in vitro. Finally, we observed that trans-neuromuscular spreading of mHTT is also occurring in vivo, from the M1 motor cortex to spinal MNs and skeletal muscles. This study is the first one where it was shown that HTTEx1Q72 propagate from neurons-to-muscle in an in vitro human-related context and mice and showing that trans-neuromuscular spreading can cause pathological-like alterations in the myotubes. This study suggests that mHTT trans-neuromuscular spreading could participate in HD-related muscular pathology in HD patients as well and therefore be considered as a novel pathological pathway.