Autologous iPSC-Derived Human Neuromuscular Junction to Model the Pathophysiology of Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a heterogeneous group of genetic neurodegenerative disorders, characterized by progressive lower limb spasticity and weakness resulting from retrograde axonal degeneration of motor neurons (MNs). Here, we generated in vitro human neuromuscular junctions (NMJs) from five HSP patient-specific induced pluripotent stem cell (hiPSC) lines, by means of microfluidic strategy, to model disease-relevant neuropathologic processes. The strength of our NMJ model lies in the generation of lower MNs and myotubes from autologous hiPSC origin, maintaining the genetic background of the HSP patient donors in both cell types and in the cellular organization due to the microfluidic devices. Three patients characterized by a mutation in the <i>SPG3a</i> gene, encoding the ATLASTIN GTPase 1 protein, and two patients with a mutation in the <i>SPG4</i> gene, encoding the SPASTIN protein, were included in this study. Differentiation of the HSP-derived lines gave rise to lower MNs that could recapitulate pathological hallmarks, such as axonal swellings with accumulation of Acetyl-α-TUBULIN and reduction of SPASTIN levels. Furthermore, NMJs from HSP-derived lines were lower in number and in contact point complexity, denoting an impaired NMJ profile, also confirmed by some alterations in genes encoding for proteins associated with microtubules and responsible for axonal transport. Considering the complexity of HSP, these patient-derived neuronal and skeletal muscle cell co-cultures offer unique tools to study the pathologic mechanisms and explore novel treatment options for rescuing axonal defects and diverse cellular processes, including membrane trafficking, intracellular motility and protein degradation in HSP.