Individuals with Down syndrome (DS) contend with deficits in skeletal muscle and motor function, the underlying causes of which are unknown and much less well studied than those of the central nervous system. Hypotonia is a universal feature of DS at birth. Later in life, adults with DS experience premature aging of the neuromuscular junction (NMJ) with reduced skeletal muscle function (sarcopenia). The mechanisms mediating sarcopenia in the DS population are not known but have a major impact on well-being of individuals with DS across their lifespan. Furthermore, there is a growing appreciation for the critical connection between physical fitness and preservation of cognitive function during aging in the general population at large, thus, we posit that maintaining and improving the integrity of the NMJ in individuals with DS will benefit both their physical and neurological trajectories. The work proposed under this Administrative Supplement will provide insights into the molecular mechanisms that contribute to altered development and aging of the NMJ in DS and pave the way for new knowledge-based therapies designed to improve and preserve muscle function. The Parent award, R01-HD101544, is an active INCLUDE (Investigation of Co-occurring conditions across the Lifespan to Understand Down syndromE) project that is funded through the end of June 2024. The proposed studies are completely in line with the underlying goals of the parent award but add an important new objective focused specifically on the NMJ. Induced pluripotent stem cells (iPSCs) derived from individuals with DS and isogenic control iPSCs will be differentiated to motor neurons and cocultured with skeletal muscle myotubes (derived either from iPSCs or primary mouse myoblasts) to establish functional NMJs (following established protocols). The cytological and electrophysiological features of the iPSC-derived NMJ (hiNMJs) will be assessed to compare the developmental, functional, and degenerative trajectories of DS hiNMJs with controls. An important strength of the hiNMJ model is that it allows us to study a well-defined synapse that requires the contribution of two very different cell types (motor neurons and muscle fibers) thereby allowing us to separate the impact of trisomy on pre-synaptic processes from its impact on those in the post-synapse. These studies enhance the goals of the parent award by developing a tractable model of DS NMJs in which we can test our underlying hypothesis that elevated metabolic activity and ROS production, coupled with a decreased capacity to remove damaged mitochondria, increases cumulative, oxidative damage over time in individuals with DS.