title: Neuromuscular junction degeneration in a human stem cell derived assembloid model of Amyotrophic Lateral Sclerosis
Creators: name: Salzinger, Andrea
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Neuromuscular Junction (NMJ)
subject: Amyotrophic Lateral Sclerosis (ALS)
subject: TDP43 Loss of Function (LoF)
subject: Human Stem Cell-Derived Model
subject: Axonal Transport

description: The neuromuscular junction (NMJ) is the synaptic connection between motor neuron (MN) endplate and skeletal muscle, transmitting neuronal signalling into muscle contraction. Several neuromuscular diseases, as well as healthy aging, are affected by NMJ degeneration and impaired synaptic transmission. To date, NMJ studies in health and disease have predominantly been carried out using in vivo animal models, however, inter-species differences within the central nervous system (CNS), and in specific the NMJ, are increasingly well recognised. This necessitates the use of human experimental models, which is now possible owing to advances in human pluripotent stem cell (hPSC) disease modelling. Therefore, I first compared two different in vitro hPSC NMJ models. (i) The neuromuscular organoid was generated from human stem cell derived neuromesodermal progenitors - a bi-potent precursor cell type facilitating the formation of both mesoderm and neuroectoderm. (ii) The neuromuscular assembloid was generated by fusion of a human stem cell derived spinal cord organoid with a human primary skeletal muscle spheroid. Both models were characterised by the presence of ISL1/2 positive motor neurons innervating fast myosin heavy chain positive skeletal muscle fibres and formation of functional neuromuscular junctions, as demonstrated by spontaneous muscle contractions. I then discuss advantages and disadvantages of each model and how they facilitate the study of human NMJs and its degeneration in a dish. I concluded to use the neuromuscular assembloid model for further experiments due to its versatility and reproducible NMJ generation. NMJ degeneration is one of the earliest known pathologies of Amyotrophic lateral sclerosis (ALS), a devastating and fatal neurodegenerative disease, characterised by motor neuron loss and subsequent muscle weakness. The mechanisms leading to NMJ degeneration and its implications on motor neuron loss, however, remain unclear. Cytoplasmic mislocalisation and aggregation of the nuclear RNA/DNA binding protein TAR DNA binding protein 43 (TDP43) is a key pathological hallmark observed in 97% of ALS patients, in both familiar and sporadic cases, with proposed toxic gain of function (GoF) and nuclear loss of function (LoF) pathomechanisms. Physiologically, TDP43 has a pleiotropic function including splicing regulation and its nuclear depletion leads to severe perturbations within gene and protein expression of downstream targets. Here, I described that TDP43 LoF within spinal cord organoids alone can trigger NMJ degeneration in a human neuromuscular assembloid model. To further understand the underlying mechanisms of NMJ degeneration, spatial transcriptomic studies were performed locally at the NMJ in neuromuscular assembloids and revealed dysregulation of mRNAs associated with key cellular pathways, such as histone acetylation, synaptic transmission, and axonal plasticity. Since NMJs are located at the distal end of the motor neuron axons, which are in some instances greater than 1m long, maintaining mitochondrial homeostasis as the local energy provider is crucial. Furthermore, mitochondrial dysfunction and impaired axonal transport have been linked to ALS. Therefore, I hypothesized that augmenting axonal transport of mitochondria by genetically ablating syntaphilin, a mitochondrial anchor protein localised within axons, could be beneficial for NMJ maintenance in ALS. Enhancing axonal transport by syntaphilin depletion significantly increased NMJ innervation in TDP43 LoF neuromuscular assembloids, which were previously characterised by temporal NMJ degeneration. This demonstrates that enhanced axonal mitochondrial transport might serve as a protective mechanism in NMJ maintenance. G4C2 repeat expansion mutation within the C9ORF72 gene is the most common genetic cause of ALS, with proposed gain of function (GoF) and loss of function (LoF) mechanisms. c9orf72 LoF in mice did not lead to spontaneous neurodegeneration, but this has not been studied in detail in other vertebrate models. Therefore, I characterised the effect of global c9orf72 loss of function in vivo on CNS cell populations in an adult zebrafish model. My results showed no alterations upon c9orf72 LoF in HB9+ MNs, GFAP+ astrocytes or 4C4+ microglia. Assessment of skeletal muscle revealed no change in NMJ innervation. These results demonstrate that c9orf72 loss alone might not trigger motor neuron death or associated pathologies in a zebrafish model. In summary, in this thesis, I (i) established and characterised a human stem cell derived in vitro model of the neuromuscular junction, (ii) showed that TDP43 loss of function leads to NMJ degeneration, (iii) presented that augmenting axonal transport of mitochondria was positively affecting NMJ maintenance and (iv) demonstrated that c9orf72 loss of function in an aged zebrafish model does not lead to abnormalities in spinal cord and NMJs. This work showcases the strength of a human in vitro NMJ model and, importantly, demonstrates that neuronal TDP43 loss of function alone can lead to NMJ degeneration by dysregulation of essential neuron-muscle crosstalk.