Titolo della tesi: Generation and characterization of 3D iPSC-derived model system for the study of Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of both upper and lower motor neurons (MNs) in the brain, brainstem and spinal cord. The mechanisms leading to MN loss are not fully understood; however, there is much evidence about the axonal degeneration as an early event in the pathogenesis. Indeed, ALS is considered a dying-back motor neuropathy, as denervation of neuromuscular endplates precedes axonal degeneration and loss of motor neuron cell bodies. The link between the disruption of neuromuscular junction (NMJ) and ALS-associated genes such as FUS, whose heterozygous mutations cause the most aggressive forms of ALS, is not still clear.
To shed light on the correlation between FUS mutations and NMJ loss, we first generated 2D hiPSC-derived neural-muscle co-cultures. By using this model system, we obtained important information about the effects of mutant FUS protein on NMJ disruption.
We also analyzed the consequences of the overexpression of HuD RNA binding protein (RBP), since it is upregulated when FUS mutant protein is present, and it has been demonstrated that it is altered in sporadic ALS patients devoid of mutations in ALS-linked genes.
However, 2D neural-muscle co-cultures have some limits, such as the lack of three-dimensional structures and interactions normally present in vivo; further, it has been recently demonstrated that the maturation of skeletal muscle is more advanced in 3D system.
The development of hPSC-derived 3D neuromuscular organoids (NMOs) has represented a great step forward for the study of neuromuscular disorders. In this complex 3D model, all components of NMJs are generated from the same progenitor population, self-organize, and form functional NMJs.
We decided to take advantage of this new model system to study in deep the alterations of development and functionality of NMJs, and to analyze defects of structure and morphology of NMOs carrying FUS mutation. Hence, by using iPSC line carrying P525L mutation of FUS and an isogenic iPSC FUSWT line, we generated NMOs.
We characterized FUSP525L and FUSWT NMOs by real-time PCR and immunofluorescence analysis at different time points of development, we performed electrophysiological analysis using a multielectrode array system (MEA), and we studied the formation of NMJ.
The results obtained suggest that 3D NMOs can be useful to study pathological mechanisms in a more complex and physiological context, and they can give insight into the possible defects of development of neural and muscular tissues and NMJ.