Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neurons (MNs) death in the spinal cord and brain, leading the loss of skeletal muscle mass (muscle atrophy). So far it has been difficult to investigate the molecular pathways of ALS because of the lack of suitable cell model system. The use of induced pluripotent stem cells (iPSC) carrying ALS mutations introduced by gene editing, represents a valuable opportunity for the study of this pathology. At the state-of-art, important improvements have been done concerning molecular processes which are involved in ALS pathology. Mutations in FUS protein have been reported in different ALS-patients. Despite its nuclear localization, FUS can shuttle between the nucleus and the cytoplasm under specific conditions. It has been reported that a hallmark of ALS disease is the abnormal cytoplasmatic accumulation of the mutated protein and NMJ degeneration has been observed as an early pathogenic event in ALS pathology. In addition, preliminary data collected in my lab highlight an aberrant increase in axon branching and growth in FUS-mutated MNs. The principal aim of my PhD project is to confirm whether the aberrant axon phenotypes observed might be involved in the neuromuscular disruption. To recapitulate the NMJ circuit formation in vitro I will take advantage from using iPSCs to obtain neural-muscle systems by 2D co-cultures and 3D organoids. The study of NMJ impairment will be conducted thanks to morphological and functional assays to verify the FUS mutation involvement in the ALS progression. Finally, the discovery of these cellular processes represents a crucial step in order to contribute to the development of more personalized future therapies.
In the last few years, iPSCs have led to important improvements for the study of neurodegenerative diseases. This innovative technology provides the opportunity to generate different cell types directly derived from patients, facilitating the development of new drugs and therapies, and overturning the concept of personalized medicine. iPSCs represent a multiple resource thanks to ability of self-renewal and differentiation in a very high number of adult cell types. Therefore, this pluripotency characteristic makes these cells suitable for increasing our knowledge about mechanisms of all those pathologies in which it is difficult to obtain neural cells from patients, including amyotrophic lateral sclerosis. Moreover, iPSCs, 2D and 3D models together provide a useful tool to generate innovative cell cultures to study NMJ degeneration observed in ALS. There is very little information about the mechanisms underlying NMJ degeneration in ALS disease and modelling these cellular interactions is a critical step to better understand the pathogenic events involved. Some recent findings revealed that traditional co-cultures of MNs and skeletal muscle cells form elementary NMJ that could be evidenced by co-localization of muscle acetylcholine receptors and presynaptic vesicles (Guo et al., 2011). Even though these models confirm the formation of synaptic structures, 2D microfluidic model system in which cells are compartmentalized, makes a greater contribution to simple co-cultures offering the possibility to better control the microenvironment of each cell component. In addition, the 2D technology provides an easier substrate for imaging assays and the high accessibility, due to the transparent components present in the devices, allows to observe hallmarks of synapse degeneration facilitating morphological studies. Moreover, this system can recapitulate the physiological spatial organization between MNs and muscle cells, preventing, at the same time, the total isolation of cell components. Furthermore, iPSCs can be differentiated in 3D organoids, in which cells can be organized to resemble the complexity of the brain's architecture to facilitate neurodegenerative diseases' study. One of the main innovations of this model is represented by the enormous potential to provide a large diversity of cell types allowing the study of cell interactions between different populations (Pasca 2018; Bordoni et al., 2018). It is crucial to consider the late onset of ALS related neurodegeneration and the consequent need to work on a system that reflects as much as possible an "aged brain". Indeed, this 3D systems shows the ability to model the earlier events of disease progression (Wang et al., 2018), but the main innovation and originality of 3D organoids is that they can be cultured for long period to recapitulate later stages of pathology, providing easier access for the analysis of cellular and molecular mechanisms underlying ALS (Martins et al., 2020). Finally, the major challenge in current organoids model is to resemble complex physiological systems. A promising approach that combines 3D organoids and microfluidic devices promotes an improvement of knowledge about intricate cellular crosstalk (Park et al., 2019). Indeed, thanks to the microfluidic control of morphogens, it is possible to emulate biochemical and mechanical environment of target organs. For this reason, it would be a novelty to develop a multiorgan model of ALS using microfluidic chip and 3D organoids to co-culture all neural-motor circuit to screen adverse effects of chemicals, drugs and environment.