hiPSC-derived 3D assembloids to investigate the neurodegenerative propagation along the corticospinal tract in ALS

• Applicant: Dr. Alberto Catanese, Ph.D.
• Subject Area: Experimental Models for the Understanding of Nervous System Diseases ; Molecular and Cellular Neurology and Neuropathology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 526123202

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease, which mainly affects neurons connecting the brain and muscles through the spinal cord (so called corticospinal tract). The specific molecular pathomechanisms underlying the vulnerability of this specific neuronal population have not been clarified yet, and an effective therapy against this disorder is still missing. The dying forward theory (mainly based on post mortem evidences) proposes a synaptic transmission of pathogenic and cytotoxic misfolded proteins (such as TDP-43) that are transported along the axons of the neurons lying within the brain (motor cortex) to the spinal motoneurons. This triggers a “neurodegenerative wave” spreading to the spinal cord, which eventually leads to muscular denervation and the patients die because of respiratory failure. Thus, it appears that alterations of the corticospinal circuits might represent a crucial entry point for a better understanding of the disease progression. The aim of this project is to investigate the specific pathologic alterations occurring in the corticospinal tract of ALS patients before neuronal loss, with particular focus on the synaptic contacts between upper and lower motoneurons. To achieve this goal, patients´ derived inducible pluripotent stem cells (iPSC) represent a bona fide model allowing to reproduce in vitro some typical disease features in a human context. We will differentiate iPSC derived from ALS patients and healthy controls into human cerebral and spinalcord organoids, and fuse them into corticospinal assembloids (CSas). These 3D human assembloids display develop functional glutamatergic synaptic contacts typical of the cortico-spinal tract. Moreover, the presence of different sub-types of glial cells makes this model the ideal one to investigate the non-cell autonomous pathomechanisms occurring in ALS. Specifically, we will focus on the synaptic abnormalities occurring over time and leading to neuronal degeneration and loss: by performing confocal and super-resolution microscopy, we aim at understanding which synaptic sub-compartment firstly shows pathological features (by monitoring the appearance of toxic protein aggregates, and aberrant clusters of synaptic proteins along the dendrites and axons). In parallel, we will monitor (using a MaxTwo MEA device) and manipulate (through optogenetics) the electrophysiological properties of ALS and control hCSA to uncover how the disease progression affects neuronal functionality. This will provide more precise entry points for the development of novel therapeutic strategies, aimed at restoring the proper synaptic composition and activity in order to increase the overall neuronal fitness in ALS.

• DFG Programme: Research Grants