FUS-ALS hiPSC-derived astrocytes impair human motor units through both gain-of-toxicity and loss-of-support mechanisms

Abstract

Background Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points. Methods In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems. Results We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/β-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs. Conclusions Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms. Graphical Abstract

The Evolution of Technology‐Driven In Vitro Models for Neurodegenerative Diseases

“…In a second work, Dittlau et al implemented their model integrating iPSCsderived astrocytes and evaluating their role on hiPSC-derived MN network and NMJs functionality. [61] They demonstrated that astrocytes have a key role in ALS pathological mechanisms, being associated with destruction of MN network and interfering with NMJ formation and functionality through several gain-of-toxicity and loss-of-support processes. In a recent work, Fornetti et al tried to reproduce a reliable NMJ model in vitro able to investigate deficiencies in the neural and muscular cross-talk behind the junction formation in 𝛼-sarcoglycanopathy (𝛼-SG), a subtype of limb-girdle muscular dystrophy.…”

“…implemented their model integrating iPSCs‐derived astrocytes and evaluating their role on hiPSC‐derived MN network and NMJs functionality. [ 61 ] They demonstrated that astrocytes have a key role in ALS pathological mechanisms, being associated with destruction of MN network and interfering with NMJ formation and functionality through several gain‐of‐toxicity and loss‐of‐support processes. In a recent work, Fornetti et al.…”

Integrative genetic and single cell RNA sequencing analysis provides new clues to the amyotrophic lateral sclerosis neurodegeneration

“…Besides, ALS-susceptibility genes and LoF pathogenicity genes exhibited high strictness in astrocytes and oligodendrocytes, both these two cell types were shown to play important roles in ALS (Ferraiuolo et al, 2016;Stoklund Dittlau et al, 2023). Excitatory, inhibitory, cerebrospinal fluid contacting neurons also required strict expressions of ALS-susceptibility genes.…”

Early nuclear phenotypes and reactive transformation in human iPSC-derived astrocytes from ALS patients withSOD1mutations

“…Previous studies have shown increased reactive transformation in astrocytes harbouring mutations in different familial ALS genes, including SOD1, C9orf72, FUS, and valosincontaining protein (VCP) (Birger et al, 2019;Taha et al, 2022;Stoklund Dittlau et al, 2023).…”