Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by motor neuron death, resulting in muscle atrophy, weakness and paralysis. In ALS, astrocytes undergo reactive transformation, ultimately contributing to neuronal death, however the causal pathways by which this occurs are poorly understood. One candidate is the transforming growth factor (TGF)-β signalling pathway, which has been found to be upregulated in ALS models and patients.
Aims
Whether TGF-β drives cell-autonomous astrocyte reactive transformation, potentially by altering astrocyte reactive sub-states, and how it may contribute to neuron death, remains unknown.
Methods
To address this, we inhibited TGF-β in hiPSC derived ALS astrocytes carrying VCP mutations, known to cause familial ALS, and performed single-cell RNA sequencing, single-cell proteomics and functional studies informed by our analysis.
Results
We identified changes in the proportion of transcriptomic reactive sub-states characterised by mitochondrial dysfunction, cytoskeletal remodelling and markers of reactive transformation, which were ameliorated following TGF-β inhibition. Single-cell mass spectrometry validated these findings, revealing a VCP mutant subpopulation featuring increased translation and proinflammatory protein expression, which was similarly abrogated by TGF-β inhibition. Finally, increased motor neuron apoptosis following treatment with astrocyte conditioned media was significantly rescued when TGF-β was inhibited in VCP mutant astrocytes.
Conclusions
Together, our analysis reveals that human VCP mutant astrocytes feature dysregulated cell-autonomous reactive sub-states, driven by TGF-β activation, which partially recapitulate aberrant astrocyte gene expression changes identified in patient postmortem single-cell studies. Additionally, inhibiting TGF-β rescues dysregulated VCP mutant sub-states and astrocyte mediated neurotoxicity. These results collectively indicate that selective TGF-β pathway inhibition in astrocytes may be a potential therapeutic approach for ALS.