Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. Although manipulation of neuronal excitability has been tested in murine and human ALS models, it is still under debate whether neuronal activity might represent a valid target for efficient therapies. In this study, we exploited a combination of transcriptomics, proteomics, optogenetics and pharmacological approaches to investigate the activity‐related pathological features of iPSC‐derived C9orf72‐mutant motoneurons (MN). We found that human ALSC9orf72 MN are characterized by accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts and a dynamic “malactivation” of the transcription factor CREB. A similar phenotype was also found in TBK1‐mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation. Notably, these alterations, along with neuronal survival, could be rescued by treating ALS‐related neurons with the K+ channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Thus, our study shows that restoring the activity‐dependent transcriptional programme and synaptic composition exerts a neuroprotective effect on ALS disease progression.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies
We considered hiPSC-derived MN from 3 patients carrying the G4C2 hexanucleotide expansion in the C9orf72 gene and 3 control lines: two obtained from healthy individuals and one in which the pathogenic C9orf72 mutation has been corrected with CRISPR technology (CorrectedC9orf72)
After 21 days in vitro (DIV), cultures from both genotypes were composed of 50% neurons (84% of which were CHAT-positive), 3% GFAP-positive astrocytes and 15% Olig2-positive cells, whereas we did not detect the presence of mature oligodendrocytes
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. A similar phenotype was found in TBK1-mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation These alterations, along with neuronal survival, could be rescued by treating ALS-related neurons with the K+ channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Broad-spectrum K+ channel blocker 4-aminopyridine (4-AP) was employed to provide proof-of-principle evidence that interventions at the level of K+ channels are sufficient to enhance MN excitability and improve MN survival (Naujock et al, 2016) and have been shown to improve electrophysiological properties, synaptic composition and survival in a mouse model of SMA as well (Simon et al, 2021) These findings strengthened the hypothesis that increasing MN firing by inhibiting K+ channels, which control fundamental currents involved in MN activity such as the A (Leroy et al, 2015) and M current (Alaburda et al, 2002), might be neuroprotective even in different MN diseases. We aimed at identifying (using ALS patients’ iPSC-derived MN) activity-dependent transcriptional programmes disrupted in ALS and affecting synaptic stability, and how to restore these by targeting K+ conductances which affect MN intrinsic excitability
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