Abstract
Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes.
Highlights
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and incurable neurodegenerative disease
We first defined differences between Valosin-containing protein (VCP) mutant and control astrocyte gene expression patterns and found 170 differentially expressed genes (FDR < 0.05), which is 11-fold more than what we previously established in terminally differentiated VCP mutant motor neurons [41]
By analysing RNA sequencing from Barbar et al human-induced pluripotent stem cell (hiPSC)-derived astrocytes stimulated to undergo reactive transformation [27], we identified 11% less retained introns in astrocytes stimulated with TNF␣, IL-1␣ and C1q compared with untreated astrocytes (2,143 versus 2,402, respectively; Supplementary Figure S3G)
Summary
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and incurable neurodegenerative disease. The pathological hallmark of ALS is cytoplasmic aggregation of the RNA-binding protein, transactive response DNA-binding protein 43 (TDP-43, encoded by TARDBP) accompanied by loss of nuclear TDP-43, in the motor neurons of >97% of ALS cases [1]. Neuroinflammation induces quiescent astrocytes to undergo dramatic changes in gene expression, morphology and function, called ‘reactive transformation’ [24,25,26]. Using inflammatory cues to mimic activated microglia, Liddelow et al profiled astrocyte gene expression changes accompanying reactive transformation and categorized them into pan-reactive, A1 (harmful) and A2 (protective) responses [24,27,28]
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