Reactive astrocytes in ALS display diminished intron retention

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. Graphical Abstract Graphical abstract Healthy astrocytes (left, blue) have prevalent nuclear intron retention in reactivity transcripts, promoting their nuclear confinement (introns shown as red rectangles). Conversely, ALS astrocytes (right, red) exhibit decreased intron retention in reactivity transcripts, enabling cytoplasmic translocation and translation upon engaging ribosomes, underlying their reactive transformation. A small number of intron retaining transcripts escape to the cytoplasm where they are degraded by nonsense mediated decay (NMD), which is enhanced in ALS astrocytes.