SETX (senataxin), the helicase mutated in AOA2 and ALS4, functions in autophagy regulation.

ABSTRACT

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.Abbreviations 3'READS 3' region extraction and deep sequencing; ACTB actin beta; ALS4 amyotrophic lateral sclerosis type 4; AOA2 ataxia with oculomotor apraxia type 2; APA alternative polyadenylation; AS alternative splicing; ATG7 autophagy-related 7; ATP6V0D2 ATPase H+ transporting V0 subunit D2; BAF bafilomycin A1; BECN1 beclin 1; ChIP chromatin IP; Chloro chloroquine; CPT camptothecin; DDR DNA damage response; DNMT1 DNA methyltransferase 1; DRIP DNA/RNA IP; DSBs double strand breaks; EBs embryoid bodies; FTD frontotemporal dementia; GABARAP GABA type A receptor-associated protein; GO gene ontology; HR homologous recombination; HTT huntingtin; IF immunofluorescence; IP immunoprecipitation; iPSCs induced pluripotent stem cells; KD knockdown; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MN motor neuron; MTORC1 mechanistic target of rapamycin kinase complex 1; PASS PolyA Site Supporting; PFA paraformaldehyde; RNAPII RNA polymerase II; SCA spinocerebellar ataxia; SETX senataxin; SMA spinal muscular atrophy; SMN1 survival of motor neuron 1, telomeric; SQSTM1/p62 sequestosome 1; TFEB transcription factor EB; TSS transcription start site; TTS transcription termination site; ULK1 unc-51 like autophagy activating kinase 1; WB western blot; WIPI2 WD repeat domain, phosphoinositide interacting 2; XRN2 5'-3' exoribonuclease 2.