Inactivation of hnRNP K by Expanded Intronic AUUCU Repeat Induces Apoptosis Via Translocation of PKCδ to Mitochondria in Spinocerebellar Ataxia 10

Abstract

We have identified a large expansion of an ATTCT repeat within intron 9 of ATXN10 on chromosome 22q13.31 as the genetic mutation of spinocerebellar ataxia type 10 (SCA10). Our subsequent studies indicated that neither a gain nor a loss of function of ataxin 10 is likely the major pathogenic mechanism of SCA10. Here, using SCA10 cells, and transfected cells and transgenic mouse brain expressing expanded intronic AUUCU repeats as disease models, we show evidence for a key pathogenic molecular mechanism of SCA10. First, we studied the fate of the mutant repeat RNA by in situ hybridization. A Cy3-(AGAAU)10 riboprobe detected expanded AUUCU repeats aggregated in foci in SCA10 cells. Pull-down and co-immunoprecipitation data suggested that expanded AUUCU repeats within the spliced intronic sequence strongly bind to hnRNP K. Co-localization of hnRNP K and the AUUCU repeat aggregates in the transgenic mouse brain and transfected cells confirmed this interaction. To examine the impact of this interaction on hnRNP K function, we performed RT–PCR analysis of a splicing-regulatory target of hnRNP K, and found diminished hnRNP K activity in SCA10 cells. Cells expressing expanded AUUCU repeats underwent apoptosis, which accompanied massive translocation of PKCδ to mitochondria and activation of caspase 3. Importantly, siRNA–mediated hnRNP K deficiency also caused the same apoptotic event in otherwise normal cells, and over-expression of hnRNP K rescued cells expressing expanded AUUCU repeats from apoptosis, suggesting that the loss of function of hnRNP K plays a key role in cell death of SCA10. These results suggest that the expanded AUUCU–repeat in the intronic RNA undergoes normal transcription and splicing, but causes apoptosis via an activation cascade involving a loss of hnRNP K activities, massive translocation of PKCδ to mitochondria, and caspase 3 activation.

CAG repeat RNA as an auxiliary toxic agent in polyglutamine disorders

“…The RNA gain-of-function mechanism initially described in DM1, 18,20,35 wherein repeat expansions are found to be toxic only at the RNA level, now also explains some aspects of pathogenesis in other non-coding expansion disorders, including FXTAS, SCA8, SCA31, HDL2 as well as SCA10 and DM2. 11,[13][14][15][16][17] SCA8 is a unique among these diseases because…”

“…The involvement of other repeat-harboring transcripts in neurodegeneration via an RNA gain-of-function mechanism has been described not only for trinucleotide repeat disorders, such as FXTAS, SCA8 and HDL2, [13][14][15] but also for DM2, SCA10 and SCA31. 9,11,16,17 In the RNA gain-of-function mechanism, the expanded repeat of a flawed RNA exerts its toxic function by sequestration and subsequent loss-of-function of RNA binding proteins, such as the muscleblind-like (MBNL) family. Resultant ribonucleoprotein complexes become trapped in the nucleus where they form microscopic bodies and become toxic.…”

Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast

“…A DNA fragment from a patient with SCA10 containing more than 500 (ATTCT) n repeats was initially cloned into the pcDNA3.1/Hygro vector as previously described (15). After we transformed the resultant plasmid into Escherichia coli, the repeat had contracted down to ≈220 units.…”

“…It may have to do with the fact that the polyadenylation signals are not as highly conserved in yeast as in higher eukaryotes (28). Notably, to date no decrease of pre-mRNA or processed mRNA for the mutant SCA10 allele has been detected in humans (15).…”

“…The SCA10 pathogenesis is not well understood. It was suggested that haploinsufficiency of the ATXN10 gene, RNA gain of function (15), or chromatin change (12,16) could contribute to the disease.…”