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.
References
[1]
Lin X, Ashizawa T (2003) SCA10 and ATTCT repeat expansion: clinical features and molecular aspects. Cytogenet Genome Res 100: 184–188.
[2]
Ashizawa T (2006) Spinocerebellar ataxia type 10: a disease caused by an expanded (ATTCT)n pentanucleotide repeat. In: Wells RD, Ashizawa T, editors. Genetic instabilities and neurological diseases. Burlington: Academic Press. pp. 433–446.
[3]
Rasmussen A, Matsuura T, Ruano L, Yescas P, Ochoa A, et al. (2001) Clinical and genetic analysis of four Mexican families with spinocerebellar ataxia type 10. Ann Neurol 50: 234–239.
[4]
Teive HA, Roa BB, Raskin S, Fang P, Arruda WO, et al. (2004) Clinical phenotype of Brazilian families with spinocerebellar ataxia 10. Neurology 63: 1509–1512.
[5]
Grewal RP, Achari M, Matsuura T, Durazo A, Tayag E, et al. (2002) Clinical features and ATTCT repeat expansion in spinocerebellar ataxia type 10. Arch Neurol 59: 1285–1290.
[6]
Matsuura T, Yamagata T, Burgess DL, Rasmussen A, Grewal RP, et al. (2000) Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10. Nat Genet 26: 191–194.
[7]
Gatchel JR, Zoghbi HY (2005) Diseases of unstable repeat expansion: mechanisms and common principles. Nat Rev Genet 6: 743–755.
[8]
Brouner JR, Willemsen R, Oostra BA (2009) Microsatellite repeat instability and neurological disease. Bioessays 31: 71–83.
[9]
Pandolfo M (2008) Friedreich ataxia. Arch Neurol 65: 1296–1303.
[10]
Sato N, Amino T, Kobayashi K, Asakawa S, Ishiguro T, et al. (2009) Spinocerebellar ataxia type 31 is associated with “inserted” penta-nucleotide repeats containing (TGGAA)n. Am J Hum Genet 85: 544–557.
[11]
Liquori CL, Ricker K, Moseley ML, Jacobsen JF, Kress W, et al. (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293: 864–867.
[12]
Mankodi A, Teng-Umnuay P, Krym M, Henderson D, Swanson M, et al. (2003) Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 54: 760–768.
[13]
Matsuura T, Fang P, Pearson CE, Jayakar P, Ashizawa T, et al. (2006) Interruptions in the expanded ATTCT repeat of spinocerebellar ataxia type 10: repeat purity as a disease modifier? Am J Hum Genet 78: 125–129.
[14]
Raskin S, Ashizawa T, Teive HA, Arruda WO, Fang P, et al. (2007) Reduced penetrance in a Brazilian family with Spinocerebellar Ataxia Type 10. Arch Neurol 64: 591–594.
[15]
Marz P, Probst A, Lang S, Schwager M, Rose-John S, et al. (2004) Ataxin-10, the spinocerebellar ataxia type 10 neurodegenerative disorder protein, is essential for survival of cerebellar neurons. J Biol Chem 279: 35542–35550.
[16]
Waragai , Nagamitsu M, Xu S, Li W, Lin YJ, et al. (2006) Ataxin 10 induces neuritogenesis via interaction with G-protein beta2 subunit. J Neurosci Res 83: 1170–1178.
[17]
Wakamiya M, Liu Y, Schuster GC, Gao R, Xu W, et al. (2006) The role of ataxin-10 in spinocerebellar ataxia type 10 pathogenesis. Neurology 67: 607–613.
[18]
Keren B, Jacquette A, Depienne C, Leite P, Durr A, et al. (2010) Evidence against haploinsufficiency of human ataxin 10 as a cause of spinocerebellar ataxia type 10. Neurogenetics 11: 273–274.
[19]
Thisted T, Lyakhov DL, Liebhaber SA (2001) Optimized RNA targets of two closely related triple KH domain proteins, heterogeneous nuclear ribonucleoprotein K and alphaCP-2KL, suggest distinct modes of RNA recognition. J Biol Chem 276: 17484–17496.
[20]
Tsukahara T, Casciato C, Helfman DM (1994) Alternative splicing of β-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5′ and 3′ splice sites of the non-muscle/smooth muscle exon 6. Nucl Acids Res 22: 2318–2325.
[21]
Expert-Bezancon A, Le Caer JP, Marie J (2002) hnRNP K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken β-tropomyosin pre mRNA. J Biol Chem 277: 16614–16623.
[22]
Lynch M, Chen L, Ravitz MJ, Mehtani S, Korenblat K, et al. (2005) hnRNP K binds a core polypyrimidine element in the eukaryotic translation initiation factor 4E (eIF4E) promoter, and its regulation of eIF4E contributes to neoplastic transformation. Mol Cell Biol 25: 6436–6453.
[23]
Moumen A, Masterson P, O'Connor MJ, Jackson SP (2005) hnRNP K: an HDM2 target and transcriptional coactivator of p53 in response to DNA damage. Cell 123: 1065–1078.
[24]
Schullery DS, Ostrowski J, Denisenko ON, Stempka L, Shnyreva M, et al. (1999) Regulated interaction of protein kinase C delta with the heterogeneous nuclear ribonucleoprotein K protein. J Biol Chem 274: 15101–15109.
[25]
Bomsztyk K, Denisenko O, Ostrowski J (2004) hnRNP K: one protein multiple processes. Bioessays 26: 629–638.
[26]
Idriss H, Kumar A, Casas-Finet JR, Guo H, Damuni Z, et al. (1994) Regulation of in vitro nucleic acid strand annealing activity of heterogeneous nuclear ribonucleoprotein protein A1 by reversible phosphorylation. Biochemistry 33: 11382–11390.
[27]
Ostrowski J, Klimek-Tomczak K, Wyrwicz LS, Mikula M, Schullery DS, et al. (2004) Heterogeneous nuclear ribonucleoprotein K enhances insulin-induced expression of mitochondrial UCP2 protein. J Biol Chem 279: 54599–54609.
[28]
Kaasinen SK, Goldstein G, Alhonem L, Janne J, Koistinaho J (2002) Induction and activation of protein kinase δ in Hippocampus and Cortex after kainic acid treatment, Exp Neurol 176: 203–212.
[29]
Nitti M, Furtaro AL, Traverso N, Odetti P, Storace D, et al. (2007) PKC delta and NADPH oxidase in AGE-induced neuronal death. Neurosci Lett 416: 261–265.
[30]
Voss OH, Kim S, Wewers MD, Doseff AI (2005) Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3. J Biol Chem 280: 17371–17379.
[31]
Brodie C, Blumberg PM (2003) Regulation of cell apoptosis by protein kinase c delta. Apoptosis 8: 19–27.
[32]
Majumder PK, Pandey P, Sun X, Cheng K, Datta R, et al. (2000) Mitochondrial translocation of protein kinase C delta in phorbol ester-induced cytochrome c release and apoptosis. J Biol Chem 275: 21793–21796.
[33]
Daughters RS, Tuttle DL, Gao W, Ikeda Y, Moseley ML, et al. (2009) RNA gain-of-function in spinocerebellar ataxia type 8. PLoS Genet 5: e1000600. doi:10.1371/journal.pgen.1000600.
[34]
Rudnicki DD, Holmes SE, Lin MW, Thornton CA, Ross CA, et al. (2007) Huntington's disease–like 2 is associated with CUG repeat-containing RNA foci. Ann Neurol 61: 272–82.
[35]
Oostra BA, Willemsen R (2009) FMR1: a gene with three faces. Biochim Biophys Acta 1790: 467–77.
[36]
Gao FH, Wu YL, Zhao M, Liu CX, Wang LS, et al. (2009) Protein Kinase C-delta mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: Involvement in apoptosis induction. Exp Cell Res 315: 3250–3258.
[37]
Jellinger KA, Stadelmann CH (2000) The enigma of cell death in neurodegenerative disorders. J Neural Transm: Suppl 6021–36.
[38]
Sumitomo M, Ohba M, Asakuma J, Asano T, Kuroki T, et al. (2002) Protein kinase Cdelta amplifies ceramide formation via mitochondrial signaling in prostate cancer cells. J Clin Invest 109: 827–836.
[39]
Sarkar PS, Chang HC, Boudi FB, Reddy S (1998) CTG repeats show bimodal amplification in E. coli Cell 95: 531–540.
[40]
Matsuura T, Ashizawa T (2002) Polymerase chain reaction amplification of expanded ATTCT repeat in spinocerebellar ataxia type 10. Ann Neurol 51: 271–272.