Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.