Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice

Abstract

Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These "deletor" mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders.

Fig. 1.

Transgene expression levels in the muscle, heart, and brain of the three transgenic Twinkle^dup (C, D, and F) and two Twinkle^AT (G, H) mouse lines. The transgene expression levels were correlated with the expression of the native Twinkle gene.

Fig. 2.

Cytochrome c oxidase activity of muscle and brain and the skeletal muscle ultrastructure. Double staining for COX/SDH activities of the quadriceps femoris of 12-month-old control (a) and Twinkle^dup mouse (b), the quadriceps femoris of 18-month-old control (c) and Twinkle^dup (d) mouse, cerebellum of 18-month-old control (e) and Twinkle^dup (f) mouse, choroid plexus of 18-month-old control (g) and Twinkle^dup (h) mouse, and indusium griseum of 18-month-old control (i) and Twinkle^dup (j) mouse (cc, corpus callosum). The arrows point to the affected cells of the transgenic mice. (k and l) Electron micrographs of ultrathin section of the quadriceps femoris of an 18-month-old Twinkle^dup mouse. (k) Normal-sized mitochondrion is pointed out by the arrow in the normal fiber (nf) and an enlarged mitochondrion in the fiber with subsarcolemmal accumulation of mitochondria (sa). In the diseased fiber (df), muscle fibrils have been replaced by abnormal large mitochondria with inclusions, peripheral cristae, and vacuoles. (l) The autophagosomes with mitochondria found from the diseased fibers are shown by arrows. (Scale bars: 25 μm, a–h; 100 μm, i and j; 1 μm, k; 200 nm, l.)

Fig. 3.

Mitochondrial DNA analysis and copy number determination. (a and c) Southern blots of muscle and brain DNA of 22-month-old control, Twinkle^WT+ mice, and Twinkle^dup F₁ mouse and an 18-month-old Twinkle^dup F₂ mice probed with mtDNA, and nuclear 18S rRNA gene. (b) Quantitation of brain mtDNA copy number of the mice against the nuclear 18S rRNA gene. (d) Overexposed Southern blot of muscle DNA shown in c. The 3-kb band found from Twinkle^dup mice is pointed by the arrow. (e) Long PCR of muscle and brain DNA of the same mice. The full-length mtDNA is indicated by the thin arrow and the most prevalent deletion molecule size of muscle mtDNA (≈3 kb) by the thick arrow.

Fig. 4.

mtDNA deletions of the muscle and brain of the Twinkle^dup mouse, determined by long PCR, cloning, and DNA sequencing. (a) Schematic diagram of mouse mtDNA showing the deleted segment in gray (see supporting information for details). Only the D-loop, 12S rRNA, and 16S rRNA were commonly spared. (b) Schematic presentation of the mouse and human mtDNA region harboring the 5′ deletion breakpoints. Black vertical lines show the 5′ deletion breakpoints identified in this study, and the asterisks below indicate how often the same 5′ breakpoint was found from dissimilar deletion molecules. The common 5′deletion breakpoint of human multiple mtDNA deletions (16070) is indicated.