Nadezhda A. Kuznetsova

Age-related changes are associated with mitochondrial dysfunction, which is often caused by the accumulation of mutations in mitochondrial DNA (mtDNA). One common model of aging and age-related diseases involves mice with a mutant DNA polymerase γ (PolG^mut) whose proofreading function is impaired, which leads to the accumulation of mutations in mtDNA. The main limitation of such a model is that introducing a mutation into the mouse’s own gene leads to the accumulation of mutations in mtDNA over several generations, making it impossible to rule out whether mtDNA mutations or compensatory effects are the cause of functional impairments such as accelerated aging. This paper describes two lines of transgenic animals with inducible expression of PolG^mut. This inducible system prevents mutation accumulation in the germline, promoting stable reproduction and reproducibility of mice, increasing experimental flexibility for various studies of mitochondrial diseases. PolG^mut activation at different stages of life and different tissues allows us to study the progression of pathological changes during mitochondrial aging over time and detect the onset of mutation accumulation. The simplicity, reproducibility, and temporal control of this system represent a significant methodological improvement for studying mitochondrial mutagenesis and the pathophysiology of aging. Using this model, we demonstrated that the most pronounced pathology in these animals is accelerated thymus involution and the accumulation of cytotoxic effector CD8+ T cells in the peripheral immune organs, while no significant abnormalities were observed in other organs and systems. These data probably indicate that mtDNA mutations primarily impair T-cell immune function.

Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations—one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (Flnc^AGA/AGA and Flnc^GA/GA) were embryonically lethal. We determined that Flnc^GA/GA embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (Flnc^GA/wt, Flnc^AGA/wt, and Flnc^GA/AGA). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (Flnc^GA/wt, Flnc^AGA/wt) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (Flnc^GA/AGA) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, Flnc^GA/AGA mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C.

Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the Flnc gene are known to cause myopathies and cardiomyopathies in humans. Here we report a generation of two mouse strains carrying filamin C mutations — one of them has a deletion of three nucleotides at position c.7418_7420, causing E>>;D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop-codon. We have shown that heterozygous animals of both strains are functionally and morphologically indistinguishable from wild type animals, while homozygosity is embryonically lethal. Interestingly, compound heterozygous mice are viable, develop normally, reach puberty and their cardiac muscle is functionally and histologically normal. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C.