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. 2013 Dec 15;22(24):4871-87.
doi: 10.1093/hmg/ddt338. Epub 2013 Jul 12.

Loss of Mitochondrial Peptidase Clpp Leads to Infertility, Hearing Loss Plus Growth Retardation via Accumulation of CLPX, mtDNA and Inflammatory Factors

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Free PMC article

Loss of Mitochondrial Peptidase Clpp Leads to Infertility, Hearing Loss Plus Growth Retardation via Accumulation of CLPX, mtDNA and Inflammatory Factors

Suzana Gispert et al. Hum Mol Genet. .
Free PMC article

Abstract

The caseinolytic peptidase P (CLPP) is conserved from bacteria to humans. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder together with the chaperone CLPX. In spite of a known relevance for the mitochondrial unfolded protein response, its substrates and tissue-specific roles are unclear in mammals. Recessive CLPP mutations were recently observed in the human Perrault variant of ovarian failure and sensorineural hearing loss. Here, a first characterization of CLPP null mice demonstrated complete female and male infertility and auditory deficits. Disrupted spermatogenesis already at the spermatid stage and ovarian follicular differentiation failure were evident. Reduced pre-/post-natal survival and marked ubiquitous growth retardation contrasted with only light impairment of movement and respiratory activities. Interestingly, the mice showed resistance to ulcerative dermatitis. Systematic expression studies detected up-regulation of other mitochondrial chaperones, accumulation of CLPX and mtDNA as well as inflammatory factors throughout tissues. T-lymphocytes in the spleen were activated. Thus, murine Clpp deletion represents a faithful Perrault model. The disease mechanism probably involves deficient clearance of mitochondrial components and inflammatory tissue destruction.

Figures

Figure 1.
Figure 1.
Two independent founder lines with CLPP null mutation show CLPX protein accumulation. (A) Scheme of the Clpp gene exon-intron structure, coding region (broad bar from ATG) and of the two independent gene-trap insertion sites F (left arrow) and G (arrow further right). (B) Absence of Clpp transcript in brain hemispheres of 5-month-old line G Clpp−/− homozygotes. The picture represents RT–PCR products with primers situated in exons 5/6 or 3/4, respectively, with lanes for three Clpp−/− versus three WT animals. Gapdh served as a loading control. (C) Absent levels of anti-CLPP immunoreactivity contrasted with elevated levels of anti-CLPX immunoreactivity (antibody HPA040262 from Sigma) in western blots of all tissues from 5-month-old three line G Clpp−/− homozygotes versus three WT animals. Porin represents a loading control of mitochondrial mass.
Figure 2.
Figure 2.
Clpp −/− mice show male and female infertility, resistance against dermatitis, reduced survival, growth and motor activity. (A) Numbers of pups born for different combinations of genotypes and sexes demonstrating male and female sterility for Clpp−/− F and G mouse lines. Clpp+/− heterozygous mice were abbreviated as het. (B) Acoustic startle tests showed absent locomotor responses to millisecond 120 dB burst noises in Clpp−/− F mice at ages 12–18 months (n = 5 versus 5). Similarly significant observations were made in aged Clpp−/− G mice and at 115 dB (data not shown). (C) Skin pathology during a defined aging period is common in mice with C57BL/6 background and was evaluated in a one-time effort for all mice housed in cages with reported cases. Twenty Clpp−/− mice (11 F line, 9 G line) and 25 matched WT controls (17 for F, 8 for G) were scored as affected by alopecia, or as affected by ulcerative dermatitis (with skin lacerations and bleeding wounds) or as unaffected. Alopecia showed similar frequencies (F line 7 WT versus 8 Clpp−/− mice, G line 1 WT versus 1 Clpp−/−), but ulcerative dermatitis exhibited significantly (P = 0.03) reduced frequencies in Clpp−/− versus WT mice (F line 4 WT versus 0 Clpp−/− mice, G line 5 WT versus 1 Clpp−/−). (D) Body weight of mice from F and G lines showing increasing deficit with advancing age (n varied between 14 and 40 animals studied per line and age; hyperbola regression best-fit compared by F-test for BMAX difference generated P-values < 0.0001 for each panel in the GraphPad software). (E) Motor analysis in open field paradigm showing significantly reduced spontaneous activity of Clpp−/− mice at age 6 months (n = 15 versus 15) for parameters HACT, MOVNO, VACT. (F) Motor coordination analysis on Rotarod showing a trend toward improved performance for Clpp−/− mice at the age of 6 months. (G) Growth curve of MEFs showing reduced population doubling (PD) with premature senescence for the Clpp−/− genotype (n = 3 Clpp−/− versus three WT, with all three WT lines showing evident spontaneous immortalization by Day 28, whereas all Clpp−/− lines went into replicative senescence followed by apoptosis at Day 32). The bar graphs illustrate mean and SEM values.
Figure 3.
Figure 3.
Male and female gonad pathology in Clpp−/− mice. (A) Light microscopy analysis of testis sections stained by H and E revealed a complete absence of mature spermatids and spermatozoa from the seminiferous tubules (see arrow) in both lines F and G at the age of 6 months. A section for line F is shown. The size bar corresponds to 100 µm. (B) Testis electron microscopy detected completed acrosome (arrow above) and axoneme (arrow below) structures of spermatids only in wild-type tissue at the age of 3 months, whereas disorganized structures were observed in age-matched Clpp−/− testis. (C) Ovary light microscopy with H and E staining revealed a reduced size of the follicular cell layer, with a higher amount of apoptotic bodies in Clpp−/− mice (see arrows). (D) The overview in H and E histology underscores the smaller size of Clpp−/− ovaries. They contained a considerably higher amount of follicles in early stages (primordial, primary, secondary and Graaf follicle), but reduced completion of the ovarian cycle (corpus luteum).
Figure 4.
Figure 4.
Activation of T-lymphocytes in the Clpp−/− spleen. Splenocytes from wild-type (illustrated by blue dots) and mutant (red dots) mice (aged until 18 months under continuous microbial exposure monitoring in shared individually ventilated housing) were characterized using multi-color flow cytometry (n = 8 Clpp−/− versus 7 WT). (A) Mutant CD8+ T cell subsets analyzed for their expression of CD44/CD62L/Ly6C showed several significant changes. (B) Mutant CD4+ T cell subsets analyzed for their expression of CD25/CD44/CD62L/Ly6C also showed several significant changes. Statistical analysis was done by using SPICE (117). Significance (alpha = 5%) in Student's t-test is indicated by +, significance in the Wilcoxon rank test by #.
Figure 5.
Figure 5.
Mitochondrial DNA copy numbers are elevated in Clpp−/− tissue. Quantitative PCR of genes encoding respiratory chain subunits in the mitochondrial DNA (mtDNA, Cox3 gene) versus nuclear DNA (nDNA, Ndufv1 gene) showed consistently elevated levels of mtDNA in Clpp−/− mice, with high significance in an initial screen of testis tissue and an independent validation in testis tissues at the age of 3 months. The bar graph illustrates mean and SEM values.

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