CLPP Depletion Causes Diplotene Arrest; Underlying Testis Mitochondrial Dysfunction Occurs with Accumulation of Perrault Proteins ERAL1, PEO1, and HARS2

Abstract

Human Perrault syndrome (PRLTS) is autosomal, recessively inherited, and characterized by ovarian insufficiency with hearing loss. Among the genetic causes are mutations of matrix peptidase CLPP, which trigger additional azoospermia. Here, we analyzed the impact of CLPP deficiency on male mouse meiosis stages. Histology, immunocytology, different OMICS and biochemical approaches, and RT-qPCR were employed in CLPP-null mouse testis. Meiotic chromosome pairing and synapsis proceeded normally. However, the foci number of the crossover marker MLH1 was slightly reduced, and foci persisted in diplotene, most likely due to premature desynapsis, associated with an accumulation of the DNA damage marker γH2AX. No meiotic M-phase cells were detected. Proteome profiles identified strong deficits of proteins involved in male meiotic prophase (HSPA2, SHCBP1L, DMRT7, and HSF5), versus an accumulation of AURKAIP1. Histone H3 cleavage, mtDNA extrusion, and cGAMP increase suggested innate immunity activation. However, the deletion of downstream STING/IFNAR failed to alleviate pathology. As markers of underlying mitochondrial pathology, we observed an accumulation of PRLTS proteins ERAL1, PEO1, and HARS2. We propose that the loss of CLPP leads to the extrusion of mitochondrial nucleotide-binding proteins to cytosol and nucleus, affecting late meiotic prophase progression, and causing cell death prior to M-phase entry. This phenotype is more severe than in mito-mice or mutator-mice.

Translation Fidelity and Respiration Deficits in CLPP-Deficient Tissues: Mechanistic Insights from Mitochondrial Complexome Profiling

“…All other ribonucleoproteins were assessed, and 16 candidates were conspicuous in all three tissues (Figure S2), with translation factors occupying a prominent place. Among the disease proteins responsible for Perrault syndrome, HARS2 was prominent because of its strong accumulation in CLPP-null tissue, as previously reported upon quantitative immunoblots [17], and here, the complexomics profile revealed a parallel distribution of CLPX and HARS2 from their monomeric sizes (precursors of 69 and 23 kDa, respectively) up to molecular sizes around 250 kDa. Similarly, a close co-migration with CLPX was documented for GFM1 from its monomeric size (84 kDa) to positions around 250 kDa, in addition to very strong accumulation (average 10-fold).…”

“…Mutations in human CLPP cause Perrault syndrome type 3 (PRLTS3), an autosomal recessive disorder characterized by complete infertility due to female primary ovarian insufficiency and male azoospermia after meiotic arrest at diplotene, with subsequent sensorineural deafness, followed by insidious neurodegeneration manifesting as ataxia, leukodystrophy, and neuropathy [16,17]. Most variants of Perrault syndrome are triggered by the failure of mitochondrial RNA processing and translation, as exemplified by other causal mutations in the tRNA-amino acid synthases HARS2 and LARS2, in the mitoribosomal factor RMND1, in the RNA degradation factor PRORP, in the nucleoid replication/repair factor TWNK, or in CLPP [18,19].…”

ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis

“…Compared with controls, the ClpP cKO spermatocytes expressed more ClpX protein, and vice versa for the ClpX cKO spermatocytes. The observation is in line with a previous study showing remarkable ClpX accumulation in many organs, including testis and ovary of ClpP deleted mice 26 , 37 . The ClpP/ClpX cKO testes were smaller than the controls.…”

Mitochondrial Unfolded Protein Response Gene Clpp Is Required for Oocyte Function and Female Fertility

“…CLPP is the key regulator of the mtUPR pathway. Mutations in the mitochondrial protease CLPP result in Perrault syndrome type 3 (PRLTS3), an autosomal recessive disorder characterized by complete infertility due to female primary ovarian insufficiency and male azoospermia after meiotic arrest at diplotene, with sensorineural deafness, ataxia, leukodystrophy, and neuropathy [11][12][13]. Previous studies have shown that global germline Clpp deficiency in female mice results in impaired oocyte maturation, abnormal formation of twocell embryos, and blastocyst development failure, ultimately leading to infertility [14,15].…”