Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) and primary ovarian insufficiency (POI) secondary to ovarian dysgenesis. However, the mutation spectrum of disease-causing genes for Perrault syndrome in the Chinese population remains poorly understood. In this study, we report on two Chinese families with Perrault syndrome type 3 caused by novel CLPP gene variants. We also conducted a comprehensive literature review of CLPP gene variants in Perrault syndrome type 3 to elucidate genotype-phenotype associations.

Using Whole Genome Sequencing (WGS) data, two pedigrees with Perrault syndrome type 3 were ascertained in the Chinese Deafness Genetics Cohort through genotype-driven analysis. Variants were validated using Sanger sequencing and copy number quantification methods. In vitro analysis of splice site variants in the CLPP gene using the minigene assay.

Two Han Chinese families were ascertained: one with compound heterozygous variants (c.270 + 1G > C and c.355A > C [p. Ile119Leu]) and the other with missense variant (c.400G > C [p. Asp134His]) together with a large deletion in CLPP. In vitro minigene assays confirmed that the c.270 + 1G > C variant causes intron 2 retention and an alternative 5' splice site in exon 2, leading to protein alteration. Among 33 Perrault syndrome type 3 patients in literature, 97% (31/32) had hearing loss, 55% (16/29) neurological disease, and 71% (15/21) females had POI. Including our 4 novel variants, 21 pathogenic CLPP gene variants have been reported, with 57% (12/21) missense and 43% (9/21) truncating variants, mainly in the ATP-dependent Clp protease proteolytic subunit. Biallelic truncating or missense plus truncating genotypes showed higher rates of neurological disease (p = 0.001), but no significant difference in hearing loss incidence compared to biallelic missense genotypes was observed.

This study highlights the challenges in diagnosing Perrault syndrome due to its genetically and clinically heterogeneity. By exploring novel variants and establishing genotype-phenotype correlations, we aim to improve the genetic diagnosis and consultation for this complex disorder.

Mitochondria are often referred to as the energy centers of the cell and are recognized as key players in signal transduction, sensing, and responding to internal and external stimuli. Under stress conditions, the mitochondrial unfolded protein response (UPRmt), a conserved mitochondrial quality control mechanism, is activated to maintain mitochondrial and cellular homeostasis. As a physiological stimulus, exercise-induced mitochondrial perturbations trigger UPRmt, coordinating mitochondria-to-nucleus communication and initiating a transcriptional program to restore mitochondrial function. The aim of this study was to evaluate the UPRmt signaling response to acute exercise in skeletal muscle. Male rats were subjected to acute treadmill exercise at 25 m/min for 60 min on a 0 % grade. Plantaris muscles were collected from both sedentary and exercise groups at various times: immediately (0), and at 1, 3, 6, 12, and 24 h post-exercise. Reactive oxygen species (ROS) production was assessed using hydrogen peroxide assay and dihydroethidium staining. Additionally, the mRNA and protein expression of UPRmt markers were measured using ELISA and real-time PCR. Mitochondrial activity was assessed using succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) staining. Our results demonstrated that acute exercise increased ROS production and upregulated UPRmt markers at both gene and protein levels. Moreover, skeletal muscle exhibited an increase in mitochondrial activity in response to exercise, as indicated by SDH and COX staining. These findings suggest that acute treadmill exercise is sufficient to induce ROS production, activate UPRmt signaling, and enhance mitochondrial activity in skeletal muscle, expanding our understanding of mitochondrial adaptations to exercise.

Sensorineural hearing loss (SNHL) is a prevalent clinical condition primarily attributed to dysfunction within various components of the auditory pathway, spanning from the inner ear to the auditory cortex. Recent research has illuminated immune and inflammation-mediated disorders of the inner ear as critical contributors to SNHL. Disruptions in the equilibrium of inflammatory mediators, chemokines, the complement system, and inflammatory vesicles within the cochlea provoke aberrations in immune cell activity, fostering a chronic pro-inflammatory milieu that detrimentally affects the structural and functional integrity of the inner ear, culminating in hearing impairment. Specific genetic mutations, especially those affecting auditory structures, play an important role in SNHL. These mutations regulate inflammatory mediators and cellular responses, thereby altering the inflammatory dynamics within the cochlea. This review delves into the pathogenesis of sensorineural hearing loss, emphasizing the impact of genetic alterations, immune responses within the inner ear, and inflammatory mediators on auditory function. It highlights the significance of Transmembrane Serine Protease 3 (TMPRSS3) and connexin gene mutations as pivotal genetic elements in SNHL, underscoring the central role of inflammatory responses in cochlear damage. Furthermore, the paper discusses the promise of gene therapy and targeted molecular interventions, underscoring the necessity for continued exploration into the specific actions of various inflammatory agents to refine personalized therapeutic strategies.

Based on a clinically staged small molecular hClpP activator ONC201, a class of imipridone derivatives was designed and synthesized. These compounds were evaluated in a protease hydrolytic assay, as well as cell growth inhibition assays in three cancer cell lines, MIA PACA-2, HCT116, and MV4-11. A number of compounds that can more potently activate hClpP and more effectively inhibit cell growth in the three cancer cell lines than ONC201 were identified. The most potent compound, ZYZ-17, activated hClpP with an EC50 value of 0.24 µM and inhibited the growth of the three cancer cell lines with IC50 values of less than 10 nM. Mechanism studies for ZYZ-17 revealed that it potently activates cellular hClpP, efficiently induces the degradation of hClpP substrates, and robustly induces apoptosis in the three cancer cell lines. Furthermore, ZYZ-17 demonstrated a promising pharmacokinetic (PK) profile and exhibited highly potent in vivo antitumor activity in a pancreatic cancer MIA PACA-2 xenograft model in BALB/c nude mice.

Maintenance of mitochondrial homeostasis requires a plethora of coordinated quality control and adaptations' mechanisms in which mitochondrial proteases play a key role. Their activation or loss of function reverberate beyond local mitochondrial biochemical and metabolic remodelling into coordinated cellular pathways and stress responses that feedback onto the mitochondrial functionality and adaptability. Mitochondrial proteolysis modulates molecular and organellar quality control, metabolic adaptations, lipid homeostasis and regulates transcriptional stress responses. Defective mitochondrial proteolysis results in disease conditions most notably, mitochondrial diseases, neurodegeneration and cancer. Here, it will be discussed how mitochondrial proteases and mitochondria stress signalling impact cellular homeostasis and determine the cellular decision to survive or die, how these processes may impact disease etiopathology, and how modulation of proteolysis may offer novel therapeutic strategies.

In mammals, the leucine-rich pentatricopeptide repeat protein (LRPPRC) and the stem-loop interacting RNA-binding protein (SLIRP) form a complex in the mitochondrial matrix that is required throughout the life cycle of most mitochondrial mRNAs. Although pathogenic mutations in the LRPPRC and SLIRP genes cause devastating human mitochondrial diseases, the in vivo function of the corresponding proteins is incompletely understood. We show here that loss of SLIRP in mice causes a decrease of complex I levels whereas other OXPHOS complexes are unaffected. We generated knock-in mice to study the in vivo interdependency of SLIRP and LRPPRC by mutating specific amino acids necessary for protein complex formation. When protein complex formation is disrupted, LRPPRC is partially degraded and SLIRP disappears. Livers from Lrpprc knock-in mice had impaired mitochondrial translation except for a marked increase in the synthesis of ATP8. Furthermore, the introduction of a heteroplasmic pathogenic mtDNA mutation (m.C5024T of the tRNAAla gene) into Slirp knockout mice causes an additive effect on mitochondrial translation leading to embryonic lethality and reduced growth of mouse embryonic fibroblasts. To summarize, we report that the LRPPRC/SLIRP protein complex is critical for maintaining normal complex I levels and that it also coordinates mitochondrial translation in a tissue-specific manner.

Ageing is accompanied by a persistent, low-level inflammation, termed "inflammageing", which contributes to the pathogenesis of age-related diseases. Mitochondria fulfil multiple roles in host immune responses, while mitochondrial dysfunction, a hallmark of ageing, has been shown to promote chronic inflammatory states by regulating the production of cytokines and chemokines. In this review, we aim to disentangle the molecular mechanisms underlying this process. We describe the role of mitochondrial signalling components such as mitochondrial DNA, mitochondrial RNA, N-formylated peptides, ROS, cardiolipin, cytochrome c, mitochondrial metabolites, potassium efflux and mitochondrial calcium in the age-related immune system activation. Furthermore, we discuss the effect of age-related decline in mitochondrial quality control mechanisms, including mitochondrial biogenesis, dynamics, mitophagy and UPRmt, in inflammatory states upon ageing. In addition, we focus on the dynamic relationship between mitochondrial dysfunction and cellular senescence and its role in regulating the secretion of pro-inflammatory molecules by senescent cells. Finally, we review the existing literature regarding mitochondrial dysfunction and inflammation in specific age-related pathological conditions, including neurodegenerative diseases (Alzheimer's and Parkinson's disease, and amyotrophic lateral sclerosis), osteoarthritis and sarcopenia.

Premature Ovarian Insufficiency (POI) is a highly heterogeneous condition characterized by ovarian dysfunction in women occurring before the age of 40, representing a significant cause of female infertility. It manifests through primary or secondary amenorrhea. While more than half of POI cases are idiopathic, genetic factors play a pivotal role in all instances with known causes, contributing to approximately 20-25% of cases. This article comprehensively reviews the genetic factors associated with POI, delineating the primary candidate genes. The discussion delves into the intricate relationship between these genes and ovarian development, elucidating the functional consequences of diverse mutations to underscore the fundamental impact of genetic effects on POI. The identified genetic factors, encompassing gene mutations and chromosomal abnormalities, are systematically classified based on whether the resulting POI is syndromic or non-syndromic. Furthermore, this paper explores the genetic interplay between mitochondrial genes, such as Required for Meiotic Nuclear Division 1 homolog Gene (RMND1), Mitochondrial Ribosomal Protein S22 Gene (MRPS22), Leucine-rich Pentapeptide Repeat Gene (LRPPRC), and non-coding RNAs, including both microRNAs and Long non-coding RNAs, with POI. The insights provided serve to consolidate and enhance our understanding of the etiology of POI, contributing to establishing a theoretical foundation for diagnosing and treating POI patients, as well as for exploring the mechanisms underlying the disease.

The global diversity of fungi has been estimated between 2 to 11 million species, of which only about 155 000 have been named. Most fungi are invisible to the unaided eye, but they represent a major component of biodiversity on our planet, and play essential ecological roles, supporting life as we know it. Although approximately 20 000 fungal genera are presently recognised, the ecology of most remains undetermined. Despite all this diversity, the mycological community actively researches some fungal genera more commonly than others. This poses an interesting question: why have some fungal genera impacted mycology and related fields more than others? To address this issue, we conducted a bibliometric analysis to identify the top 100 most cited fungal genera. A thorough database search of the Web of Science, Google Scholar, and PubMed was performed to establish which genera are most cited. The most cited 10 genera are Saccharomyces, Candida, Aspergillus, Fusarium, Penicillium, Trichoderma, Botrytis, Pichia, Cryptococcus and Alternaria. Case studies are presented for the 100 most cited genera with general background, notes on their ecology and economic significance and important research advances. This paper provides a historic overview of scientific research of these genera and the prospect for further research. Citation: Bhunjun CS, Chen YJ, Phukhamsakda C, Boekhout T, Groenewald JZ, McKenzie EHC, Francisco EC, Frisvad JC, Groenewald M, Hurdeal VG, Luangsa-ard J, Perrone G, Visagie CM, Bai FY, Błaszkowski J, Braun U, de Souza FA, de Queiroz MB, Dutta AK, Gonkhom D, Goto BT, Guarnaccia V, Hagen F, Houbraken J, Lachance MA, Li JJ, Luo KY, Magurno F, Mongkolsamrit S, Robert V, Roy N, Tibpromma S, Wanasinghe DN, Wang DQ, Wei DP, Zhao CL, Aiphuk W, Ajayi-Oyetunde O, Arantes TD, Araujo JC, Begerow D, Bakhshi M, Barbosa RN, Behrens FH, Bensch K, Bezerra JDP, Bilański P, Bradley CA, Bubner B, Burgess TI, Buyck B, Čadež N, Cai L, Calaça FJS, Campbell LJ, Chaverri P, Chen YY, Chethana KWT, Coetzee B, Costa MM, Chen Q, Custódio FA, Dai YC, Damm U, de Azevedo Santiago ALCM, De Miccolis Angelini RM, Dijksterhuis J, Dissanayake AJ, Doilom M, Dong W, Alvarez-Duarte E, Fischer M, Gajanayake AJ, Gené J, Gomdola D, Gomes AAM, Hausner G, He MQ, Hou L, Iturrieta-González I, Jami F, Jankowiak R, Jayawardena RS, Kandemir H, Kiss L, Kobmoo N, Kowalski T, Landi L, Lin CG, Liu JK, Liu XB, Loizides M, Luangharn T, Maharachchikumbura SSN, Makhathini Mkhwanazi GJ, Manawasinghe IS, Marin-Felix Y, McTaggart AR, Moreau PA, Morozova OV, Mostert L, Osiewacz HD, Pem D, Phookamsak R, Pollastro S, Pordel A, Poyntner C, Phillips AJL, Phonemany M, Promputtha I, Rathnayaka AR, Rodrigues AM, Romanazzi G, Rothmann L, Salgado-Salazar C, Sandoval-Denis M, Saupe SJ, Scholler M, Scott P, Shivas RG, Silar P, Souza-Motta CM, Silva-Filho AGS, Spies CFJ, Stchigel AM, Sterflinger K, Summerbell RC, Svetasheva TY, Takamatsu S, Theelen B, Theodoro RC, Thines M, Thongklang N, Torres R, Turchetti B, van den Brule T, Wang XW, Wartchow F, Welti S, Wijesinghe SN, Wu F, Xu R, Yang ZL, Yilmaz N, Yurkov A, Zhao L, Zhao RL, Zhou N, Hyde KD, Crous PW (2024). What are the 100 most cited fungal genera? Studies in Mycology 108: 1-411. doi: 10.3114/sim.2024.108.01.

Significance: Mitochondria are eukaryotic organelles with various essential functions. They are both the source and the targets of reactive oxygen species (ROS). Different branches of a mitochondrial quality control system (mQCS), such as ROS balancing, degradation of damaged proteins, or whole mitochondria, can mitigate the adverse effects of ROS stress. However, the capacity of mQCS is limited. Overwhelming this capacity leads to dysfunctions and aging. Strategies to interfere into mitochondria-dependent human aging with the aim to increase the healthy period of life, the health span, rely on the precise knowledge of mitochondrial functions. Experimental models such as Podospora anserina, a filamentous fungus with a clear mitochondrial aging etiology, proved to be instrumental to reach this goal. Recent Advances: Investigations of the P. anserina mQCS revealed that it is constituted by a complex network of different branches. Moreover, mitochondrial architecture and lipid homeostasis emerged to affect aging. Critical Issues: The regulation of the mQCS is only incompletely understood. Details about the involved signaling molecules and interacting pathways remain to be elucidated. Moreover, most of the currently generated experimental data were generated in well-controlled experiments that do not reflect the constantly changing natural life conditions and bear the danger to miss relevant aspects leading to incorrect conclusions. Future Directions: In P. anserina, the precise impact of redox signaling as well as of molecular damaging for aging remains to be defined. Moreover, natural fluctuation of environmental conditions needs to be considered to generate a realistic picture of aging mechanisms as they developed during evolution.

The integrated stress response (ISR) triggered in response to various cellular stress enables mammalian cells to effectively cope with diverse stressful conditions while maintaining their normal functions. Four kinases (PERK, PKR, GCN2, and HRI) of ISR regulate ISR signaling and intracellular protein translation via mediating the phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51. Early ISR creates an opportunity for cells to repair themselves and restore homeostasis. This effect, however, is reversed in the late stages of ISR. Currently, some studies have shown the non-negligible impact of ISR on diseases such as ischemic diseases, cognitive impairment, metabolic syndrome, cancer, vanishing white matter, etc. Hence, artificial regulation of ISR and its signaling with ISR modulators becomes a promising therapeutic strategy for relieving disease symptoms and improving clinical outcomes. Here, we provide an overview of the essential mechanisms of ISR and describe the ISR-related pathways in organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. Meanwhile, the regulatory effects of ISR modulators and their potential application in various diseases are also enumerated.

LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the peptidase CLPP also act in the matrix, especially during stress periods, but their substrates are poorly defined. Mammalian CLPP deletion triggers infertility, deafness, growth retardation, and cGAS-STING-activated cytosolic innate immunity. CLPX mutations impair heme biosynthesis and heavy metal homeostasis. CLPP and CLPX are conserved from bacteria to humans, despite their secondary role in proteolysis. Based on recent proteomic-metabolomic evidence from knockout mice and patient cells, we propose that CLPP acts on phase-separated ribonucleoprotein granules and CLPX on multi-enzyme condensates as first-aid systems near the inner mitochondrial membrane. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, mitochondrial RNA granules and nucleoids, and the D-foci-mediated degradation of toxic double-stranded mtRNA/mtDNA. Unfolding multi-enzyme condensates, CLPX maximizes PLP-dependent delta-transamination and rescues malformed nascent peptides. Overall, their actions occur in granules with multivalent or hydrophobic interactions, separated from the aqueous phase. Thus, the role of CLPXP in the matrix is compartment-selective, as other mitochondrial peptidases: MPPs at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space.

Mitochondria play a central role in cellular metabolism producing the necessary ATP through oxidative phosphorylation. As a remnant of their prokaryotic past, mitochondria contain their own genome, which encodes 13 subunits of the oxidative phosphorylation system, as well as the tRNAs and rRNAs necessary for their translation in the organelle. Mitochondrial protein synthesis depends on the import of a vast array of nuclear-encoded proteins including the mitochondrial ribosome protein components, translation factors, aminoacyl-tRNA synthetases or assembly factors among others. Cryo-EM studies have improved our understanding of the composition of the mitochondrial ribosome and the factors required for mitochondrial protein synthesis and the advances in next-generation sequencing techniques have allowed for the identification of a growing number of genes involved in mitochondrial pathologies with a defective translation. These disorders are often multisystemic, affecting those tissues with a higher energy demand, and often present with neurodegenerative phenotypes. In this article, we review the known proteins required for mitochondrial translation, the disorders that derive from a defective mitochondrial protein synthesis and the animal models that have been established for their study.

Mitochondria are pleiotropic organelles central to an array of cellular pathways including metabolism, signal transduction, and programmed cell death. Mitochondria are also key drivers of mammalian immune responses, functioning as scaffolds for innate immune signaling, governing metabolic switches required for immune cell activation, and releasing agonists that promote inflammation. Mitochondrial DNA (mtDNA) is a potent immunostimulatory agonist, triggering pro-inflammatory and type I interferon responses in a host of mammalian cell types. Here we review recent advances in how mtDNA is detected by nucleic acid sensors of the innate immune system upon release into the cytoplasm and extracellular space. We also discuss how the interplay between mtDNA release and sensing impacts cellular innate immune endpoints relevant to health and disease.

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.

The etiology of hearing impairment is multifactorial, with contributions from both genetic and environmental factors. Although genetic studies have yielded valuable insights into the development and function of the auditory system, the contribution of gene products and their interaction with alternate environmental factors for the maintenance and development of auditory function requires further elaboration. In this review, we provide an overview of the current knowledge on the role of redox dysregulation as the converging factor between genetic and environmental factor-dependent development of hearing loss, with a focus on understanding the interaction of oxidative stress with the physical components of the peripheral auditory system in auditory disfunction. The potential involvement of molecular factors linked to auditory function in driving redox imbalance is an important promoter of the development of hearing loss over time.

Mitochondria plays an essential role in regulating cellular metabolic homeostasis, proliferation/differentiation, and cell death. Mitochondrial dysfunction is implicated in many age-related pathologies. Evidence supports that the dysfunction of mitochondria and the decline of mitochondrial DNA copy number negatively affect ovarian aging. However, the mechanism of ovarian aging is still unclear. Treatment methods, including antioxidant applications, mitochondrial transplantation, emerging biomaterials, and advanced technologies, are being used to improve mitochondrial function and restore oocyte quality. This article reviews key evidence and research updates on mitochondrial damage in the pathogenesis of ovarian aging, emphasizing that mitochondrial damage may accelerate and lead to cellular senescence and ovarian aging, as well as exploring potential methods for using mitochondrial mechanisms to slow down aging and improve oocyte quality.

Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins. The mitochondrial protease complex (ClpXP), which consists of caseinolytic mitochondrial matrix peptidase X (ClpX) and caseinolytic protease P (ClpP) and mediates the degradation of misfolded, damaged, and oxidized proteins, is essential for maintaining mitochondrial homeostasis. ClpXP has been implicated in meiosis regulation, but its precise role is currently unknown. In this study, we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X ( ClpxcKO ) mouse model to investigate the function of ClpX in meiosis. We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis. Specifically, it caused asynapsis of homologous chromosomes and impaired meiotic recombination, resulting in meiotic arrest in the zygotene-to-pachytene transition phase. The loss of ClpX compromised the double-strand break (DSB) repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1 (RAD51) to DSB sites. This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in ClpxcKO spermatocytes, as discerned by electron microscopy. Furthermore, ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in ClpxcKO spermatocytes. Taken together, we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing, synapsis, and recombination in spermatocytes during meiotic prophase I.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Caseinolytic peptidase P (CLPP) plays a central role in mitochondrial unfolded protein response (mtUPR) by promoting the breakdown of misfolded proteins and setting in motion a cascade of reactions to re-establish protein homeostasis. Global germline deletion of Clpp in mice results in female infertility and accelerated follicular depletion. Telomeres are tandem repeats of 5'-TTAGGG-3' sequences found at the ends of the chromosomes. Telomeres are essential for maintaining chromosome stability during somatic cell division and their shortening is associated with cellular senescence and aging. In this study, we asked whether the infertility and ovarian aging phenotype caused by global germline deletion of Clpp is associated with somatic aging, and tested telomere length in tissues of young and aging mice. We found that impaired mtUPR caused by the lack of CLPP is associated with accelerated telomere shortening in both oocytes and somatic cells of aging mice. In addition, expression of several genes that maintain telomere integrity was decreased, and double-strand DNA breaks were increased in telomeric regions. Our results highlight how impaired mtUPR can affect telomere integrity and demonstrate a link between loss of mitochondrial protein hemostasis, infertility, and somatic aging.

Mitochondrial unfolded protein stress response (mtUPR) plays a critical role in regulating cellular and metabolic stress response and helps maintain protein homeostasis. Caseinolytic peptidase P (CLPP) is one of the key regulators of mtUPR and promotes unfolded protein degradation. Previous studies demonstrated that global deletion of Clpp resulted in female infertility, whereas no impairment was found in the mouse model with targeted deletion of Clpp in cumulus/granulosa cells. These results suggest the need to delineate the function of Clpp in oocytes. In this study, we aimed to further explore the role of mtUPR in female reproductive competence and senescence using a mouse model. Oocyte-specific targeted deletion of Clpp in mice resulted in female subfertility associated with metabolic and functional abnormalities in oocytes, thus highlighting the importance of CLPP-mediated protein homeostasis in oocyte competence and reproductive function.

The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.

Premature ovarian insufficiency (POI) is a clinical syndrome of ovarian dysfunction characterized by cessation of menstruation occurring before the age of 40 years. The genetic causes of idiopathic POI remain unclear. Here we recruited a POI patient from a consanguineous family to screen for potential pathogenic variants associated with POI. Genetic variants of the pedigree were screened using whole-exome sequencing analysis and validated through direct Sanger sequencing. A homozygous variant in TUFM (c.524G>C: p.Gly175Ala) was identified in this family. TUFM (Tu translation elongation factor, mitochondrial) is a nuclear-encoded mitochondrial protein translation elongation factor that plays a critical role in maintaining normal mitochondrial function. The variant position was highly conserved among species and predicted to be disease causing. Our in vitro functional studies demonstrated that this variant causes decreased TUFM protein expression, leading to mitochondrial dysfunction and impaired autophagy activation. Moreover, we found that mice with targeted Tufm variant recapitulated the phenotypes of human POI. Thus, this is the first report of a homozygous pathogenic TUFM variant in POI. Our findings highlighted the essential role of mitochondrial genes in folliculogenesis and ovarian function maintenance.

Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the critical roles of ClpP and ClpX in regulating meiosis and spermatogenesis.

Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions remain elusive. In this study, we demonstrate that mice deficient for the mitochondrial intra-membrane rhomboid protease PARL, a recently reported model of the mitochondrial encephalopathy Leigh syndrome, develop early testicular atrophy caused by a complete arrest of spermatogenesis during meiotic prophase I, followed by degeneration and death of arrested spermatocytes. This process is independent of neurodegeneration. Interestingly, genetic modifications of PINK1, PGAM5, and TTC19 - three major substrates of PARL with important roles in mitochondrial homeostasis - fail to reproduce or modify this severe phenotype, indicating that the spermatogenic arrest arises from distinct molecular pathways. We further observed severe abnormalities in mitochondrial ultrastructure in PARL-deficient spermatocytes, along with prominent electron transfer chain defects, disrupted coenzyme Q (CoQ) biosynthesis, and metabolic rewiring. These mitochondrial defects are associated with a germ cell-specific decrease in GPX4 expression leading arrested spermatocytes to ferroptosis - a regulated cell death modality characterized by uncontrolled lipid peroxidation. Our results suggest that mitochondrial defects induced by PARL depletion act as an initiating trigger for ferroptosis in primary spermatocytes through simultaneous effects on GPX4 and CoQ - two major inhibitors of ferroptosis. These findings shed new light on the potential role of ferroptosis in the pathogenesis of mitochondrial diseases and male infertility warranting further investigation.

Breast cancer is the most frequently diagnosed malignancy worldwide and the leading cause of cancer mortality in women. Despite the recent development of new therapeutics including targeted therapies and immunotherapy, triple-negative breast cancer remains an aggressive form of breast cancer, and thus improved treatments are needed. In recent decades, it has become increasingly clear that breast cancers harbor metabolic plasticity that is controlled by mitochondria. A myriad of studies provide evidence that mitochondria are essential to breast cancer progression. Mitochondria in breast cancers are widely reprogrammed to enhance energy production and biosynthesis of macromolecules required for tumor growth. In this review, we will discuss the current understanding of mitochondrial roles in breast cancers and elucidate why mitochondria are a rational therapeutic target. We will then outline the status of the use of mitochondria-targeting drugs in breast cancers, and highlight ClpP agonists as emerging mitochondria-targeting drugs with a unique mechanism of action. We also illustrate possible drug combination strategies and challenges in the future breast cancer clinic.

Premature ovarian insufficiency (POI) is characterized by early loss of ovarian function before the age of 40 years. It is confirmed to have a strong and indispensable genetic component. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a key inducer of mitochondrial protein quality control for the clearance of misfolded or damaged proteins, which is necessary to maintain mitochondrial function. Previous findings have shown that the variation in CLPP is closely related to the occurrence of POI, which is consistent with our findings. This study identified a novel CLPP missense variant (c.628G > A) in a woman with POI who presented with secondary amenorrhea, ovarian dysfunction, and primary infertility. The variant was located in exon 5 and resulted in a change from alanine to threonine (p.Ala210Thr). Importantly, Clpp was mainly localized in the cytoplasm of mouse ovarian granulosa cells and oocytes, and was relatively highly expressed in granulosa cells. Moreover, the overexpression of c.628G > A variant in human ovarian granulosa cells decreased the proliferative capacity. Functional experiments revealed that the inhibition of CLPP decreased the content and activity of oxidative respiratory chain complex IV by affecting the degradation of aggregated or misfolded COX5A, leading to the accumulation of reactive oxygen species and reduction of mitochondrial membrane potential, ultimately activating the intrinsic apoptotic pathways. The present study demonstrated that CLPP affected the apoptosis of granulosa cells, which might be one of the mechanisms by which CLPP aberrations led to the development of POI.

Mitochondria, the main provider of energy in eukaryotic cells, contains more than 1000 different proteins and is closely related to the development of cells. However, damaged proteins impair mitochondrial function, further contributing to several human diseases. Evidence shows mitochondrial proteases are critically important for protein maintenance. Most importantly, quality control enzymes exert a crucial role in the modulation of mitochondrial functions by degrading misfolded, aged, or superfluous proteins. Interestingly, cancer cells thrive under stress conditions that damage proteins, so targeting mitochondrial quality control proteases serves as a novel regulator for cancer cells. Not only that, mitochondrial quality control proteases have been shown to affect mitochondrial dynamics by regulating the morphology of optic atrophy 1 (OPA1), which is closely related to the occurrence and progression of cancer. In this review, we introduce mitochondrial quality control proteases as promising targets and related modulators in cancer therapy with a focus on caseinolytic protease P (ClpP), Lon protease (LonP1), high-temperature requirement protein A2 (HrtA2), and OMA-1. Further, we summarize our current knowledge of the advances in clinical trials for modulators of mitochondrial quality control proteases. Overall, the content proposed above serves to suggest directions for the development of novel antitumor drugs.

Chemotherapy is extensively used to treat cancers and is often associated with ovarian damage and leads to premature ovarian insufficiency and infertility, while the role of mitochondria during ovarian damage with chemotherapy remains unknown. This study used a mouse model with oocyte-specific deletion of mitochondrial stress response gene Caseinolytic peptidase P (Clpp) to investigate mitochondrial homeostasis in oocytes from mice receiving a chemotherapeutic drug cyclophosphamide (CTX). We found that oocyte-specific deletion of Clpp reduced fecundity of the mice at advanced age. The deletion led to meiotic defects with elevated abnormal spindle rate and aneuploidy rate with impaired mitochondrial function in the MII oocytes from 8-week-old mice. Upon CTX treatment at 8-week-old, the oocyte competence and folliculogenesis from the oocyte-specific Clpp knockout mice was further deteriorated with dramatic impairment of mitochondrial distribution and function including elevated ROS level, decreased mitochondrial membrane potential, respiratory chain activity and ATP production. Taken together, the results indicate that that ClpP was required for oocyte competence during maturation and early folliculogenesis, and its deficiency deteriorate cyclophosphamide-induced ovarian damage.

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.

Reproductive aging is on the rise globally and inseparable from the entire aging process. An extreme form of reproductive aging is premature ovarian insufficiency (POI), which to date has mostly been of idiopathic etiology, thus hampering further clinical applications and associated with enormous socioeconomic and personal costs. In the field of reproduction, the important functional role of inflammation-induced ovarian deterioration and therapeutic strategies to prevent ovarian aging and increase its function are current research hotspots. This review discusses the general pathophysiology and relative causes of POI and comprehensively describes the association between the aging features of POI and infertility. Next, various preclinical studies of stem cell therapies with potential for POI treatment and their molecular mechanisms are described, with particular emphasis on the use of human induced pluripotent stem cell (hiPSC) technology in the current scenario. Finally, the progress made in the development of hiPSC technology as a POI research tool for engineering more mature and functional organoids suitable as an alternative therapy to restore infertility provides new insights into therapeutic vulnerability, and perspectives on this exciting research on stem cells and the derived exosomes towards more effective POI diagnosis and treatment are also discussed.

Ovarian cells, including oocytes, granulosa/cumulus cells, theca cells, and stromal cells, contain abundant mitochondria, which play indispensable roles in the processes of ovarian follicle development. Ovarian function is closely controlled by mitochondrial proteostasis and mitostasis. While mitochondrial proteostasis and mitostasis are disturbed by several factors, leading to dysfunction of ovarian function and initiating the mitochondrial unfolded protein response (UPRmt) and mitophagy to maintain or recover ovarian function and mitochondrial function, clear interactions between the 2 pathways in the ovary have not been fully elucidated. Here, we comprehensively summarize the molecular networks or regulatory mechanisms behind further mitochondrial research in the ovary. This review provides novel insights into the interactions between the UPRmt and mitophagy in ovarian functions.