Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA

Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.

A Rare Case of Perrault Syndrome with Auditory Neuropathy Spectrum Disorder: Cochlear Implantation Treatment and Literature Review

Perrault syndrome (PRLTS) is a rare autosomal recessive disorder characterised by ovarian failure in females and sensorineural hearing loss (SNHL) in both genders. In the present paper we describe a child affected by PRLTS3, due to CLPP homozygous mutations, presenting auditory neuropathy spectrum disorder (ANSD) with bilateral progressive SNHL. This is the first case reported in the literature of an ANSD in PRLTS3. CLPP is a nuclear encoded mitochondrial protease directed at the mitochondrial matrix. It is encoded on chromosome 19. This protease participates in mitochondrial protein quality control by degrading misfolded or damaged proteins, thus maintaining the normal metabolic function of the cell. In PRLTS3, the peptidase activity of CLPP is suppressed. Neurological impairments involved in PRLTS3 suggest that the pathogenic mutations in CLPP might trigger a mitochondrial dysfunction. A comprehensive description of the clinical and audiological presentation, as well as the issues related to cochlear implant (CI) procedure and the results, are addressed and discussed. A brief review of the literature on this topic is also provided.

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX With Its Interactors at the Nucleoid and RNA Granule

Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brain. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with POLDIP2 as nucleoid component, LRPPRC as mitochondrial poly(A) mRNA granule element, GFM1 (in mouse also GRSF1) as tRNA processing factors. Only in mouse, accumulated ClpX, GFM1 and GRSF1 appeared in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2 and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids, and show nucleoid enlargement in human as a key consequence.

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Two novel likely pathogenic variants of HARS2 identified in a Chinese family with sensorineural hearing loss

Mutations in HARS2 are one of the genetic causes of Perrault syndrome, characterized by sensorineural hearing loss (SNHL) and ovarian dysfunction. Here, we identified two novel putative pathogenic variants of HARS2 in a Chinese family with sensorineural hearing loss including two affected male siblings, c.349G > A (p.Asp117Asn) and c.908 T > C (p.Leu303Pro), through targeted next-generation sequencing methods. The two affected siblings (13 and 11 years old) presented with early-onset, rapidly progressive SNHL. The affected siblings did not have any inner ear malformations or delays in gross motor development. Combined with preexisting clinical reports, Perrault syndrome may be latent in some families with non-syndromic deafness associated with HARS2 mutations. The definitive diagnosis of Perrault syndrome based on clinical features alone is a challenge in sporadic males, and preadolescent females with no signs of POI. Our findings further expanded the existing spectrum of HARS2 variants and Perrault syndrome phenotypes, which will assist in molecular diagnosis and genetic counselling of patients with HARS2 mutations.

Two novel likely pathogenic variants of HARS2 identified in a Chinese family with Perrault syndrome

Mutations in HARS2 are one of the genetic causes of Perrault syndrome, characterized by sensorineural hearing loss (SNHL) and ovarian dysfunction. Here, we identified two novel putative pathogenic variants of HARS2 in a Chinese family with sensorineural hearing loss including two affected male siblings, c.349G>A (p.Asp117Asn) and c.908T>C (p.Leu303Pro), through targeted next-generation sequencing methods. The two affected siblings (13 and 11 years old) presented with early-onset, rapidly progressive SNHL. The affected siblings did not have any inner ear malformations or delays in gross motor development. Combined with preexisting clinical reports, Perrault syndrome may be latent in some families with non-syndromic deafness associated with HARS2 mutations. The definitive diagnosis of Perrault syndrome based on clinical features alone is a challenge in sporadic males, and preadolescent females with no signs of POI. Our findings further expanded the existing spectrum of HARS2 variants and Perrault syndrome phenotypes, which will assist in molecular diagnosis and genetic counselling of patients with HARS2 mutations.

Two Novel Pathogenic Variants Confirm RMND1 Causative Role in Perrault Syndrome with Renal Involvement

RMND1 (required for meiotic nuclear division 1 homolog) pathogenic variants are known to cause combined oxidative phosphorylation deficiency (COXPD11), a severe multisystem disorder. In one patient, a homozygous RMND1 pathogenic variant, with an established role in COXPD11, was associated with a Perrault-like syndrome. We performed a thorough clinical investigation and applied a targeted multigene hearing loss panel to reveal the cause of hearing loss, ovarian dysfunction (two cardinal features of Perrault syndrome) and chronic kidney disease in two adult female siblings. Two compound heterozygous missense variants, c.583G>A (p.Gly195Arg) and c.818A>C (p.Tyr273Ser), not previously associated with disease, were identified in RMND1 in both patients, and their segregation with disease was confirmed in family members. The patients have no neurological or intellectual impairment, and nephrological evaluation predicts a benign course of kidney disease. Our study presents the mildest, so far reported, RMND1-related phenotype and delivers the first independent confirmation that RMND1 is causally involved in the development of Perrault syndrome with renal involvement. This highlights the importance of including RMND1 to the list of Perrault syndrome causative factors and provides new insight into the clinical manifestation of RMND1 deficiency.