Biology subjects

Prudent, J.

Publications and source records attributed to Prudent, J..

12 recordsLinked to original sources

bioRxiv2026

CLN6 is a glycerophosphoglycerol epimerase in bis(monoacylglycero)phosphate biosynthesis

Lysosomal catabolism depends on bis(monoacylglycero)phosphate (BMP), a phospholipid with atypical S,S stereochemistry of unknown biosynthetic origin. CLN8 and CLN5 catalyze sequential acylation steps in BMP synthesis, and their loss of function causes Batten disease, a group of inherited neurodegenerative lysosomal storage disorders. Here we identify CLN6, a biochemically uncharacterized endoplasmic reticulum protein whose loss also causes Batten disease, as the calcium-dependent glycerophosphoglycerol epimerase that establishes BMP stereochemistry upstream of CLN8. CLN6 deficiency depletes BMP in cells, mice, and patient lysosomes, and reduces lysosomal hydrolase levels. BMP-precursor supplementation bypasses CLN6, restores BMP and increases hydrolase abundance in CLN6-deficient cells and mice. These findings place three Batten disease proteins in a single biosynthetic pathway and reveal BMP deficiency as a reversible cause of disrupted lysosomal hydrolase homeostasis.

biochemistry↗

bioRxiv2026

Single-nucleoid imaging in whole cells defines the dynamics of the mtDNA life cycle

The mitochondrial genome (mtDNA) is essential for oxidative phosphorylation, and mammalian cells typically contain hundreds or thousands of copies. Although specific aspects of mtDNA replication and degradation have been explored, a complete account of the mtDNA life cycle has remained elusive, particularly for nondividing cells. Using super-resolution and 4D lattice light-sheet imaging, we quantified the full mtDNA life cycle in quiescent primary human cells. We show that cells maintain steady state by replicating and degrading 1.5 {+/-} 0.2% of total mtDNA content each hour, a remarkably rapid flux. Younger mtDNA molecules are closer to the nucleus, spared from degradation, and serve as foci for further replication events. Non-proliferative cells regulate nucleoid density within the mitochondrial network, and the mitochondrial membrane potential sustains mtDNA copy number. These findings provide a foundational understanding of the dynamics underlying mtDNA homeostasis and a mechanism explaining how mutations accumulate in aging and disease.

cell biology↗

bioRxiv2025

MitoPerturb-Seq identifies common and gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy

Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.

genomics↗

bioRxiv2023

Protein thiol alterations drive aberrant phase separation in aging

Cellular homeostasis relies on precise regulation through chemical processes, such as protein posttranslational modifications (PTM) and physical processes, such as biomolecular condensation. Aging disrupts this balance, increasing susceptibility to diseases and death. However, the mechanisms behind age-related pathogenesis remain elusive. In this study, we dissected various cysteine PTMs and their impact on protein-mediated biomolecular condensation in aging brain. Our findings reveal that aging is associated with significant remodeling of cysteine PTMs, which impacts protein ability to participate in liquid-liquid phase separation (LLPS). Specifically, aging leads to an increase in protein sulfenylation and sulfonylation, which promotes LLPS and through conformational change increases the propensity of proteins to aggregate. Protein persulfidation, a protective thiol modification, prevents this by causing condensate dissolution. We demonstrate that age-induced alterations in cysteine PTMs influence the LLPS properties of synapsin-1 and G3BP2, resulting in disruptions in neurotransmitter release and stress granule formation, respectively. Additionally, our study uncovers that GAPDH is susceptible to LLPS and cysteine sulfonylation exacerbates its transition from condensates to aggregates. Mice deficient in cystathionine gamma-lyase, a pro-longevity gene that regulates intracellular persulfide levels, exhibit a shorter lifespan and spontaneous development of neurofibrillary tangles.

biochemistry↗

bioRxiv2023

Preserved striatal innervation and motor function despite severe loss of nigral dopamine neurons following mitochondrial dysfunction induced by mtDNA mutations

Degeneration of dopamine neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinsons disease (PD). In idiopathic cases, high levels of mitochondrial DNA (mtDNA) mutations associated with mitochondrial dysfunction are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E-variant of the mitochondrial helicase Twinkle in dopamine neurons, leading to accelerated mtDNA ageing. K320E-TwinkleDaN mice showed normal motor function at 20 months of age, although already [~]70% of nigral dopamine neurons had perished. The remaining neuron population still preserved [~]75% of axon terminals in the dorsal striatum, which enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of surviving nigral dopamine neurons. We conclude that a small population of substantia nigra neurons can adapt to mtDNA mutations and maintain motor control in mice, holding chances for new treatment strategies in PD patients.

neuroscience↗

bioRxiv2022

ESYT1 tethers the endoplasmic reticulum to mitochondria and is required for mitochondrial lipid and calcium homeostasis

Mitochondria interact with the endoplasmic reticulum (ER) at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). MERCs are crucial for a myriad of physiological functions including lipid synthesis and transport, and calcium signaling. Alterations in the structure, composition or regulation of MERCs contribute to the aetiology of many pathologies including neurodegenerative and metabolic diseases. The proteins mediating the formation of MERCs have been extensively studied in yeast, where the ER-mitochondria encounter structure (ERMES) complex mediates the transport of lipids between the ER and mitochondria via three lipid binding SMP-domain proteins. However, none of the SMP proteins of the ERMES complex have orthologues in mammals suggesting that alternate pathways have evolved in metazoans. Combining proximity labelling (BioID), confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain containing protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. The deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs, indicating that the ESYT1-SYN2JBP complex plays a role in tethering ER and mitochondria. Loss of this complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by re-expression of wild-type ESYT1 as well as an artificial mitochondria-ER tether. Together, these results reveal a novel function of ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.

cell biology↗

bioRxiv2022

Loss of Mfn1 but not Mfn2 enhances adipogenesis

ObjectiveA biallelic missense mutation in mitofusin 2 (MFN2) causes multiple symmetric lipomatosis and partial lipodystrophy, implicating disruption of mitochondrial fusion or interaction with other organelles in adipocyte differentiation, growth and/or survival. In this study, we aimed to document the impact of loss of mitofusin 1 (Mfn1) or 2 (Mfn2) on adipogenesis in cultured cells. MethodsWe characterised adipocyte differentiation of wildtype (WT), Mfn1-/- and Mfn2-/- mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes in which Mfn1 or 2 levels were reduced using siRNA. ResultsMfn1-/- MEFs displayed striking fragmentation of the mitochondrial network, with surprisingly enhanced propensity to differentiate into adipocytes, as assessed by lipid accumulation, expression of adipocyte markers (Plin1, Fabp4, Glut4, Adipoq), and insulin-stimulated glucose uptake. RNA sequencing revealed a corresponding pro-adipogenic transcriptional profile including Pparg upregulation. Mfn2-/- MEFs also had a disrupted mitochondrial morphology, but in contrast to Mfn1-/- MEFs they showed reduced expression of adipocyte markers and no increase in insulin-stimulated glucose uptake. Mfn1 and Mfn2 siRNA mediated knockdown studies in 3T3-L1 adipocytes generally replicated these findings. ConclusionsLoss of Mfn1 but not Mfn2 in cultured pre-adipocyte models is pro-adipogenic. This suggests distinct, non-redundant roles for the two mitofusin orthologues in adipocyte differentiation.

cell biology↗

bioRxiv2022

A mouse model of human mitofusin 2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin-2. How this selective perturbation of mitochondrial function leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation by isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation perturbs mitochondrial morphology and activates the integrated stress response selectively in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

cell biology↗

bioRxiv2022

PERK-ATAD3A interaction protects mitochondrial proteins synthesis during ER stress

Widespread repression of protein synthesis rates is a key feature of Endoplasmic Reticulum (ER) stress, mediated by the ER sensor kinase PERK. While select transcripts escape this repression, global translational down-regulation impacts crucial protein levels in all cellular compartments, beyond the ER. How the cell manages this paradox is unclear. PERK has a unique cytoplasmic loop within its kinase domain that binds PERKs target, eIF2. We identified the mitochondrial protein, ATAD3A, as a new interactor of the loop, binding to a highly conserved region within it. During ER stress, increased interaction between ATAD3A and PERK attenuates PERK signalling to eIF2, removing the translational block on several mitochondrial proteins. This occurs at novel context-dependent, mitochondria-ER contact sites. The interaction provides a previously unknown mechanism for fine-tuning translational repression at a local level, mitigating the impact of ER stress on mitochondria. Further, it represents a new target for selective modulation of PERK-eIF2 signalling in diseases from cancer to neurodegeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/501280v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@f74252org.highwire.dtl.DTLVardef@1234633org.highwire.dtl.DTLVardef@d5dc9aorg.highwire.dtl.DTLVardef@8606fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations

Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.

biochemistry↗

bioRxiv2020

Decreasing pdzd8-mediated mitochondrial-ER contacts in neurons improves fitness by increasing mitophagy

The complex cellular architecture of neurons combined with their longevity makes maintaining a healthy mitochondrial network particularly important and challenging. One of the many roles of mitochondrial-ER contact sites (MERCs) is to mediate mitochondrial quality control through regulating mitochondrial turn over. Pdzd8 is a newly discovered MERC protein, the organismal functions of which have not yet been explored. Here we identify and provide the first functional characterization of the Drosophila melanogaster ortholog of Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion and reduces lifespan. We also show that depletion of pdzd8 rescues the locomotor defects characterizing an Alzheimers disease (AD) fly model over-expressing Amyloid{beta}1-42 (A{beta}42) and prolongs the survival of flies fed with mitochondrial toxins. Together, our results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

cell biology↗

bioRxiv2020

In vivo and in vitro mechanistic characterization of a clinically relevant PolγA mutation

Mutations in POLG, encoding POL{gamma}A, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generated a POLGA449T/A449T mouse model, which reproduces the most common human recessive mutation of POLG, encoding the A467T change, and dissected the mechanisms underlying pathogenicity. We show that the A449T mutation impairs DNA binding and mtDNA synthesis activities of POL{gamma} in vivo and in vitro. Interestingly, the A467T mutation also strongly impairs interactions with POL{gamma}B, the homodimeric accessory subunit of holo-POL{gamma}. This allows the free POL{gamma}A to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POL{gamma}A, which in turn exacerbates the molecular phenotypes of PolgA449T/A449T mice. Importantly, we validated this mechanism for other mutations affecting the interaction between the two POL{gamma} subunits. We suggest that LONP1 dependent degradation of POL{gamma}A can be exploited as a target for the development of future therapies.

genetics↗