Mitochondrial Maintenance Research Articles

Novel DNM1L variants impair mitochondrial dynamics through divergent mechanisms.

Imbalances in mitochondrial and peroxisomal dynamics are associated with a spectrum of human neurological disorders. Mitochondrial and peroxisomal fission both involve dynamin-related protein 1 (DRP1) oligomerisation and membrane constriction, although the precise biophysical mechanisms by which distinct DRP1 variants affect the assembly and activity of different DRP1 domains remains largely unexplored. We analysed four unreported de novo heterozygous variants in the dynamin-1-like gene DNM1L affecting different highly conserved DRP1 domains, leading to developmental delay, seizures, hypotonia, and/or rare cardiac complications in infancy. Single-nucleotide DRP1 stalk domain variants were found to correlate with more severe clinical phenotypes, with in vitro recombinant human DRP1 mutants demonstrating greater impairments in protein oligomerisation, DRP1-peroxisomal recruitment, and both mitochondrial and peroxisomal hyperfusion compared to GTPase or GTPase-effector domain variants. Importantly, we identified a novel mechanism of pathogenesis, where a p.Arg710Gly variant uncouples DRP1 assembly from assembly-stimulated GTP hydrolysis, providing mechanistic insight into how assembly-state information is transmitted to the GTPase domain. Together, these data reveal that discrete, pathological DNM1L variants impair mitochondrial network maintenance by divergent mechanisms.

A Metabolomic Approach and Traditional Physical Assessments to Compare U22 Soccer Players According to Their Competitive Level.

Simple SummaryDifferent physical tests are applied in the sports context for athlete selection and training prescriptions. Recently, the metabolomics approach has been used to understand biological systems; it has shown high potential to answer integrated questions, including with regard to soccer. Our study is the first to combine traditional physical assessment protocols with metabolomics to detail and compare the physiological profiles of two soccer teams (under-22 soccer players) at different competitive levels. Using anthropometric measurements and the critical velocity model, no differences were observed between elite and non-elite soccer players. However, the metabolomic analysis was able to differentiate among the fasting serum metabolic profiles of athletes of different competitive levels. Our results suggest that the application of metabolomics combined with traditional physical assessment protocols can improve characterizations of athletes, strengthening the differentiation of the physiological profiles of soccer teams. Thus, the metabolomic approach can contribute by offering additional data in individual characterizations of athletes, improving training programs, and consequently, increasing the performance of players during competitions.The purpose of this study was to use traditional physical assessments combined with a metabolomic approach to compare the anthropometric, physical fitness level, and serum fasting metabolic profile among U22 soccer players at different competitive levels. In the experimental design, two teams of male U22 soccer were evaluated (non-elite = 20 athletes, competing in a regional division; elite = 16 athletes, competing in the first division of the national U22 youth league). Earlobe blood samples were collected, and metabolites were extracted after overnight fasting (12 h). Untargeted metabolomics through Liquid Chromatograph Mass Spectrometry (LC-MS) analysis and anthropometric evaluation were performed. Critical velocity was applied to determine aerobic (CV) and anaerobic (ARC) capacity. Height (non-elite = 174.4 ± 7.0 cm; elite = 176.5 ± 7.0 cm), body mass index (non-elite = 22.1 ± 2.4 kg/m2; elite = 21.9 ± 2.3 kg/m2), body mass (non-elite = 67.1 ± 8.8 kg; elite = 68.5 ± 10.1 kg), lean body mass (non-elite = 59.3 ± 7.1 kg; elite = 61.1 ± 7.9 kg), body fat (non-elite = 7.8 ± 2.4 kg; elite = 7.3 ± 2.4 kg), body fat percentage (non-elite = 11.4 ± 2.4%; elite = 10.5 ± 1.7%), hematocrit (non-elite = 50.2 ± 4.0%; elite = 51.0 ± 4.0%), CV (non-elite = 3.1 ± 0.4 m/s; elite = 3.0 ± 0.2 m/s), and ARC (non-elite = 129.6 ± 55.7 m; elite = 161.5 ± 61.0 m) showed no significant differences between the elite and non-elite teams, while the multivariate Partial Least Squares Discriminant Analysis (PLS-DA) model revealed a separation between the elite and non-elite athletes. Nineteen metabolites with importance for projection (VIP) >1.0 were annotated as belonging to the glycerolipid, sterol lipid, fatty acyl, flavonoid, and glycerophospholipid classes. Metabolites with a high relative abundance in the elite group were related in the literature to a better level of aerobic power, greater efficiency in the recovery process, and improvement of mood, immunity, decision making, and accuracy, in addition to acting in mitochondrial preservation and electron transport chain maintenance. In conclusion, although classical physical assessments were not able to distinguish the teams at different competitive levels, the metabolomics approach successfully indicated differences between the fasting metabolic profiles of elite and non-elite teams.

TWNK in Parkinson's Disease: A Movement Disorder and Mitochondrial Disease Center Perspective Study.

BackgroundParkinsonian features have been described in patients harboring variants in nuclear genes encoding for proteins involved in mitochondrial DNA maintenance, such as TWNK.ObjectivesThe aim was to screen for TWNK variants in an Italian cohort of Parkinson's disease (PD) patients and to assess the occurrence of parkinsonism in patients presenting with TWNK‐related autosomal dominant progressive external ophthalmoplegia (TWNK‐adPEO).MethodsGenomic DNA of 263 consecutively collected PD patients who underwent diagnostic genetic testing was analyzed with a targeted custom gene panel including TWNK, as well as genes causative of monogenic PD. Genetic and clinical data of 18 TWNK‐adPEO patients with parkinsonism were retrospectively analyzed.ResultsSix of 263 PD patients (2%), presenting either with isolated PD (n = 4) or in combination with bilateral ptosis (n = 2), carried TWNK likely pathogenic variants. Among 18 TWNK‐adPEO patients, 5 (28%) had parkinsonism.ConclusionsWe show candidate TWNK variants occurring in PD without PEO. This finding will require further confirmatory studies. © 2022 Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Novel TOP3A Variant Associated With Mitochondrial Disease

ObjectivesTopoisomerase III alpha plays a key role in the dissolution of double Holliday junctions and is required for mitochondrial DNA (mtDNA) replication and maintenance. Sequence variants in the TOP3A gene have been associated with the Bloom syndrome–like disorder and described in an adult patient with progressive external ophthalmoplegia. The purpose of this report is to expand the clinical phenotype of the TOP3A-related diseases and clarify the role of this gene in primary mitochondrial disorders.MethodsA 44-year-old woman was referred to our hospital because of exercise intolerance and creatine kinase increase. Muscle biopsy and a targeted next-generation sequencing (NGS) analysis were performed.ResultsA histopathologic assessment documented a mitochondrial myopathy, and a molecular analysis revealed a novel homozygous variant in the TOP3A gene associated with multiple mtDNA deletions.DiscussionThis case suggests that TOP3A is one of the several nuclear genes associated with mtDNA maintenance disorder and expands the spectrum of its associated phenotypes, ranging from a clinical condition defined Bloom syndrome–like disorder to canonical mitochondrial syndromes.

Evidence for mitochondrial Lonp1 expression in the nucleus

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress.

Abstract 2283: SLC25A46 shows potential as a novel genetic regulator in breast cancer development

Abstract SLC25A46 is a part of the mitochondrial solute carrier family (SLC25) and acts as a transporter transferring lipids between the mitochondria and endoplasmic reticulum space. SLC25A46 is expressed in the mitochondrial outer membrane, where it plays a significant role in regulating mitochondrial dynamics, cristae maintenance, and mitochondrial respiratory function. SLC25A46 prevents the formation of hyperfilamentous mitochondria and interacts with novel mitochondrial fusion proteins such as MFN-2 and OPA1 and the cristae organizing complex, MICOS. Notably, research has shown that a first-in-class, small molecule-specific OPA1 inhibitor can be used pharmacologically to curtail tumor growth; however, the OPA1 inhibitor did not explore how the interacting partner, SLC25A46, was affected by such inhibition. Indeed, novel targets are needed for advanced breast cancers as a means of therapeutic interventions. Considering the critical role of SLC25A46, we sought to investigate novel mitochondrial proteins that are understudied in breast cancer. We applied a statistical framework to calculate predicted gene expression for SLC25A46 in 70,349 patients of European descent in BioVU, the Vanderbilt University Medical Center (VUMC) biorepository linked to de-identified electronic health records (EHRs). A genetically predicted gene expression model for SLC25A46 was trained on directly measured RNA-seq data across 17 tissues from the Genotype-Tissue Expression (GTEx) Project. We then fitted a multivariable logistic regression model to test SLC25A46 predicted expression against 1,704 phenotypes for each tissue. We observed that patients with high predicted expression of SLC25A46 were more likely to receive diagnosis of ‘abnormal findings in mammograms’ (P=2.76E-06, OR=2.26) in adipose subcutaneous tissues. Likewise, we found yet another significant association with ‘lump or mass in breast’ (P=8.9E-06, OR=2.61) in adipose subcutaneous tissues. These results pass their individual Bonferroni correction that was applied separately for each tissue, but fell short of passing a strict multiple testing threshold that accounted for all tissues. Given the association between SLC25A46 and abnormal phenotypes that are commonly coded for patients in relation to breast cancer, we conducted a linear regression model with SLC25A46 and ionized calcium and found a modest association (P=0.03, OR=1.02). Elevated serum levels of calcium are frequently observed in advanced breast cancer. Additionally, proteins that interact with SLC25A46 such as OPA1, are overexpressed in breast cancer. We further constructed a polygene score to determine the effects of these genes in all breast cancer phenotypes. Together, these data suggest that SLC25A46 has implications in breast cancer as a novel target in cancer development. Citation Format: Heather K. Beasley, Ky'Era V. Actkins, Tyne Miller-Flemming, Bret C. Mobley, Lea Davis, Antentor O. Hinton, Nancy J. Cox. SLC25A46 shows potential as a novel genetic regulator in breast cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2283.

SQSTM1/p62 in intrahepatic cholangiocarcinoma promotes tumor progression via epithelial-mesenchymal transition and mitochondrial function maintenance.

SQSTM1/p62 is a selective autophagy receptor that regulates multiple signaling pathways participating in the initiation and progression of tumors. Metastasis is still the main cause for intrahepatic cholangiocarcinoma (ICC)-associated mortality. Hence, this study aimed to explore the mechanism of p62 promoting the progression of ICC. Western blotting and immunohistochemical analyses were conducted to detect the expression level of protein p62 in ICC tissues and its correlation with prognosis. Subsequently, the loss-of-function experiments in vitro and in vivo were performed to define the role of p62 in ICC cell proliferation, invasion, and metastasis. Then, the effect of p62 knockdown on mitochondrial function and mitophagy was evaluated by measuring the oxygen consumption rate, and using immunofluorescence and western blotting analyses. The expression of p62 was significantly upregulated in ICC specimens compared with normal tissues. We further illustrated that p62 expression positively correlated with lymph node metastasis and poor prognosis. The loss-of-function assays revealed that p62 not only promoted ICC cell proliferation, migration, and invasive capacities in vitro, but also induced lung metastasis in the xenograft mouse model. Mechanistically, high expression of p62-induced epithelial-mesenchymal transition (EMT) with the upregulation of Snail, vimentin, N-cadherin, and downregulation of E-cadherin. Moreover, the autophagy-dependent function of p62 might play a vital role in maintaining the mitochondrial function of ICC by mitophagy which might further promote EMT. These data provided new evidence for the mechanism by which abundant p62 expression promoted ICC progression, suggesting a promising therapeutic target for antimetastatic strategies in patients with ICC.

Modelling autosomal dominant optic atrophy associated with OPA1 variants in iPSC-derived retinal ganglion cells.

Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterized by the preferential loss of retinal ganglion cells (RGCs), resulting in optic nerve degeneration and progressive bilateral central vision loss. More than 60% of genetically confirmed patients with DOA carry variants in the nuclear OPA1 gene, which encodes for a ubiquitously expressed, mitochondrial GTPase protein. OPA1 has diverse functions within the mitochondrial network, facilitating inner membrane fusion and cristae modelling, regulating mitochondrial DNA maintenance and coordinating mitochondrial bioenergetics. There are currently no licensed disease-modifying therapies for DOA and the disease mechanisms driving RGC degeneration are poorly understood. Here, we describe the generation of isogenic, heterozygous OPA1 null induced pluripotent stem cell (iPSC) (OPA1+/−) through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of a control cell line, in conjunction with the generation of DOA patient-derived iPSC carrying OPA1 variants, namely, the c.2708_2711delTTAG variant (DOA iPSC), and previously reported missense variant iPSC line (c.1334G>A, DOA plus [DOA]+ iPSC) and CRISPR/Cas9 corrected controls. A two-dimensional (2D) differentiation protocol was used to study the effect of OPA1 variants on iPSC-RGC differentiation and mitochondrial function. OPA1+/−, DOA and DOA+ iPSC showed no differentiation deficit compared to control iPSC lines, exhibiting comparable expression of all relevant markers at each stage of differentiation. OPA1+/− and OPA1 variant iPSC-RGCs exhibited impaired mitochondrial homeostasis, with reduced bioenergetic output and compromised mitochondrial DNA maintenance. These data highlight mitochondrial deficits associated with OPA1 dysfunction in human iPSC-RGCs, and establish a platform to study disease mechanisms that contribute to RGC loss in DOA, as well as potential therapeutic interventions.

A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer

The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2•–) -generating drug β-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, β-Lapachone did not generate mitochondrial O2•– but rather SOD2 loss enhanced the efficacy of β-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized cells to β-Lapachone, demonstrating that these effects may be translated to increase ROS sensitivity therapeutically.

Clinical Manifestations and Molecular Backgrounds of Parkinson's Disease Regarding Genes Identified From Familial and Population Studies.

Over the past 20 years, numerous robust analyses have identified over 20 genes related to familial Parkinson's disease (PD), thereby uncovering its molecular underpinnings and giving rise to more sophisticated approaches to investigate its pathogenesis. α-Synuclein is a major component of Lewy bodies (LBs) and behaves in a prion-like manner. The discovery of α-Synuclein enables an in-depth understanding of the pathology behind the generation of LBs and dopaminergic neuronal loss. Understanding the pathophysiological roles of genes identified from PD families is uncovering the molecular mechanisms, such as defects in dopamine biosynthesis and metabolism, excessive oxidative stress, dysfunction of mitochondrial maintenance, and abnormalities in the autophagy–lysosome pathway, involved in PD pathogenesis. This review summarizes the current knowledge on familial PD genes detected by both single-gene analyses obeying the Mendelian inheritance and meta-analyses of genome-wide association studies (GWAS) from genome libraries of PD. Studying the functional role of these genes might potentially elucidate the pathological mechanisms underlying familial PD and sporadic PD and stimulate future investigations to decipher the common pathways between the diseases.

The combination of NRF1 and Nrf2 activators in myoblasts stimulate mechanisms of proteostasis without changes in mitochondrial respiration

Persistent oxidative stress contributes to hallmarks of aging, including impaired proteostasis and mitochondrial dysfunction, while acute oxidative challenges resolved swiftly contribute to beneficial adaptations. Adaptive homeostasis is where acute exposures to sub-toxic stimuli kindle transient expansion of responses necessary to reestablish homeostasis. Elucidating mechanisms underlying adaptive homeostasis will provide novel targets for healthspan extension. Nuclear erythroid-related factor 2 (Nrf2) is a key regulator of cytoprotective gene transcription for redox homeostasis; nuclear respiratory factor 1 (NRF1) is a transcription factor that regulates expression of genes necessary for mitochondrial function. Regulation of both is compromised with advancing age. We hypothesized that NRF1 (NRF1a) and Nrf2 (Nrf2a) activators might improve adaptive homeostasis in C2C12 myoblasts by promoting mitochondrial proteome maintenance and function. Using stable isotope tracing, we assessed protein synthesis over a 16-hr treatment with NRF1a, Nrf2a, or both, with and without a hydrogen peroxide (H 2 O 2 ) stress. We assessed mitochondrial function using high-resolution respirometry. Co-treatment of NRF1a and Nrf2a under H 2 O 2 stress favored proteostatic maintenance ( p <0.05). H 2 O 2 stress decreased mitochondrial respiration and this decrease was not altered by NRF1a/Nrf2a co-treatment. These results suggest that simultaneously targeting Nrf2 and NRF1 may be a viable approach for reestablishing mitochondrial protein homeostasis following a stress, but that this adaptation may not improve respiratory capacity.

Cholesterol accumulation induced by acetylated LDL exposure modifies the enzymatic activities of the TCA cycle without impairing the respiratory chain functionality in macrophages

The unregulated uptake of modified low-density lipoproteins (LDL) by macrophages leads to foam cell formation, promoting atherosclerotic plaque progression. The cholesterol efflux capacity of macrophages by the ATP-Binding Cassette transporters depends on the ATP mitochondrial production. Therefore, the mitochondrial function maintenance is crucial in limiting foam cell formation. Thus, we aimed to investigate the mechanisms involved in the mitochondrial dysfunction that may occur in cholesterol-laden macrophages.We incubated THP-1 macrophages with acetylated LDL (acLDL) to obtain cholesterol-laden cells or with mildly oxidized LDL (oxLDL) to generate cholesterol- and oxidized lipids-laden cells. Cellular cholesterol content was measured in each condition. Mitochondrial function was evaluated by measurement of several markers of energetic metabolism, oxidative phosphorylation, oxidative stress, mitochondrial biogenesis and dynamics.OxLDL-exposed macrophages exhibited a significantly reduced mitochondrial respiration and complexes I and III activities, associated to an oxidative stress state and a reduced mitochondrial DNA copy number. Meanwhile, acLDL-exposed macrophages featured an efficient oxidative phosphorylation despite the decreased activities of aconitase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.Our study revealed that mitochondrial function was differently impacted according to the nature of modified LDL. Exposure to cholesterol and oxidized lipids carried by oxLDL leads to a mitochondrial dysfunction in macrophages, affecting the mitochondrial respiratory chain functional capacity, whereas the cellular cholesterol enrichment induced by acLDL exposure results in a tricarboxylic acid cycle shunt while maintaining mitochondrial energetic production, reflecting a metabolic adaptation to cholesterol intake. These new mechanistic insights are of direct relevance to the understanding of the mitochondrial dysfunction in foam cells.

P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7-/- CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Polymerase ζ Is Involved in Mitochondrial DNA Maintenance Processes in Concert with APE1 Activity.

Mitochondrial DNA (mtDNA) damaged by reactive oxygen species (ROS) triggers so far poorly understood processes of mtDNA maintenance that are coordinated by a complex interplay among DNA repair, DNA degradation, and DNA replication. This study was designed to identify the proteins involved in mtDNA maintenance by applying a special long-range PCR, reflecting mtDNA integrity in the minor arc. A siRNA screening of literature-based candidates was performed under conditions of enforced oxidative phosphorylation revealing the functional group of polymerases and therein polymerase ζ (POLZ) as top hits. Thus, POLZ knockdown caused mtDNA accumulation, which required the activity of the base excision repair (BER) nuclease APE1, and was followed by compensatory mtDNA replication determined by the single-cell mitochondrial in situ hybridization protocol (mTRIP). Quenching reactive oxygen species (ROS) in mitochondria unveiled an additional, ROS-independent involvement of POLZ in the formation of a typical deletion in the minor arc region. Together with data demonstrating the localization of POLZ in mitochondria, we suggest that POLZ plays a significant role in mtDNA turnover, particularly under conditions of oxidative stress.

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction.

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.

The Role of ATF5 in Mitochondrial Maintenance, Biogenesis and UPRmt Signaling Following Acute Exercise in Skeletal Muscle

Maintenance of the mitochondrial protein folding environment is essential for organellar and cellular homeostasis. Over 99% of mitochondrial proteins require import into mitochondria, followed by their folding and intraorganellar sorting. Mitochondrial stress can result in the accretion of misfolded proteins, establishing a requirement for mitochondrial protein quality control (MQC) strategies. The Mitochondrial Unfolded Protein Response (UPRmt) is a compartment‐specific MQC mechanism that increases the expression of protective enzymes by Activating Transcription Factor 5 (ATF5) to restore mitochondrial function. Contractile activity during acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis. However, the roles of ATF5 and the UPRmtin basal mitochondrial maintenance and exercise‐induced UPRmt signaling in skeletal muscle are not known. To investigate this, we subjected WT and whole‐body ATF5 KO mice to a bout of acute exercise and collected skeletal muscle tissues immediately after. ATF5 KO animals exhibited 2‐fold increases in phosphorylated JNK protein levels, indicative of enhanced stress signaling. Interestingly, in KO muscle, PGC‐1a protein was enhanced by 50% and 40% in nuclear and cytosolic compartments, respectively, suggesting an increased drive toward mitochondrial biogenesis in the absence of ATF5. Muscle from these animals also displayed a more abundant, but dysfunctional, mitochondrial pool, with a 20% increase in mitochondrial content, 30‐40% reductions in respiration, and a 20% increase in ROS emissions, corresponding with no changes in exercise performance. The UPRmt proteins mtHSP70 and LONP were upregulated 20‐30% in KO muscle, while ATF4 mRNA was upregulated 2.5‐3.7‐fold, along with an 8% increase in its nuclear localization. Furthermore, KO muscle showed an impaired UPRmt mRNA response to acute exercise, suggesting a regulatory role for ATF5 in the maintenance of a high‐quality mitochondrial pool, and in mediating the transcription of UPRmt genes during exercise.

SUMO 2 the rescue: how SUMO2 regulates the mitochondria via Drp1 modification

Mitochondria are dynamic powerhouses of cells and their fission and fusion must be tightly regulated for normal cell function. Mitochondrial fission is mediated by the GTPase, Drp1, and mis‐regulation of Drp1 leads to mitochondrial hyper‐fragmentation, a known marker of disease. It has been reported that Drp1 is modified by SUMO1 and SUMO2/3, however, the mechanisms of Drp1 regulation by individual SUMO paralogs remain to be fully understood. Here, we have used CRISPR/Cas9 derived SUMO1 and SUMO2 knockout (KO) cell lines, to perform a systematic investigation of paralog‐specific effects on mitochondrial maintenance and function. In contrast to expectations, we observed multiple mitochondrial defects specifically in SUMO2 KO, but not SUMO1 KO, cells. SUMO2 KO cells had reduced mitochondrial activity based on results of MTT assays. By immunofluorescence microscopy, we also observed an increase in mitochondrial fragmentation in SUMO2 KO cells. Paradoxically, increased fragmentation occurred despite reduced levels of Drp1 protein expression and sequestration of Drp1 in large cytosolic foci. Taken together, our findings indicate that SUMO2 plays a non‐redundant, paralog‐specific role in regulating mitochondrial function, in part through effects on Drp1. We anticipate that a more detailed understanding of the molecular effects of SUMO2 on Drp1 may lead to novel therapeutic approaches to treat or prevent ischemic injury, neurodegeneration, and other mitochondrial‐related maladies.

The combination of NRF1 and Nrf2 activators in myoblasts stimulate mechanisms of proteostasis without changes in mitochondrial respiration

Persistent oxidative stress contributes to hallmarks of aging, including impaired proteostasis and mitochondrial dysfunction, while acute oxidative challenges resolved swiftly contribute to beneficial adaptations. Adaptive homeostasis is where acute exposures to sub‐toxic stimuli kindle transient expansion of responses necessary to reestablish homeostasis. Elucidating mechanisms underlying adaptive homeostasis will provide novel targets for healthspan extension. Nrf2 is a key regulator of cytoprotective gene transcription for redox homeostasis; NRF1 is a transcription factor that regulates expression of genes necessary for mitochondrial function. Regulation of both is compromised with advancing age. We hypothesized that NRF1 (NRF1a) and Nrf2 (Nrf2a) activators might improve adaptive homeostasis in response to an oxidative challenge by promoting mitochondrial proteome maintenance and function in C2C12 myoblasts. Using deuterium stable isotope tracing, we assessed protein synthesis over a 16‐hr treatment with a CON (DMSO), NRF1a, Nrf2a, or both, with and without a hydrogen peroxide (H2O2) stress. We assessed mitochondrial function using high‐resolution respirometry. Co‐treatment of NRF1a and Nrf2a under H2O2 stress favored proteostatic maintenance (p&lt;0.05) through maintained protein synthesis (p&gt;0.05), but a decrease in cell proliferation (p&lt;0.05). H2O2 stress significantly decreased mitochondrial maximal respiration, but this decrease was not rescued by NRF1a/Nrf2a co‐treatment indicating the same ATP availability. Interestingly, there were no differences in submaximal ADP stimulated respiration or ADP sensitivity between CON, H2O2, or the NRF1a/Nrf2a co‐treatment (p&gt;0.05). Further, there were no differences in endogenous redox capacity or mitochondrial protein content between treatment groups. These results suggest that simultaneously targeting NRF1 and Nrf2 may be a viable approach for reestablishing mitochondrial protein homeostasis following a stress, but that this adaptation may not improve respiratory capacity.

P2RX7 enhances tumor control by CD8+ T cells in adoptive cell therapy

Abstract Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes generation of memory populations following acute infections and is critical for responses to chronic infection. Despite these findings, recent studies have indicated that P2RX7 may limit the efficacy of anti-tumor responses. We show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T cell adoptive transfer model. Tumor specific P2rx7−/− CD8+ T cells exhibited impaired mitochondrial maintenance and function, but do not display signs of overt exhaustion or poor survival early in the anti-tumor response. However, as tumor burden increases the frequency P2RX7 deficient CD8+ T cells progressively declines relative to WT CD8+ T cells within the tumor. This was associated with worsening mitochondrial function and increased expression of some markers of T cell exhaustion among P2rx7−/− CD8+ T cells. Extending these studies, we found that transient in vitro stimulation of P2RX7 using the ATP analog BzATP led to enhanced B16 tumor control by CD8+ T cells. The improved ability of BzATP stimulated CD8+ T cells to prolong survival of tumor bearing mice was accompanied by enhanced mitochondrial function of tumor specific CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and indicates the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy. Supported by grants F30 award from the NIH (1F30CA250321-01)

Parkin Deficiency Impairs Mitochondrial DNA Dynamics and Propagates Inflammation

Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional mitochondria. New mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins, and recent studies suggest that parkin influences this process at both levels. In addition, parkin was shown to prevent mitochondrial membrane permeability, impeding mitochondrial DNA (mtDNA) escape and subsequent neuroinflammation. However, parkin's regulatory roles independent of mitophagy are not well described in patient-derived neurons. We sought to investigate parkin's role in preventing neuronal mtDNA dyshomeostasis, release, and glial activation at the endogenous level. We generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from PD patients with parkin (PRKN) mutations and healthy controls. Live-cell imaging, proteomic, mtDNA integrity, and gene expression analyses were employed to investigate mitochondrial biogenesis and genome maintenance. To assess neuroinflammation, we performed single-nuclei RNA sequencing in postmortem tissue and quantified interleukin expression in mtDNA/lipopolysaccharides (LPS)-treated iPSC-derived neuron-microglia co-cultures. Neurons from patients with PRKN mutations revealed deficits in the mitochondrial biogenesis pathway, resulting in mtDNA dyshomeostasis. Moreover, the energy sensor sirtuin 1, which controls mitochondrial biogenesis and clearance, was downregulated in parkin-deficient cells. Linking mtDNA disintegration to neuroinflammation, in postmortem midbrain with PRKN mutations, we confirmed mtDNA dyshomeostasis and detected an upregulation of microglia overexpressing proinflammatory cytokines. Finally, parkin-deficient neuron-microglia co-cultures elicited an enhanced immune response when exposed to mtDNA/LPS. Our findings suggest that parkin coregulates mitophagy, mitochondrial biogenesis, and mtDNA maintenance pathways, thereby protecting midbrain neurons from neuroinflammation and degeneration. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.