Mitochondrial Architecture Research Articles

Reduced bioenergetics and mitochondrial fragmentation in human primary cytotrophoblasts induced by an EGFR-targeting chemical mixture

Exposures to complex environmental chemical mixtures during pregnancy reach and target the feto-placental unit. This study investigates the influence of environmental chemical mixtures on placental bioenergetics. Recognizing the essential role of the epidermal growth factor receptor (EGFR) in placental development and its role in stimulating glycolysis and mitochondrial respiration in trophoblast cells, we explored the effects of chemicals known to disrupt EGFR signaling on cellular energy production. Human primary cytotrophoblasts (hCTBs) and a first-trimester extravillous trophoblast cell line (HTR-8/SVneo) were exposed to a mixture of EGFR-interfering chemicals, including atrazine, bisphenol S, niclosamide, PCB-126, PCB-153, and trans-nonachlor. An RNA sequencing approach revealed that the mixture altered the transcriptional signature of genes involved in cellular energetics. Next, the impact of the mixture on cellular bioenergetics was evaluated using a combination of mitochondrial and glycolytic stress tests, ATP production, glucose consumption, lactate synthesis, and super-resolution imaging. The chemical mixture did not alter basal oxygen consumption but diminished the maximum respiratory capacity in a dose-dependent manner, indicating a disruption of mitochondrial function. The respiratory capacity and ATP production were increased by EGF, while the Chem-Mix reduced both EGF- and non-EGF-mediated oxygen consumption rate in hCTBs. A similar pattern was observed in the glycolytic medium acidification, with EGF increasing the acidification, and the Chem-Mix blocking EGF-induced glycolytic acidification. Furthermore, direct stochastic optical reconstruction microscopy (dSTORM) imaging demonstrated that the Chem-Mix led to a reduction of the mitochondrial network architecture, with findings supported by a decrease in the abundance of OPA1, a mitochondrial membrane GTPase involved in mitochondrial fusion. In conclusion, we demonstrated that a mixture of EGFR-disrupting chemicals alters mitochondrial remodeling, resulting in disturbed cellular bioenergetics, reducing the capacity of human cytotrophoblast cells to generate energy. Future studies should investigate the mechanism by which mitochondrial dynamics are disrupted and the pathological significance of these findings.

Complete mitochondrial genome of the terrestrial isopod Cubaris murina Brandt, 1833: new family gene order and novel tRNA secondary structures

ABSTRACT Among isopods (order Isopoda), species belonging to the suborder Oniscidea are the most successful colonisers of terrestrial habitats and the largest family in this order is Armadillidae. In this study, the complete mitochondrial genome of an armadillid isopod, Cubaris murina, was assembled and characterised in detail for the first time. The gene order and secondary structure of transfer RNA (tRNA) genes were compared among isopods. The 14,212-bp-long mitogenome consists of 13 protein-coding genes, 20 tRNAs, two ribosomal RNAs, and four non-coding regions (NCRs), each longer than 100 bp. Two tRNA genes (trnG and trnI) were missing and several other tRNA genes exhibited secondary structures that departed from the typical cloverleaf pattern. Deletions and nucleotide insertions in the T- and D-arms and mismatches in the acceptor stem were commonly observed in these tRNAs. Notably, the mitogenome of C. murina exhibited a new gene order containing four tRNA rearrangements, and differences in the location of the NCRs were detected when compared to those reported for other isopods, including oniscid species. Interestingly, the second NCR had a similar number of secondary structures and repetitive elements to those reported in the control region of other crustaceans. The novel gene order, tRNA secondary structures, and NCR organisation reported herein for the mitogenome of C. murina confirms the hyper-variability of mitochondrial genomes in isopods. This study is the first to characterise in detail the mitogenome of the invasive isopod C. murina, belonging to the species-rich family Armadillidae within the Isopoda, and advances our understanding of mitochondrial genome architecture in isopods.

Poration of mitochondrial membranes by amyloidogenic peptides and other biological toxins.

Mitochondria are essential organelles known to serve broad functions, including in cellular metabolism, calcium buffering, signaling pathways and the regulation of apoptotic cell death. Maintaining the integrity of the outer (OMM) and inner mitochondrial membranes (IMM) is vital for mitochondrial health. Cardiolipin (CL), a unique dimeric glycerophospholipid, is the signature lipid of energy-converting membranes. It plays a significant role in maintaining mitochondrial architecture and function, stabilizing protein complexes and facilitating efficient oxidative phosphorylation (OXPHOS) whilst regulating cytochrome c release from mitochondria. CL is especially enriched in the IMM and at sites of contact between the OMM and IMM. Disorders of protein misfolding, such as Alzheimer's and Parkinson's diseases, involve amyloidogenic peptides like amyloid-β, tau and α-synuclein, which form metastable toxic oligomeric species that interact with biological membranes. Electrophysiological studies have shown that these oligomers form ion-conducting nanopores in membranes mimicking the IMM's phospholipid composition. Poration of mitochondrial membranes disrupts the ionic balance, causing osmotic swelling, loss of the voltage potential across the IMM, release of pro-apoptogenic factors, and leads to cell death. The interaction between CL and amyloid oligomers appears to favour their membrane insertion and pore formation, directly implicating CL in amyloid toxicity. Additionally, pore formation in mitochondrial membranes is not limited to amyloid proteins and peptides; other biological peptides, as diverse as the pro-apoptotic Bcl-2 family members, gasdermin proteins, cobra venom cardiotoxins and bacterial pathogenic toxins, have all been described to punch holes in mitochondria, contributing to cell death processes. Collectively, these findings underscore the vulnerability of mitochondria and the involvement of CL in various pathogenic mechanisms, emphasizing the need for further research on targeting CL-amyloid interactions to mitigate mitochondrial dysfunction.

The crossroad between mitochondrial architecture and metabolism

The crossroad between mitochondrial architecture and metabolism

Exploring Mitochondrial Heterogeneity and Evolutionary Dynamics in Thelephora ganbajun through Population Genomics.

Limited exploration in fungal mitochondrial genetics has uncovered diverse inheritance modes. The mitochondrial genomes are inherited uniparentally in the majority of sexual eukaryotes, our discovery of persistent mitochondrial heterogeneity within the natural population of the basidiomycete fungus Thelephora ganbajun represents a significant advance in understanding mitochondrial inheritance and evolution in eukaryotes. Here, we present a comprehensive analysis by sequencing and assembling the complete mitogenomes of 40 samples exhibiting diverse cox1 heterogeneity patterns from various geographical origins. Additionally, we identified heterogeneous variants in the nad5 gene, which, similar to cox1, displayed variability across multiple copies. Notably, our study reveals a distinct prevalence of introns and homing endonucleases in these heterogeneous genes. Furthermore, we detected potential instances of horizontal gene transfer involving homing endonucleases. Population genomic analyses underscore regional variations in mitochondrial genome composition among natural samples exhibiting heterogeneity. Thus, polymorphisms in heterogeneous genes, introns, and homing endonucleases significantly influence mitochondrial structure, structural variation, and evolutionary dynamics in this species. This study contributes valuable insights into mitochondrial genome architecture, population dynamics, and the evolutionary implications of mitochondrial heterogeneity in sexual eukaryotes.

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes.

The liver, the largest internal organ and a metabolic hub, undergoes significant declines due to aging, affecting mitochondrial function and increasing the risk of systemic liver diseases. How the mitochondrial three-dimensional (3D) structure changes in the liver across aging, and the biological mechanisms regulating such changes confers remain unclear. In this study, we employed Serial Block Face-Scanning Electron Microscopy (SBF-SEM) to achieve high-resolution 3D reconstructions of murine liver mitochondria to observe diverse phenotypes and structural alterations that occur with age, marked by a reduction in size and complexity. We also show concomitant metabolomic and lipidomic changes in aged samples. Aged human samples reflected altered disease risk. To find potential regulators of this change, we examined the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex, which plays a crucial role in maintaining mitochondrial architecture. We observe that the MICOS complex is lost during aging, but not Sam50. Sam50 is a component of the sorting and assembly machinery (SAM) complex that acts in tandem with the MICOS complex to modulate cristae morphology. In murine models subjected to a high-fat diet, there is a marked depletion of the mitochondrial protein SAM50. This reduction in Sam50 expression may heighten the susceptibility to liver disease, as our human biobank studies corroborate that Sam50 plays a genetically regulated role in the predisposition to multiple liver diseases. We further show that changes in mitochondrial calcium dysregulation and oxidative stress accompany the disruption of the MICOS complex. Together, we establish that a decrease in mitochondrial complexity and dysregulated metabolism occur with murine liver aging. While these changes are partially be regulated by age-related loss of the MICOS complex, the confluence of a murine high-fat diet can also cause loss of Sam50, which contributes to liver diseases. In summary, our study reveals potential regulators that affect age-related changes in mitochondrial structure and metabolism, which can be targeted in future therapeutic techniques.

The Whole Mitochondrial Genome Sequence of Dendrobium loddigesii Rolfe, an Endangered Orchid Species in China, Reveals a Complex Multi-Chromosome Structure.

Dendrobium loddigesii is a precious traditional Chinese medicine with high medicinal and ornamental value. However, the characterization of its mitochondrial genome is still pending. Here, we assembled the complete mitochondrial genome of D. loddigesii and discovered that its genome possessed a complex multi-chromosome structure. The mitogenome of D. loddigesii consisted of 17 circular subgenomes, ranging in size from 16,323 bp to 56,781 bp. The total length of the mitogenome was 513,356 bp, with a GC content of 43.41%. The mitogenome contained 70 genes, comprising 36 protein-coding genes (PCGs), 31 tRNA genes, and 3 rRNA genes. Furthermore, we detected 403 repeat sequences as well as identified 482 RNA-editing sites and 8154 codons across all PCGs. Following the sequence similarity analysis, 27 fragments exhibiting homology to both the mitogenome and chloroplast genome were discovered, accounting for 9.86% mitogenome of D. loddigesii. Synteny analysis revealed numerous sequence rearrangements in D. loddigesii and the mitogenomes of related species. Phylogenetic analysis strongly supported that D. loddigesii and D. Amplum formed a single clade with 100% bootstrap support. The outcomes will significantly augment the orchid mitochondrial genome database, offering profound insights into Dendrobium's intricate mitochondrial genome architecture.

Mitochondrial Elongation and ROS-Mediated Apoptosis in Prostate Cancer Cells under Therapy with Apalutamide and Complex I Inhibitor.

Metabolic reprogramming and mitochondrial dynamics are pivotal in prostate cancer (PCa) progression and treatment resistance, making them essential targets for therapeutic intervention. In this study, we investigated the effects of the androgen receptor antagonist apalutamide (ARN) and the mitochondrial electron transport chain complex I inhibitor IACS-010759 (IACS) on the mitochondrial network architecture and dynamics in PCa cells. Treatment with ARN and/or IACS induced significant changes in mitochondrial morphology, particularly elongation, in androgen-sensitive PCa cells. Additionally, ARN and IACS modulated the mitochondrial fission and fusion processes, indicating a convergence of metabolic and androgen-signaling pathways in shaping mitochondrial function. Notably, the combination treatment with ARN and IACS resulted in increased apoptotic cell death and mitochondrial oxidative stress selectively in the androgen-sensitive PCa cells. Our findings highlight the therapeutic potential of targeting mitochondrial metabolism in prostate cancer and emphasize the need for further mechanistic understanding to optimize treatment strategies and improve patient outcomes.

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes.

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2.

Regulation of mitochondrial network architecture and function in mesenchymal stem cells by micropatterned surfaces

Abstract Mitochondrial network architecture, which is closely related to mitochondrial function, is mechanically sensitive and regulated by multiple stimuli. However, the effects of microtopographic cues on mitochondria remain poorly defined. Herein, polycaprolactone (PCL) surfaces were used as models to investigate how micropatterns regulate mitochondrial network architecture and function in rat adipose-derived stem cells (rASCs). It was found that large pit (LP) induced rASCs to form larger and more complex mitochondrial networks. Consistently, the expression of key genes related to mitochondrial dynamics revealed that mitochondrial fusion (MFN1 and MFN2) and midzone fission (DRP1 and MFF) were increased in rASCs on LP. In contrast, the middle pit (MP) enhanced mitochondrial biogenesis, as evidenced by the larger mitochondrial area and higher expression of PGC-1. Both LP and MP promoted ATP production in rASCs. It is likely that LP increased ATP levels through modulating mitochondrial network architecture while MP stimulated mitochondria biogenesis to do so. Our study clarified the regulation of micropatterned surfaces on mitochondria, highlighting the potential of LP and MP as a simple platform to stimulate mitochondria and the subsequent cellular function of MSCs.

Comparative analysis of mitochondrial genomes in Ceratocystis fimbriata complex across diverse hosts

The decline ofAcacia mangiumWilld. in Malaysia, especially in Sabah since 2010, is primarily due to Ceratocystiswilt and canker disease (CWCD) caused by theCeratocystis fimbriataEllis & Halst. complex. This study was aimed to investigate the mitochondrial genome architecture of two differentC. fimbriatacomplex isolates from Malaysia: one fromA. mangiumin Pahang (FRIM1162) and another fromEucalyptus pellitain Sarawak (FRIM1441). This research employed Next-Generation Sequencing (NGS) to contrast genomes from diverse hosts with nine additional mitochondrial sequences, identifying significant genetic diversity and mutational hotspots in the mitochondrial genome alignment. The mitochondrial genome-based phylogenetic analysis revealed a significant genetic relationship between the studied isolates and theC. fimbriatacomplex in the South American Subclade, indicating that theC. fimbriatacomplex discovered in Malaysia isC. manginecans. The comparative mitochondrial genome demonstrates the adaptability of the complex due to mobile genetic components and genomic rearrangements in the studiedfungal isolates. This research enhances our knowledge of the genetic diversity and evolutionary patterns within theC. fimbriatacomplex, aiding in a deeper understanding of fungal disease development and host adaption processes. The acquired insights are crucial for creating specific management strategies for CWCD, improving the overall understanding of fungal disease evolution and control.

A dynamin superfamily-like pseudoenzyme coordinates with MICOS to promote cristae architecture

Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial contact site and cristae organizing system (MICOS) complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae-shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS to promote normal mitochondrial morphology and respiratory function. Mmc1 is a distant relative of the dynamin superfamily of proteins (DSPs), GTPases, which are well established to shape and remodel membranes. Similar to DSPs, Mmc1 self-associates and forms high-molecular-weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting that it does not dynamically remodel membranes. These data are consistent with the model that Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.

Sleeve Gastrectomy Reduces Oxidative Stress and Reverses Mitochondrial Dysfunction Associated with Metabolic Syndrome.

Previous studies have detected mitochondrial alterations in tissues of individuals with obesity and type 2 diabetes mellitus (T2DM). Metabolic surgery could be an effective treatment to improve mitochondrial morphology and reduce oxidative stress (OS). An experimental study was carried out using 48 male Wistar rats, divided into 6 groups (n = 8): control (C), induced Metabolic Syndrome (MS); intervention with sleeve gastrectomy (SG), MS + SG with 6weeks postoperatively (MS + SG6), MS + SG with 12weeks postoperatively (MS + SG12), and MS + SG with 24weeks postoperatively (MS + SG24). Biochemical markers indicative of MS (glycemia, cholesterol, and triglyceride levels) and oxidative stress markers (nitric oxide levels, Superoxide dismutase and Myeloperoxidase activity) were determined. To study mitochondrial morphology, tissue sections of the thoracic aorta, stomach, liver, heart, and kidney were observed by electron microscopy. MS group exhibited elevated glycemic values and dyslipidemia. SG and MS + SG groups showed improvements in glycemia and lipid profiles compared to MS. OS biomarkers indicated reduced oxidative stress in SG and MS + SG groups compared to MS. Electron microscopy revealed mitochondrial alterations in MS. SG group showed no changes compared to the control. MS + SG6 and MS + SG12 groups showed a recovery of mitochondrial morphology until reaching images similar to the control in MS + SG24. Metabolic surgery could improve mitochondrial function by restoring mitochondrial morphology and architecture and, consequently, reducing systemic oxidative stress and remitting associated metabolic alterations.

Complete mitochondrial genome of Angelica dahurica and its implications on evolutionary analysis of complex mitochondrial genome architecture in Apiaceae.

Angelica dahurica is a kind of Chinese traditional herbs with economic and ornament value, widely distributed in China. Despite its significance, there have been limited comprehensive investigations on the genome of A. dahurica, particularly regarding mitochondrial genomes. To investigate the conversion between mitochondrial genome and chloroplast genome, a complete and circular mitochondrial genome was assembled using Oxford Nanopore Technologies (ONT) long reads. The mitochondrial genome of A. dahurica had a length of 228,315 base pairs (bp) with 45.06% GC content. The mitochondrial genome encodes 56 genes, including 34 protein-coding genes, 19 tRNA genes and 3 rRNA genes. Moreover, we discovered that 9 homologous large fragments between chloroplast genome and mitochondrial genome based on sequence similarity. This is the first report for A. dahurica mitochondrial genome, which could provide an insight for communication between plastid genome, and also give a reference genome for medicinal plants within the Angelica family.

Abstract SY35-02: Spatial mapping of mitochondrial networks in lung cancer

Abstract Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional profiling of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform comprised of positron emission tomography (PET) imaging, respirometry and 3-dimensional scanning block-face electron microscopy (3D SBEM). Interestingly, the diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumors align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumor cells. In tumors with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. In contrast, we discovered that in tumors with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated i) peri-nuclear localization of mitochondria, ii) structural remodeling of cristae and iii) mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumors. Structure-and-function studies defining the relationship between mitochondrial architecture and metabolic dependencies may hold promise as strategies to profile metabolic liabilities unique to lung cancer subtypes. We anticipate that coupling PET imaging with 3D SBEM will have dynamic applications beyond that of lung cancer and enrich our understanding of how mitochondrial bioenergetics impact human disease. Citation Format: David B. Shackelford. Spatial mapping of mitochondrial networks in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY35-02.

Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Abstract 383: Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment

Abstract Background: Mitophagy is a pivotal cellular process crucial for maintaining homeostasis in hypoxic conditions, selectively eliminating dysfunctional mitochondria through autophagy. This process is implicated in various diseases, including cancer. Non-Small Cell Lung Cancer (NSCLC), known for its prevalence and aggressiveness, often encounters hypoxic microenvironments. Mic19, a core protein of the mitochondrial cristae membrane, plays vital roles in maintaining mitochondrial architecture and function. However, its involvement in mitophagy in hypoxic NSCLC and the underlying molecular mechanisms remain inadequately explored. Methods: NSCLC cells were cultured under conditions with 1% O2 oxygen concentration to simulate a hypoxic microenvironment. Protein expression levels of Mic19 were analyzed using Western Blot and immunofluorescence (IF). Cellular proliferation and metastasis capability were assessed through CCK8 and transwell experiments. Immunoprecipitation experiments were conducted to validate the interaction between Mic19 and key mitophagy regulatory proteins. IF, confocal microscopy, ATP measurement, oxygen consumption rate (OCR), mitochondrial membrane potential, and transmission electron microscopy were employed to assess the formation of mitochondrial cristae and the level of mitophagy. Results: In hypoxic conditions, Mic19 exhibited up-regulation in NSCLC cells. Subsequent in vitro experiments confirmed Mic19's pivotal role in promoting the growth and metastasis of lung cancer cell lines. Through clinical data analysis, Mic19 emerged as an independent prognostic indicator for NSCLC. Inhibition of Mic19 expression exacerbated the disruption of mitochondrial morphology and structure in lung cancer cells, accompanied by the loss of mitochondrial cristae. Notably, Mic19 expression closely correlated with ATP and OCR production in tumor cells. Assessment of mitochondrial membrane potential and transmission electron microscopy revealed Mic19 as a critical downstream target of hypoxia, mediating mitophagy. Overall, these findings not only underscore the significance of Mic19 in regulating mitochondrial dynamics but also highlight its multifaceted role in tumor progression, positioning Mic19 as a promising therapeutic target and prognostic biomarker for NSCLC. Conclusion: This study provides novel insights into the mechanistic understanding of hypoxia-induced mitophagy in NSCLC, emphasizing the regulatory role of Mic19. Mic19 emerges as a potential therapeutic target for future interventions in NSCLC patients. Citation Format: Hua Huang, Chen Ding, Zhanrui Zhang, Di Wu, Chen Chen, Yongwen Li, Hongyu Liu, Jun Chen. Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 383.

Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

Targeting of mitochondrial fission through natural flavanones elicits anti-myeloma activity.

Mitochondrial alterations, often dependent on unbalanced mitochondrial dynamics, feature in the pathobiology of human cancers, including multiple myeloma (MM). Flavanones are natural flavonoids endowed with mitochondrial targeting activities. Herein, we investigated the capability of Hesperetin (Hes) and Naringenin (Nar), two aglycones of Hesperidin and Naringin flavanone glycosides, to selectively target Drp1, a pivotal regulator of mitochondrial dynamics, prompting anti-MM activity. Molecular docking analyses were performed on the crystallographic structure of Dynamin-1-like protein (Drp1), using Hes and Nar molecular structures. Cell viability and apoptosis were assessed in MM cell lines, or in co-culture systems with primary bone marrow stromal cells, using Cell Titer Glo and Annexin V-7AAD staining, respectively; clonogenicity was determined using methylcellulose colony assays. Transcriptomic analyses were carried out using the Ion AmpliSeq™ platform; mRNA and protein expression levels were determined by quantitative RT-PCR and western blotting, respectively. Mitochondrial architecture was assessed by transmission electron microscopy. Real time measurement of oxygen consumption was performed by high resolution respirometry in living cells. In vivo anti-tumor activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. Hes and Nar were found to accommodate within the GTPase binding site of Drp1, and to inhibit Drp1 expression and activity, leading to hyperfused mitochondria with reduced OXPHOS. In vitro, Hes and Nar reduced MM clonogenicity and viability, even in the presence of patient-derived bone marrow stromal cells, triggering ER stress and apoptosis. Interestingly, Hes and Nar rewired MM cell metabolism through the down-regulation of master transcriptional activators (SREBF-1, c-MYC) of lipogenesis genes. An extract of Tacle, a Citrus variety rich in Hesperidin and Naringin, was capable to recapitulate the phenotypic and molecular perturbations of each flavanone, triggering anti-MM activity in vivo. Hes and Nar inhibit proliferation, rewire the metabolism and induce apoptosis of MM cells via antagonism of the mitochondrial fission driver Drp1. These results provide a framework for the development of natural anti-MM therapeutics targeting aberrant mitochondrial dependencies.

Impact of Mitochondrial Architecture, Function, Redox Homeostasis, and Quality Control on Organismic Aging: Lessons from a Fungal Model System.

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.