Altered Mitochondrial Dynamics Research Articles

The overexpression of human amylin in pancreatic β cells facilitate the appearance of amylin aggregates in the kidney contributing to diabetic nephropathy.

Diabetic nephropathy is one of the most frequent complications of diabetic patients and is the leading cause of end-stage renal disease worldwide. The complex physiopathology of this complication raises a challenge in the development of effective medical treatments. Therefore, a better understanding of this disease is necessary for producing more targeted therapies. In this work we propose human amylin as a possible mediator in the development of diabetic nephropathy. Islet amyloid polypeptide or amylin is a hormone co-secreted with insulin. The human isoform has the ability to fold and form amyloid aggregates in the pancreas of patients with type 2 diabetes mellitus, disrupting cellular homeostasis due to its ability to form pores in lipid bilayers. It has been described that hIAPP can be secreted and exported in extracellular vesicles outside the pancreas, being a plausible connecting mechanism between the β-cell and other peripheral tissues such as the kidney. Here, we demonstrate that tubular, podocytes and mesangial cells can incorporate hIAPP coming from β-cells. Then, this hIAPP can form aggregates inside these kidney cells, contributing to its failure. In order to study the consequences in vivo, we found amylin aggregates in the kidney of mice overexpressing hIAPP after feeding a high fat diet. In addition, we observed an increase in glomerulosclerosis index and inflammation. Specifically, there were significant changes in signalling pathways directly involved in the diabetic nephropathy such as an increased in mTORC1 signaling pathway, an alteration in mitochondrial dynamics and an increased in endoplasmic reticulum stress. All these results demonstrate the importance of hIAPP in the kidney and its possible contribution in the development of diabetic nephropathy.

Comprehensive atlas of mitochondrial distribution and dynamics during oocyte maturation in mouse models

BackgroundOocytes, the largest cells in mammals, harbor numerous mitochondria within their cytoplasm. These highly dynamic organelles are crucial for providing energy resources and serving as central regulators during oogenesis. Mitochondrial dynamics ensure proper energy distribution for various cellular processes involved in oocyte maturation. Previous studies have used alterations in mitochondrial distribution as a biomarker to assess the oocyte health. However, there are discrepancies between studies regarding mitochondrial distribution profiles in healthy oocytes. Consequently, a comprehensive mitochondrial distribution profile in oocytes during maturation has not been fully characterized. Additionally, there is a lack of objective, quantitative methods to evaluate alterations in mitochondrial distribution profiles in oocytes.MethodsThis study aims to provide an in-depth overview of mitochondrial distribution profiles in mouse oocytes at different maturation stages: germinal vesicle (GV) stage, metaphase I (MI), and mature metaphase II (MII). Freshly collected mouse GV, MI and MII oocytes were stained with MitoTracker Red. Confocal microscopy was used to obtain images of mitochondrial distribution profiles in these oocytes. Using the Imaris software, we reconstructed three-dimensional (3D) surface renderings of each oocyte and quantitatively illustrated the mitochondrial distribution profiles.ResultsAt the GV stage, mitochondria in oocytes were evenly distributed throughout the ooplasm. As oocytes progressed to MI and MII stages, mitochondria aggregated and formed clusters, the mean size of mitochondrial clusters and the proportions of clustered mitochondria increased along with the maturation of oocytes.ConclusionsOur findings reveal that mitochondria in mouse oocytes are highly dynamic, undergoing significant reorganizations during oocyte maturation. We for the first time provided comprehensive mitochondrial distribution profiles in mouse oocytes at the GV, MI and MII stages. These mitochondrial distribution profiles were further quantitatively evaluated. Our methods provide an objective and standardized approach for evaluating alterations in mitochondrial dynamics, which can be used as biomarkers to monitor oocyte conditions during maturation.

Mitochondrial Dysfunction as a Potential Mechanism Mediating Cardiac Comorbidities in Parkinson’s Disease

Individuals diagnosed with Parkinson’s disease (PD) often exhibit heightened susceptibility to cardiac dysfunction, reflecting a complex interaction between these conditions. The involvement of mitochondrial dysfunction in the development and progression of cardiac dysfunction and PD suggests a plausible commonality in some aspects of their molecular pathogenesis, potentially contributing to the prevalence of cardiac issues in PD. Mitochondria, crucial organelles responsible for energy production and cellular regulation, play important roles in tissues with high energetic demands, such as neurons and cardiac cells. Mitochondrial dysfunction can occur in different and non-mutually exclusive ways; however, some mechanisms include alterations in mitochondrial dynamics, compromised bioenergetics, biogenesis deficits, oxidative stress, impaired mitophagy, and disrupted calcium balance. It is plausible that these factors contribute to the increased prevalence of cardiac dysfunction in PD, suggesting mitochondrial health as a potential target for therapeutic intervention. This review provides an overview of the physiological mechanisms underlying mitochondrial quality control systems. It summarises the diverse roles of mitochondria in brain and heart function, highlighting shared pathways potentially exhibiting dysfunction and driving cardiac comorbidities in PD. By highlighting strategies to mitigate dysfunction associated with mitochondrial impairment in cardiac and neural tissues, our review aims to provide new perspectives on therapeutic approaches.

Drp1 acetylation mediated by CDK5-AMPK-GCN5L1 axis promotes cerebral ischemic injury via facilitating mitochondrial fission

The aberrant acetylation of mitochondrial proteins is involved in the pathogenesis of multiple diseases including neurodegenerative diseases and cerebral ischemic injury. Previous studies have shown that depletion of mitochondrial NAD+, which is necessary for mitochondrial deacetylase activity, leads to decreased activity of mitochondrial deacetylase and thus causes hyperacetylation of mitochondrial proteins in ischemic brain tissues, which results in altered mitochondrial dynamics. However, it remains largely unknown about how mitochondrial dynamics-related protein Drp1 is acetylated in ischemic neuronal cells and brain tissues. Here, we showed that Drp1 and GCN5L1 expression was up-regulated in OGD-treated neuronal cells and ischemic brain tissues induced by dMCAO, accompanied by the increased mitochondrial fission, mtROS accumulation, and cell apoptosis. Further, we confirmed that ischemia/hypoxia promoted Drp1 interaction with GCN5L1 in neuronal cells and brain tissues. GCN5L1 knockdown attenuated, while its overexpression enhanced Drp1 acetylation and mitochondrial fission, indicating that GCN5L1 plays a crucial role in ischemia/hypoxia-induced mitochondrial fission by acetylating Drp1. Mechanistically, ischemia/hypoxia induced Drp1 phosphorylation by CDK5 upregulation-mediated activation of AMPK in neuronal cells, which in turn facilitated the interaction of GCN5L1 with Drp1, thus enhancing Drp1 acetylation and mitochondrial fission. Accordingly, inhibition of AMPK alleviated ischemia/hypoxia- induced Drp1 acetylation and mitochondrial fission and protected brain tissues from ischemic damage. These findings provide a novel insight into the functional roles of GCN5L1 in regulating Drp1 acetylation and identify a previously unrecognized CDK5-AMPK-GCN5L1 pathway that mediates the acetylation of Drp1 in ischemic brain tissues.

A self-assembled layer on titanium surface via silane coupling of chlorogenic acid and strontium chelate improves the osteogenic potential in a high-glucose environment

Bioactive coatings on titanium surfaces represent a crucial strategy to enhance the success rate of titanium implants in diabetic patients. In this study, a chelate comprising chlorogenic acid (CGA) and strontium (Sr) was synthesized and subsequently immobilized onto titanium surface through aminoethyl-aminopropyltrimethoxysilane (AEAPTMS) coupling, wherein AEAPTMS was selected from four silanes based on quantum chemistry analysis. This novel coating was characterized by XPS, SEM, contact angle measurements, and friction experiments, and its effects on cell adhesion, proliferation, osteogenic differentiation, and mitochondrial function in a high-glucose environment were evaluated by comparison with the surface treatments of none, alkali thermal treatment, and physically immersed CGA&Sr solution. It was proven that CGA&Sr could be grafted onto the titanium surface by AEAPTMS coupling, via Si-O bonds and -NH-C = O- bonds at the two ends of AEAPTMS. Furthermore, it provided satisfactory friction resistance and hydrophilicity, as well as improvement in proliferation, adhesion, and osteogenic mineralization of MC3T3-E1 cells in a high-glucose environment. The AEAPTMS-CGA&Sr coating significantly enhanced the osteogenic potential of cells by upregulating the expression levels of key osteogenesis-related genes such as—OCN, OSX, ALP, Runx2, and COL-1 and promoted the secretion of osteogenesis-related proteins. Mitochondrial dynamics analysis showed that CGA&Sr effectively restored hyperglycemia-induced alterations in mitochondrial dynamics and function by modulating the downregulation of mitochondrial fission genes Fis1 and Drp1, upregulating fusion genes Mfn1 and Mfn2, promoting recovery of mitochondrial membrane potential, and eliminating reactive oxygen species. The study results suggested that the AEAPTMS-CGA&Sr coating might be a promising strategy for modifying titanium implants for tolerance to a high-glucose environment.

Characterization of fission and fusion mitochondrial dynamics in HD fibroblasts according to patient's severity status

Huntington's Disease (HD) is an inheritable neurodegenerative condition caused by an expanded CAG trinucleotide repeat in the HTT gene with a direct correlation between CAG repeats expansion and disease severity with earlier onset-of- disease. Previously we have shown that primary skin fibroblasts from HD patients exhibit unique phenotype disease features, including distinct nuclear morphology and perturbed actin cap linked with cell motility, that are correlated with the HD patient disease severity. Here we provide further evidence that mitochondrial fission-fusion morphology balance dynamics, classified using a custom image-based high-content analysis (HCA) machine learning tool, that improved correlation with HD severity status. This mitochondrial phenotype is supported by appropriate changes in fission-fusion biomarkers (Drp1, MFN1, MFN2, VAT1) levels in the HD patients' fibroblasts. These findings collectively point towards a dysregulation in mitochondrial dynamics, where both fission and fusion processes may be disrupted in HD cells compared to healthy controls. This study shows for the first time a methodology that enables identification of HD phenotype before patient's disease onset (Premanifest). Therefore, we believe that this tool holds a potential for improving precision in HD patient's diagnostics bearing the potential to evaluate alterations in mitochondrial dynamics throughout the progression of HD, offering valuable insights into the molecular mechanisms and drug therapy evaluation underlying biological differences in any disease stage.

Functional Food Mixture Extricates D-Galactose-Induced Skeletal Muscle Impairment in Rats.

Aging-related muscle atrophy/sarcopenia is the most common type of muscle impairment that affects the quality of life. In the current study, we examined the effect of a functional food mixture of amla, turmeric, black pepper, cinnamon, and ginger on D-galactose-induced muscle alterations in rats. Wistar rats were randomly divided into three groups: Control (C), D-galactose (G), and D-galactose + functional food mixture intervention (G + I). Rats in group-G and -G + I were injected with D-galactose (300 mg/kg/day) for 90 days. After 3 months of the experimental period, the rats were sacrificed to collect gastrocnemius muscle. Group-G rats showed elevated levels of inflammatory cytokines (TNFα and NF-kB), atrogenes (atrogin-1 and MuRF1), decreased insulin/IGF1 signaling (decreased AKT phosphorylation), altered mitochondrial dynamics (increased fission and decreased fusion proteins), increased apoptotic mediators (Bax/Bcl-2, and caspase-3), and decreased muscle cell cross-sectional area when compared with group-C (p < 0.05). Interestingly, supplementation with the functional food mixture prevented galactose-induced alterations in the muscle. The observed anti-inflammatory, insulin-sensitizing, mitochondria-protective, and antiapoptotic effects of the functional food could be the underlying mechanisms in displaying positive effects against galactose-induced muscle atrophy and, hence, may be useful for the prevention of age-related muscle disorders.

BET inhibitors (BETi) influence oxidative phosphorylation metabolism by affecting mitochondrial dynamics leading to alterations in apoptotic pathways in triple-negative breast cancer (TNBC) cells.

Repressing BET proteins' function using bromodomain inhibitors (BETi) has been shown to elicit antitumor effects by regulating the transcription of genes downstream of BRD4. We previously showed that BETi promoted cell death of triple-negative breast cancer (TNBC) cells. Here, we proved that BETi induce altered mitochondrial dynamics fitness in TNBC cells falling in cell death. We demonstrated that BETi treatment downregulated the expression of BCL-2, and proteins involved in mitochondrial fission and increased fused mitochondria. Impaired mitochondrial fission affected oxidative phosphorylation (OXPHOS) inducing the expression of OXPHOS-related genes, SDHa and ATP5a, and increased cell death. Consistently, the amount of mitochondrial DNA and mitochondrial membrane potential (∆Ψm) increased in BETi-treated cells compared to control cells. Lastly, BETi in combination with Metformin reduced cell growth. Our results indicate that mitochondrial dynamics and OXPHOS metabolism support breast cancer proliferation and represent novel BETi downstream targets in TNBC cells.

Drp1 depletion protects against ferroptotic cell death by preserving mitochondrial integrity and redox homeostasis

Mitochondria are highly dynamic organelles which undergo constant fusion and fission as part of the mitochondrial quality control. In genetic diseases and age-related neurodegenerative disorders, altered mitochondrial fission-fusion dynamics have been linked to impaired mitochondrial quality control, disrupted organelle integrity and function, thereby promoting neural dysfunction and death. The key enzyme regulating mitochondrial fission is the GTPase Dynamin-related Protein 1 (Drp1), which is also considered as a key player in mitochondrial pathways of regulated cell death. In particular, increasing evidence suggests a role for impaired mitochondrial dynamics and integrity in ferroptosis, which is an iron-dependent oxidative cell death pathway with relevance in neurodegeneration. In this study, we demonstrate that CRISPR/Cas9-mediated genetic depletion of Drp1 exerted protective effects against oxidative cell death by ferroptosis through preserved mitochondrial integrity and maintained redox homeostasis. Knockout of Drp1 resulted in mitochondrial elongation, attenuated ferroptosis-mediated impairment of mitochondrial membrane potential, and stabilized iron trafficking and intracellular iron storage. In addition, Drp1 deficiency exerted metabolic effects, with reduced basal and maximal mitochondrial respiration and a metabolic shift towards glycolysis. These metabolic effects further alleviated the mitochondrial contribution to detrimental ROS production thereby significantly enhancing neural cell resilience against ferroptosis. Taken together, this study highlights the key role of Drp1 in mitochondrial pathways of ferroptosis and expose the regulator of mitochondrial dynamics as a potential therapeutic target in neurological diseases involving oxidative dysregulation.

Unraveling mitochondrial dysfunction: comprehensive perspectives on its impact on neurodegenerative diseases.

Neurodegenerative diseases represent a significant challenge to modern medicine, with their complex etiology and progressive nature posing hurdles to effective treatment strategies. Among the various contributing factors, mitochondrial dysfunction has emerged as a pivotal player in the pathogenesis of several neurodegenerative disorders. This review paper provides a comprehensive overview of how mitochondrial impairment contributes to the development of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, driven by bioenergetic defects, biogenesis impairment, alterations inmitochondrial dynamics (such as fusion or fission), disruptions in calcium buffering, lipid metabolism dysregulation and mitophagy dysfunction. It also covers current therapeutic interventions targeting mitochondrial dysfunction in these diseases.

Japanese encephalitis virus infection causes reactive oxygen species-mediated skeletal muscle damage.

Skeletal muscle wasting is a clinically proven pathology associated with Japanese encephalitis virus (JEV) infection; however, underlying factors that govern skeletal muscle damage are yet to be explored. The current study aims to investigate the pathobiology of skeletal muscle damage using a mouse model of JEV infection. Our study reveals a significant increment in viral copy number in skeletal muscle post-JEV infection, which is associated with enhanced skeletal muscle cell death. Molecular and biochemical analysis confirms NOX2-dependent generation of reactive oxygen species, leading to autophagy flux inhibition and cell apoptosis. Along with this, an alteration in mitochondrial dynamics (change in fusion and fission process) and a decrease in the total number of mitochondria copies were found during JEV disease progression. The study represents the initial evidence of skeletal muscle damage caused by JEV and provides insights into potential avenues for therapeutic advancement.

Mitochondria mediated inhibitory effect of Nyctanthes arbor-tristis (L.) flower extract against breast adenocarcinoma and T-cell lymphoma: An in vitro and in vivo study

Ethnopharmacological relevanceThe flowers of Nyctanthes arbor-tristis (L.) heals mouth ulcers. Its tinctures promote gastric secretions, and improve lung expectoration when taken orally. It has traditionally been used to treats scabies and other skin problems. The leaves of NAT(L.) plant are used in Ayurvedic medicine to treat sciatica, chronic fever, rheumatism, internal worm infections, and as a laxative, diaphoretic, and diuretic. The bark used in treatment of snakebite and bronchitis. In addition to traditional uses, pharmacologically this plant has potent antimalarial, antiarthritic, anticancer and antidiabetic activity. However, the mechanistic antiproliferative potentials of NAT(L.) flower as anticancer therapeutics has not yet been explored. Aim of the studyThe current study is based on a broad range of scientific literature that highlights the nutritional and therapeutic benefits of NAT (L.). Present investigation was carried out to determine the therapeutic efficacy of NAT (L.) against breast adenocarcinoma cells and T-cell lymphoma. Materials and methodsThe ethyl-acetate extract of NAT(L.) was tested against breast cancer cells to assess the anticancer potential. To evaluate apoptosis, intracellular ROS levels and mitochondrial dynamics, fluorescence microscopy and flow cytometry were employed. Additionally, cell cycle analysis and western blotting were also performed. Furthermore, in vivo antitumor efficacy of flower extracts was investigated in T-cell lymphoma-bearing BALB/c mice model. ResultsOur present study revealed that NAT (L.) exert anticancer activity against breast cancer cells effectively at IC50 320 μg/ml while having less impact on normal cells with IC50 more than 480 μg/ml. Fluorescence imaging showed that NAT (L.) treatment elicits a concentration-dependent rise in the occurrence of apoptotic cell deaths with altered mitochondrial dynamics and was subsequently confirmed by flow cytometry. Further, flow cytometric analysis delineates ethyl acetate flower extract exposure promotes arrest of cells in S phase of the cell cycle. The differential expression of apoptotic proteins such as Bax, Bcl-2, cleaved PARP-1, cleaved caspase 3, Cytochrome-c, p53 and VEGF A were influenced by NAT (L.) treatment. The in vivo antitumor activity study delineates that NAT(L.) therapy significantly increased the life span of T-cell lymphoma bearing mice while reducing tumor load and belly size growth pattern without causing significant other distinct side effects as evident by histopathological studies. ConclusionOur current findings unveil that NAT(L.) ethyl acetate flower extract potentially induces mitochondrial pathway of apoptosis, promote cell cycle arrest, reduces tumor load of mice, enhances survivability and could be a promising agent against the triple negative breast cancer and lymphoma.

SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a ‘highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19.

Abstract PO4-28-06: A Singleminded-2s/Sirtuin interac4on in the regula4on of mitochondrial dynamics across normal mammary development and ER+ BC progression

Abstract Characterizing the mechanisms underlying the regulation of cellular differentiation and mitochondrial dynamics are critical to understanding normal development and breast cancer in the mammary gland. Our previous studies have highlighted the significance of Singleminded-2s (Sim2s), a member of the bHLH/PAS family, in regulating mitochondrial dynamics during mammary gland development and the progression of estrogen receptor-positive (ER+) breast cancer. Sim2s is temporally regulated, with maximal expression occurring during mid-lactation. Cross-fostered pups nursed by miceover-expressing Sim2s under the mouse mammary tumor virus (MMTV-Sim2s) display significantly higher weights by mid-lactation compared to pups nursed by control dams. Overexpression of Sim2s leads to alterations in mitochondrial morphology and dynamics, characterized by increased OPA1 expression and decreased DRP1 levels, resulting in enhanced mitochondrial fusion and elongation. Furthermore, we have extended our investigations to an ER+ breast cancer cell line (MCF7). We have identified SIM2s as a tumor suppressor expressed in mammary epithelial cells, known to inhibit epithelial-mesenchymal transition (EMT) and metastasis. In our previous studies, we discovered that loss of SIM2s expression in the MCF7 cell line promotes mitochondrial fragmentation, as evidenced by a decrease in OPA1 expression and an increase in DRP1 levels. In conjunction with these findings, literature suggests that sirtuins also play a role in the regulation of mitochondrial dynamics similar to Sim2s. Specifically, there is an upregulation of Sirtuin 1 in cancer when Sim2 is lost, while Sirtuin 3 increases during differentiation and normal development progression. Therefore, our aim is to elucidate the potential interaction between Singleminded-2s and sirtuins, a family of NAD+-dependent deacetylases, in the regulation of mitochondrial dynamics. We hypothesize that Sim2s may modulate mitochondrial function through its association with sirtuins, thereby influencing normal mammary gland development and breast cancer progression. Citation Format: Hannah Carter. A Singleminded-2s/Sirtuin interac4on in the regula4on of mitochondrial dynamics across normal mammary development and ER+ BC progression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-06.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

Abstract 259: Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) patients who experience relapse have a dismal prognosis, with 5-year overall survival rates of 10% for adults and 36% for children. Relapse is attributed to cytotoxic chemotherapy’s inability to target Leukemia Initiating Cells (LICs) that have the potential to re-populate the cancer. There is a need for novel therapeutic strategies to target LIC self-renewal in T-ALL with improved safety profiles and better chances of transition to clinical development. This project utilized a transgenic zebrafish model that accurately recapitulates the most aggressive form of the human T-ALL. This model offered several advantages, including a high frequency of LICs and suitability for drug testing. Over 770 FDA-approved drugs were screened, in vivo using &amp;gt;2,500 syngeneic CG1 zebrafish. Further secondary screening in human T-ALL cells narrowed down the potential hits into 4 compounds that specifically interfere with self-renewal. Finally, the hit drug Amiloride was confirmed by a limiting dilation assay (LDA), resulting in a more than 4-fold decrease in the LIC frequency (p=0.0026). The top hit, Amiloride, which reduced the frequency of LICs in zebrafish and human T-ALL cells, is an inhibitor of the Sodium Hydrogen Exachanger-1 (NHE1). NHE1 has been previously linked to the proliferation, migration, and drug resistance of different cancers. In addition, Amiloride treatment was found to impair mitochondrial function in cancer cells, offering a possible mechanism for the inhibition of LICs. Research has not yet investigated how NHE1, and the altered mitochondrial dynamics related to its inhibition affect the development and persistence of LICs in T-ALL. In our human T-ALL cells, we found that Amiloride significantly reduced mitochondrial basal respiration (p=0.0074), ATP production (p=0.0008) and increased the expression of genes associated with mitochondrial stress such as TFAM, PINK1, PARKIN and MT-CO1. Experiments using shRNA mediated knock down of the NHE1 in human T-ALL cells are underway and will be followed by investigating the impact of NHE1 inhibition on primary patient-derived T-ALL cells in a mouse model. Overall, this project showcases of the power of the zebrafish in cancer research in an efficient and accurate large-scale screen for drugs that impact LIC self-renewal. We have identified Amiloride as a top hit that reduces the frequency of LIC in vivo and in vitro. Finally, we are continuing to investigate the role of the NHE1 in self-renewal in human T-ALL and the associated mitochondrial alterations. Citation Format: Majd Al-Hamaly, Yelena Chernyavskaya, Emma Arvin, Logan Turner, Meghan Haney, Jessica Blackburn. Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells [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 259.

Disturbances in mitochondrial bioenergetics and control quality and unbalanced redox homeostasis in the liver of a mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a lysosomal storage disease caused by mutations in the gene encoding the enzyme iduronate 2-sulfatase (IDS) and biochemically characterized by the accumulation of glycosaminoglycans (GAGs) in different tissues. It is a multisystemic disorder that presents liver abnormalities, the pathophysiology of which is not yet established. In the present study, we evaluated bioenergetics, redox homeostasis, and mitochondrial dynamics in the liver of 6-month-old MPS II mice (IDS-). Our findings show a decrease in the activity of α-ketoglutarate dehydrogenase and an increase in the activities of succinate dehydrogenase and malate dehydrogenase. The activity of mitochondrial complex I was also increased whereas the other complex activities were not affected. In contrast, mitochondrial respiration, membrane potential, ATP production, and calcium retention capacity were not altered. Furthermore, malondialdehyde levels and 2',7'-dichlorofluorescein oxidation were increased in the liver of MPS II mice, indicating lipid peroxidation and increased ROS levels, respectively. Sulfhydryl and reduced glutathione levels, as well as glutathione S-transferase, glutathione peroxidase (GPx), superoxide dismutase, and catalase activities were also increased. Finally, the levels of proteins involved in mitochondrial mass and dynamics were decreased in knockout mice liver. Taken together, these data suggest that alterations in energy metabolism, redox homeostasis, and mitochondrial dynamics can be involved in the pathophysiology of liver abnormalities observed in MPS II.

Role of Mitochondrial Dysfunction in Related Diseases-A Review

Background: Mitochondrial dysfunction (MD) is increasingly recognized for its role in a wide array of diseases, extending beyond primary mitochondrial diseases (PMDs) to include secondary mitochondrial diseases (SMDs) and a broad spectrum of neurodegenerative, cardiovascular, metabolic, and oncological disorders. This growing awareness underscores the need for a comprehensive review of the mechanisms underlying MD, its clinical implications, and emerging therapeutic strategies. Objective: To synthesize current knowledge on the pathogenesis of diseases associated with MD, delineate the distinction between PMDs and SMDs, explore the genetic and environmental factors contributing to MD, and assess the potential of novel therapeutic approaches targeting mitochondrial dysfunction. Methods: A narrative review was conducted through a structured search of PubMed, Scopus, Web of Science, and Google Scholar, focusing on articles published up to April 2023. Keywords related to mitochondrial dysfunction and its implications across various diseases were used to identify relevant studies. Inclusion criteria targeted original research, reviews, and meta-analyses published in English that contributed to understanding MD's role in disease pathogenesis and treatment. Data extraction and synthesis were performed to highlight key findings, mechanisms, and therapeutic strategies. Results: The review emphasizes the critical role of mitochondrial dysfunction in the pathogenesis of a wide range of diseases. It identifies specific mtDNA mutations contributing to PMDs and highlights how SMDs arise from mitochondrial impairment secondary to other conditions. The synthesis of findings points to oxidative stress, impaired ATP production, and altered mitochondrial dynamics as central to MD's impact on cellular and systemic health. Additionally, the review explores emerging therapies, including mitochondrial-targeted antioxidants, gene therapy, and metabolic interventions, underscoring their potential in managing MD-related diseases. Conclusion: Mitochondrial dysfunction is a pivotal factor in the etiology of numerous diseases, with genetic mutations and environmental influences contributing to its development and progression. Understanding these complex mechanisms is essential for devising effective therapeutic interventions. While significant advances have been made, further research is needed to fully exploit the therapeutic potential of targeting mitochondrial dysfunction.

Mitochondrial Dysfunction and Metabolic Reprogramming in Obesity and Asthma.

Mitochondrial dysfunction and metabolic reprogramming have been extensively studied in many disorders ranging from cardiovascular to neurodegenerative disease. Obesity has previously been associated with mitochondrial fragmentation, dysregulated glycolysis, and oxidative phosphorylation, as well as increased reactive oxygen species production. Current treatments focus on reducing cellular stress to restore homeostasis through the use of antioxidants or alterations of mitochondrial dynamics. This review focuses on the role of mitochondrial dysfunction in obesity particularly for those suffering from asthma and examines mitochondrial transfer from mesenchymal stem cells to restore function as a potential therapy. Mitochondrial targeted therapy to restore healthy metabolism may provide a unique approach to alleviate dysregulation in individuals with this unique endotype.

Anthocyanins and their metabolites promote white adipose tissue beiging by regulating mitochondria thermogenesis and dynamics

High-fat diet (HFD) consumption and excess nutrient availability can cause alterations in mitochondrial function and dynamics. We previously showed that anthocyanins (AC) decreased HFD-induced body weight gain and fat deposition. This study investigated: i) the capacity of AC to mitigate HFD-induced alterations in mitochondrial dynamics, biogenesis, and thermogenesis in mouse subcutaneous white adipose tissue (sWAT), and ii) the underlying mechanisms of action of cyanidin-3-O-glucoside (C3G), delphinidin-3-O-glucoside (D3G), and their gut metabolites on mitochondria function/dynamics in 3T3-L1 adipocytes treated with palmitate. Mice were fed control or HFD diets, added or not with 40 mg AC/kg body weight (BW). Compared to control and AC-supplemented mice, HFD-fed mice had fewer sWAT mitochondria that presented alterations of their architecture. AC supplementation prevented HFD-induced decrease of proteins involved in mitochondria biogenesis (PPARγ, PRDM16 and PGC-1α), and thermogenesis (UCP-1), and decreased AMPK phosphorylation. AC supplementation also restored the alterations in sWAT mitochondrial dynamics (Drp-1, OPA1, MNF-2, and Fis-1) and mitophagy (BNIP3L/NIX) caused by HFD consumption. In mature 3T3-L1, C3G, D3G, and their metabolites protocatechuic acid (PCA), 4-hydroxybenzaldehyde (HB), and gallic acid (GA) differentially affected palmitate-mediated decreased cAMP, PKA, AMPK, and SIRT-1 signaling pathways. C3G, D3G, and metabolites also prevented palmitate-mediated decreased expression of PPARγ, PRDM16, PGC-1α, and UCP1. Results suggest that consumption of select AC, i.e. cyanidin and delphinidin, could promote sWAT mitochondriogenesis and improve mitochondria dynamics in the context of HFD/obesity-induced dysmetabolism in part by regulating PKA, AMPK, and SIRT-1 signaling pathways.