Mitochondrial Cristae Morphology Research Articles

Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models

BackgroundRetinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR).MethodsAn in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted.ResultsAPI treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission.ConclusionAPI protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

Differential Mitochondrial, Oxidative Stress and Inflammatory Responses to SARS-CoV-2 Spike Protein Receptor Binding Domain in Human Lung Microvascular, Coronary Artery Endothelial and Bronchial Epithelial Cells.

Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1β in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD.

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue (Adv. Biology 1/2024)

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue (Adv. Biology 1/2024)

Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients.

Proteinuria is a common and important clinical manifestation of chronic kidney disease (CKD) and an independent risk factor for the progression of kidney disease. As a component of the glomerular filtration barrier (GFB), podocyte plays a key role in the pathogenesis of glomerular diseases and proteinuria. However, the pathophysiology of glomerular diseases associated with mitochondrial function is incompletely understood. Here, we identified three novel mutations in MTX2, encoding a membrane protein in mitochondria, associated with multisystem manifestations including nephrotic proteinuria and kidney injury in two Chinese patients. Conditional podocyte-specific Mtx2 knockout (Pod-Mtx2-KO) mice present a series of podocyte and glomerular abnormalities from 8 weeks to old age, including microalbuminuria, glomerular mesangial hyperplasia, fusion and effacement of foot process. MTX2 deficiency impaired podocyte functions in vitro, manifested by reductions of adhesion, migration and endocytosis, which were further restored by overexpression of MTX2. Moreover, MTX2 defects led to abnormal mitochondrial structure and dysfunction, evidenced with defects of complex I and III, increased production of reactive oxygen species (ROS), and decreased protein levels of Sam50-CHCHD3-Mitofilin axis in the mitochondrial intermembrane space bridging (MIB) complex which is responsible for maintaining mitochondrial cristae morphology. Collectively, these findings reveal that the normal expression of MTX2 in glomerulus plays an important role in the adhesion, migration, endocytosis, proliferation and other physiological functions of podocytes, which may be realized by maintaining the morphological structure and function of mitochondria. Abnormal expression of MTX2 can lead to mitochondrial dysfunction and structural abnormalities by Sam50-CHCHD3-Mitofilin axis in podocyte, which further induces podocyte injury, glomerular lesions and proteinuria.

Lasmiditan restores mitochondrial quality control mechanisms and accelerates renal recovery after ischemia–reperfusion injury

BackgroundMitochondrial dysfunction is a well-established result of acute kidney injury (AKI). Previously, we identified that 5-hydroxytryptamine 1F (5-HT1F) receptor agonism with lasmiditan induces mitochondrial biogenesis (MB) and improves renal vasculature and function in an AKI mouse model. We hypothesize that lasmiditan also modulates mitochondrial dynamics and mitophagy in a mouse model of AKI. MethodsMale mice were subjected to renal ischemia/reperfusion (I/R) and treated daily with lasmiditan (0.3 mg/kg) or vehicle beginning 24 h after injury for 3 or 6d. Serum creatinine was measured to estimate glomerular filtration. Electron microscopy was used to assess mitochondrial morphology and mitophagy. Mitochondrial-related protein were confirmed with immunoblotting. Mitochondrial function was assessed with ATP measurements. ResultsLasmiditan treatment improved mitochondrial and kidney recovery as early as 3d post-AKI, as evidenced by increased ATP, and decreased serum creatinine, respectively. Electron micrographs of renal cortices revealed that lasmiditan also decreased mitochondrial damage and increased mitochondrial area and size by 6d after I/R injury. Additionally, lasmiditan treatment increased mitolysosomes by 3d, indicating induction of mitophagy. Phosphorylation of mitophagy-related proteins were also increased in the renal cortices of lasmiditan-treated AKI mice 3d after I/R injury, whereas fusion-related proteins were increased at 6d after I/R injury. ConclusionThese data reveal that lasmiditan accelerates renal recovery, restores normal mitochondrial membrane and cristae morphology, decreases excessive mitochondrial fission, and accelerates mitophagy post-AKI in a time-dependent manner, establishing mitochondrial function and recovery from AKI.

Shenqi Dihuang Decoction inhibits high-glucose induced ferroptosis of renal tubular epithelial cells via Nrf2/HO-1/GPX4 pathway

This study aims to explore the effects of Shenqi Dihuang Decoction on high-glucose induced ferroptosis and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4) axis in human renal tubular epithelial cells(HK-2) and to clarify the underlying mechanism. The cell injury model was established by exposing HK-2 to high glucose, and the Shenqi Dihuang Decoction-medicated serum was prepared. The optimal concentration and intervention time of Shenqi Dihuang Decoction were determined. HK-2 were divided into normal, high glucose, and low-, medium-, and high-dose Shenqi Dihuang Decoction groups. After interventions, the cell proliferation rate in each group was determined and the cell morphology and mitochondrial ultrastructure were observed. Then, the levels of intracellular reactive oxygen species(ROS), ferrous ion(Fe~(2+)), glutathione(GSH), and malondialdehyde(MDA) and the protein levels of Nrf2, HO-1, GPX4, and xCT were measured. The optimal concentration and intervention time of Shenqi Dihuang Decoction-medicated serum were determined to be 10% and 24 h, respectively. Compared with the high glucose group, high-dose Shenqi Dihuang Decoction promoted the proliferation of HK-2. The cells in the low-, medium-, and high-dose Shenqi Dihuang Decoction groups presented tight arrangement, an increased cell count, improved morphology from a spindle-fiber shape to a cobblestone shape, and improved morphology and structure of mitochondrial membrane and cristae, compared with those in the high glucose group. Meanwhile, all the doses of Shenqi Dihuang Decoction inhibited ROS elevation to mitigate the peroxidation damage, lowered the Fe~(2+) and MDA levels and elevated the GSH level to inhibit lipid peroxidation, and activated the antioxidant pathway to upregulate the protein levels of Nrf2, HO-1, xCT, and GPX4. In conclusion, Shenqi Dihuang Decoction-medicated serum can inhibit high-glucose induced ferroptosis of HK-2 in vitro, which involves the antioxidant effect and the activation of the Nrf2/HO-1/GPX4 pathway.

A X-linked nonsense APOO/MIC26 variant causes a lethal mitochondrial disease with progeria-like phenotypes.

APOO/MIC26 is a subunit of the MICOS complex required for mitochondrial cristae morphology and function. Here, we report a novel variant of the APOO/MIC26 gene that causes a severe mitochondrial disease with overall progeria-like phenotypes in two patients. Both patients developed partial agenesis of the corpus callosum, bilateral congenital cataract, hypothyroidism, and severe immune deficiencies. The patients died at an early age of 12 or 18 months. Exome sequencing revealed a mutation (NM_024122.5): c.532G>T (p.E178*) in the APOO/MIC26 gene that causes a nonsense mutation leading to the loss of 20 C-terminal amino acids. This mutation resulted in a highly unstable and degradation prone MIC26 protein, yet the remaining minute amounts of mutant MIC26 correctly localized to mitochondria and interacted physically with other MICOS subunits. MIC26 KO cells expressing MIC26 harboring the respective APOO/MIC26 mutation showed mitochondria with perturbed cristae architecture and fragmented morphology resembling MIC26 KO cells. We conclude that the novel mutation found in the APOO/MIC26 gene is a loss-of-function mutation impairing mitochondrial morphology and cristae morphogenesis.

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue.

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, it is hypothesized that significant morphological changes in BAT mitochondria and cristae will be present with aging. A quantitative 3D electron microscopy approach is developed to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, the 3D morphology of mitochondrial cristae is investigated in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, an increase is found in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging.

ATAD3A: A Key Regulator of Mitochondria-Associated Diseases.

Mitochondrial membrane protein ATAD3A is a member of the AAA-domain-containing ATPases superfamily. It is important for the maintenance of mitochondrial DNA, structure, and function. In recent years, an increasing number of ATAD3A mutations have been identified in patients with neurological symptoms. Many of these mutations disrupt mitochondrial structure, function, and dynamics and are lethal to patients at a young age. Here, we summarize the current understanding of the relationship between ATAD3A and mitochondria, including the interaction of ATAD3A with mitochondrial DNA and mitochondrial/ER proteins, the regulation of ATAD3A in cholesterol mitochondrial trafficking, and the effect of known ATAD3A mutations on mitochondrial function. In the current review, we revealed that the oligomerization and interaction of ATAD3A with other mitochondrial/ER proteins are vital for its various functions. Despite affecting different domains of the protein, nearly all documented mutations observed in ATAD3A exhibit either loss-of-function or dominant-negative effects, potentially leading to disruption in the dimerization of ATAD3A; autophagy; mitophagy; alteration in mitochondrial number, size, and cristae morphology; and diminished activity of mitochondrial respiratory chain complexes I, IV, and V. These findings imply that ATAD3A plays a critical role in mitochondrial dynamics, which can be readily perturbed by ATAD3A mutation variants.

Mitochondrial dynamics and cristae shape changes during metabolic reprogramming.

The architecture of the mitochondrial network and cristae critically impact cell differentiation and identity. Cells undergoing metabolic reprogramming to aerobic glycolysis (Warburg effect), such as immune cells, stem cells, and cancer cells, go through controlled modifications in mitochondrial architecture, which is critical for achieving the resulting cellular phenotype. Recent studies in immunometabolism have shown that the manipulation of mitochondrial network dynamics and cristae shape directly affects T cell phenotype and macrophage polarization through altering energy metabolism. Similar manipulations also alter the specific metabolic phenotypes that accompany somatic reprogramming, stem cell differentiation, and cancer cells. The modulation of OXPHOS activity, accompanied by changes in metabolite signaling, ROS generation, and ATP levels is the shared underlying mechanism. The plasticity of mitochondrial architecture is particularly vital for metabolic reprogramming. Consequently, failure to adapt the appropriate mitochondrial morphology often compromises the differentiation and identity of the cell. Immune, stem, and tumor cells exhibit striking similarities in their coordination of mitochondrial morphology with metabolic pathways. However, although many general unifying principles can be observed, their validity is not absolute, and the mechanistic links thus need to be further explored. Better knowledge of the molecular mechanisms involved and their relationships to both mitochondrial network and cristae morphology will not only further deepen our understanding of energy metabolism but may also contribute to improved therapeutic manipulation of cell viability, differentiation, proliferation, and identity in many different cell types.

Mitochondrial Cristae Morphology Reflecting Metabolism, Superoxide Formation, Redox Homeostasis, and Pathology.

Significance: Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Recent Advances: Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. Critical Issues: The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. Future Directions: To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer via the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases.

Maturases and Group II Introns in the Mitochondrial Genomes of the Deepest Jakobid Branch.

Ophirinina is a recently described suborder of jakobid protists (Excavata) with only one described species to date, Ophirina amphinema. Despite the acquisition and analysis of massive transcriptomic and mitogenomic sequence data from O. amphinema, its phylogenetic position among excavates remained inconclusive, branching as sister group either to all Jakobida or to all Discoba. From a morphological perspective, it has not only several typical jakobid features but also unusual traits for this group, including the morphology of mitochondrial cristae (sac-shaped to flattened-curved cristae) and the presence of two flagellar vanes. In this study, we have isolated, morphologically characterized, and sequenced genome and transcriptome data of two new Ophirinina species: Ophirina chinija sp. nov. and Agogonia voluta gen. et sp. nov. Ophirina chinija differs from O. amphinema in having rounded cell ends, subapically emerging flagella and a posterior cell protrusion. The much more distantly related A. voluta has several unique ultrastructural characteristics, including sac-shaped mitochondrial cristae and a complex "B" fiber. Phylogenomic analyses with a large conserved-marker dataset supported the monophyly of Ophirina and Agogonia within the Ophirinina and, more importantly, resolved the conflicting position of ophirinids as the sister clade to all other jakobids. The characterization of the mitochondrial genomes showed that Agogonia differs from all known gene-rich jakobid mitogenomes by the presence of two group II introns and their corresponding maturase protein genes. A phylogenetic analysis of the diversity of known maturases confirmed that the Agogonia proteins are highly divergent from each other and define distant families among the prokaryotic and eukaryotic maturases. This opens the intriguing possibility that, compared to other jakobids, Ophirinina may have retained additional mitochondrial elements that may help to understand the early diversification of eukaryotes and the evolution of mitochondria.

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ̊C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1∆ cells grown on synthetic media at both 30 ̊C and 37 ̊C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1∆gpd1∆ double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.

Super-resolution imaging of mitochondrial cristae using a more hydrophobic far-red Si-rhodamine probe.

Mitochondrial probe SiRPFA was synthesized by attaching a long perfluoroalkyl chain on Si-rhodamine cationic dye. High lipophilicity endowed SiRPFA with mitochondrial membrane potential independent properties. Under stimulated emission depletion microscopy, SiRPFA clearly revealed changes in mitochondrial cristae morphology during autophagy induced by starvation or apoptosis.

Cardiac sympathetic innervation is an unrecognized disease target in autosomal dominant optic atrophy

The Optic Atrophy-1 protein (Opa1) controls mitochondrial (mt) fusion and cristae morphology, ATP production, maintenance of mtDNA, and apoptosis. Mutations in Opa1 gene cause Autosomal Dominant Optic Atrophy (ADOA), a neurodegenerative disease characterized by “retinal ganglion cell” death and visual loss. Recently, ADOA has been described as a “multi-systemic disease” also featuring myopathy, peripheral neuropathy, Parkinsonism and cardiac rhythm alterations. Sympathetic neurons (SNs) are the main extrinsic regulators of heart activity in basal and stress conditions, and their homeostasis strikingly depend on the correct mitochondrial dynamics. We here determined the effect of ADOA genetics on cardiac sympathetic innervation. The majority of Opa1 gene mutations result in truncated forms of the protein, leading to reduction in its cellular content. To study the impact of Opa1 haploinsufficiency on cardiac innervation, we generated transgenic mice with SN-specific Opa1 deletion (TH-Opa1+/− mice), which, in heterozygosity, causes 50% reduction of protein expression. Heart function of TH-Opa1+/− mice was assessed by ECHO and ECG telemetry. Cardiac innervation was evaluated by confocal IF, and whole-mount IF in tissue clarified heart blocks. In vitro assays achieved insights on the effects of Opa1 haploinsufficiency on SNs. SNs were also evaluated in skin biopsies from ADOA patients, and age- and sex-matched controls. We here found that SN density is significantly reduced in the skin of ADOA patients, indicating thatSNs are additional, previously neglected, targets of ADOA. To isolate the effects of Opa1 haploinsufficiency in SNs, we analyzed hearts of adult and aged TH-Opa1+/− mice, which showed intact morphology and contractile function. Looking at cardiac innervation, confocal IF and 3-D reconstruction of the SN network revealed a significant reduction in innervation density, which appeared evident already in adult mice, and worsened during ageing. Consistently, TH-Opa1+/− mice showed decreased Heart Rate Variability and increased incidence of arrhythmic beats. In vitro assays demonstrated the causal role of Opa1 haploinsufficiency in affecting SNs, by compromising TrkA/NGF signaling, which is key to sustain neuronal viability. Our data demonstrates that Opa1 haploinsufficiency also affects peripheral autonomic innervation, thus explaining extra-retinal symptoms experienced by ADOA patients.

CD84 Is a Therapeutically Targetable Driver of Leukemogenesis Via Disruption of Energy Supply in Acute Myeloid Leukemia

CD84 is a member of the signaling lymphocyte activation molecule (SLAM) family; it forms homophilic dimers by self-association. CD84 is reported as an important survival receptor in chronic lymphocytic leukemia and its activation upregulates PD-L1 expression on leukemia cells. We and others recently reported that CD84 is highly expressed on myeloid derived suppressor cells, but its function in myeloid malignancies has yet to be investigated. Using publicly available microarray data (GSE13509), we found that CD84 is overexpressed on acute myeloid leukemia (AML) cells compared to normal hematopoietic counterparts (AML, n=542 vs. normal, n=73, p<0.0001). and that its upregulation is associated with poor prognosis in AML patients (GSE10358; p=0.0105). Knocking down CD84 expression in AML cell lines (THP1 and HEL) by shRNAs caused a substantial inhibition of cell viability and extended THP1-luciferase xenografted mice survival in vivo (median survival: shCtrl, 38 days vs shCD84, 53 days, p=0.0015, n=5). Moreover, we observed that CD84 depletion in AML primary patient cells dramatically induced cell apoptosis (shCtrl: 13.0±1.84% vs shCD84: 29.33±1.3%; n=3; p=0.02) and inhibited colony formation (shCtrl: 68.44±11.65 vs shCD84: 15.56±6.32; n=3; p=0.01). In light of highly expressed CD84 in MLL-r AML and inv(16) AML, we employed MLL-AF9 and CBFB-MYH11(CM) AML mouse models, and found that CD84 knockdown robustly dampened cell growth and decreased colony-forming units in vitro. In both models, bone marrow (BM) transplantation shown that CD84 deficiency significantly decreased leukemic engraftment in BM (MLL-AF9: shCtrl: 95.92±0.61% vs shCD84: 76.42±4.11%, p=0.0016, n=5/group; inv(16): shCtrl: 39.02±2.98% vs shCD84: 13.66±1.24%, p<0.0001, n=5/group), spleen, and peripheral blood (PB). In AML patient derived xenografts (PDX), we observed AML burden was markedly reduced in BM (shCtrl:51.22±10.29% vs shCD84: 2.304±0.16%; n=5/group, p=0.0014) and spleen (shCtrl: 8.790±2.989% vs shCD84: 0.8420±0.13%; n=5/group, p=0.0255) in the CD84 knockdown group. Mechanistically, CD84 deletion depressed mTOR signaling, induced mitochondrial stress, and disrupted energy supply chains based on RNA-seq analysis. We demonstrated CD84 deletion caused mitochondrial dysfunction as indicated by decreasing oxidation phosphorylation (shCtrl vs shCD84, p<0.0001) and fatty acid oxidation (shCtrl vs shCD84, p=0.0004). Next, we determined that CD84 deletion caused disruption of mitochondrial matrix morphology and loss of mitochondrial cristae in AML. We also observed a dramatic loss of mitochondrial membrane potential upon CD84 deletion. We generated a novel murine-human chimeric anti-human CD84 monoclonal antibody (mAb) with high affinity and specificity. In the presence of NK cells, anti-CD84 mAb induced strong antibody-dependent cell-mediated cytotoxicity in CD84-expressing AML lines (n=2) and primary samples (n=2), but not in healthy donor CD34+ cells (n=2). Next, the luciferase-expressing THP1 was transplanted into NSG mice. Mice with comparable tumor burden were randomized into 2 groups: control human IgG or anti-CD84 mAb (5mg/kg, i.v, bid, n=6/group), every week together with 3x106 human PB mononuclear cells (PBMCs) as effector cells (for 4 weeks). Anti-CD84 mAb significantly suppressed leukemia development and extended median survival (IgG: 30 days vs anti-CD84 mAb: undefined, p=0.0018). However, without PBMCs, human IgG or anti-CD84 mAb did not induce significant differences in tumor burden or median survival (n=5/group; p=0.5082). Finally, we developed two AML PDXs in NSG mice. Following engraftment >1% in PB, mice were divided into 2 groups (n=6/group). We maintained the same treatment strategy as applied above. In both AML PDXs, the BM engraftment of CD45+CD33+ cells markedly decreased in anti-CD84 mAb treated mice (AML #1: IgG: 94.25± 0.41% vs anti-CD84 mAb: 71.52±3.96%, p= 0.0002; AML #2: IgG: 88.74±0.7% vs anti-CD84 mAb: 53.98±5.61%, p=0.0003). Anti-CD84mAb treatments in secondary transplantation of BM harvested from the first treatment groups resulted in further significant reduction in BM engraftment (IgG: 86.48± 0.67% vs anti-CD84 mAb: 29.74±6.02%, n=5, p<0.0001), and extended survival (IgG, 28 days vs anti-CD84 mAbs, 36 days, p=0.0026, n=5) following third BM transplantation. The results indicated our pharmacological targeting of CD84 inhibits leukemogenesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of Danzhi Xiaoyao Powder on behavior and mitochondrial morphology and function of anxiety model rats

Danzhi Xiaoyao Powder is a classical prescription for anxiety. This study aims to analyze the effect of this medicine on mitochondrial morphology and function of anxiety rats and explore the mechanism of it against anxiety. Specifically, uncertain empty bottle drinking water stimulation(21 days) was employed to induce anxiety in rats. The elevated plus-maze test and open field test were respectively performed on the 7 th, the 14 th, and the 21 st days of the stimulation, so as to detect the anxiety-related protein index brain-derived neurotrophic factor(BDNF) and evaluate the anxiety level of animals. On this basis, the effect of this prescription on anxiety rats was preliminarily evaluated. After the behavioral test on the 21 st day, rats were killed and the brain tissues were separated for the observation of the mitochondrial morphology and the determination of mitochondrial function-related indicators and the adenosine 5'-monophosphate-activated protein kinase(AMPK) level. The results showed that Danzhi Xiaoxiao Powder could alleviate the anxiety-like behavior of rats, significantly increase the percentage of time in open arm in elevated plus-maze test and the ration of activity time in the central area of the field, dose-dependently raise the activity levels of respiratory chain complex Ⅰ,Ⅱ,Ⅲ and Ⅳ and the adenosine triphosphate(ATP) content, and elevate the levels of BDNF and phosphorylated AMPK(p-AMPK). Clear structure and intact morphology of mitochondrial cristae in medial prefrontal cortex cells and amygdala were observed in the Danzhi Xiaoyao Powder group. In summary, Danzhi Xiaoyao Powder exerts therapeutic effect on anxiety, and the mechanism is the likelihood that p-AMPK protects the structure and maintains the function of mitochondria.

Abstract P3088: The Histone Lysine Methyltransferase SMYD1a Protects Heart From Ischemic Injury By Regulating OPA1 Mediated Cristae Remodeling And Supercomplex Formation

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of Smyd1 in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including Ppargc1a , and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to &gt;50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of Ppargc1a and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.

Mitofilin downregulation/Loss favors the initiation and development of dilated cardiomyopathy that progresses to acute heart failure and cardiac arrest

RationalHeart failure (HF) is a clinical syndrome characterized by a constellation of symptoms that are caused by a structural and/or functional cardiac abnormality, ultimately resulting in reduced cardiac output and/or elevated intracardiac pressures. Despite the fact that many cardiovascular diseases end in HF, the condition too often fails to attract the awareness and emphasis it deserves. Worryingly, the mechanisms leading to HF are still not clearly defined. Therefore, research to decipher mechanisms of cardiac decompensation in HF with reduced ejection fraction (HFrEF) and to identify new treatments is urgently needed.ObjectiveThe objective of this study is to determine the role of the mitochondrial inner membrane protein (Mitofilin/Mic60), which plays a crucial role in maintaining mitochondrial cristae morphology, downregulation and loss in the onset and progression of HF and in increasing the prevalence of sudden death in the mouse.Material and methodsWe developed a Mitofilin‐floxed mouse model and crossed this mouse with a transgenic mouse that expresses Tamoxifen inducible Myh6‐Cre recombinase to generate a cardiomyocyte‐specific Mitofilin KO (cMitofilin‐/‐) mouse. Kaplan Meier Curve was used to determine the rate of mice survival, mitochondrial integrity was measured by analyzing cristae morphology and ROS production was assessed using amplex red method. The calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore opening was measured using calcium green dye. Cardiac function was measured by echocardiography and Mic60 mRNA assessed by qRT‐PCR.ResultsWe found an dramatic increase in sudden cardiac death in cMitofilin‐/‐ mice within 5 days post Tamoxifen treatment. Mitofilinfl+/fl++Cre(+) mice treated TAM (cMitofilin‐/‐) displayed significantly increase in cardiac size and reduction of cardiac function indicative of acute dilated cardiomyopathy (DCM) compared to vehicle. cMitofilin‐/‐mice showed mitochondrial cristae disruption and dysfunction including reduced mitochondrial CRC and increased ROS production compared to WT. We observed that full‐body Mitofilin‐/‐ mice do not survive after multiple crossing of full‐body heterozygote Mitofilin+/‐ mice, suggesting that a single allele expression of Mitofilin is sufficient to rescue the DCM phenotype. To determine the role of Mitofilin downregulation in DCM, we compared Mitofilin levels in WT mice treated with Doxorubicin (DOX) versus vehicle. DOX is a highly effective anticancer drug that causes acute ventricular dysfunction, and may induce late‐onset cardiomyopathy and HF. We found a dramatic reduction of Mitofilin in DOX‐treated animals that exhibited a higher death rate versus controls; this finding was associated with an increase in mitochondrial oxidative stress and dysfunction. Furthermore, mice treated with DOX displayed a decrease in Mitofilin mRNA, suggesting reduced Mitofilin expression during HF is at least in part mediated by Mitofilin transcriptional dysregulation. Interestingly, Mitofilin overexpression in H9c2 cardiomyocytes prevented DOX‐induced increase in cell survival.Conclusion. Together, these results indicate that Mitofilin downregulation/loss induces the onset and progression of DCM that is associated with HF and cardiac arrest. Our results provide a better understanding of mitochondrial‐dependent mechanism of DCM and represent a novel potential therapeutic target against DCM and HF.

From the Structural and (Dys)Function of ATP Synthase to Deficiency in Age-Related Diseases.

The ATP synthase is a mitochondrial inner membrane complex whose function is essential for cell bioenergy, being responsible for the conversion of ADP into ATP and playing a role in mitochondrial cristae morphology organization. The enzyme is composed of 18 protein subunits, 16 nuclear DNA (nDNA) encoded and two mitochondrial DNA (mtDNA) encoded, organized in two domains, FO and F1. Pathogenetic variants in genes encoding structural subunits or assembly factors are responsible for fatal human diseases. Emerging evidence also underlines the role of ATP-synthase in neurodegenerative diseases as Parkinson’s, Alzheimer’s, and motor neuron diseases such as Amyotrophic Lateral Sclerosis. Post-translational modification, epigenetic modulation of ATP gene expression and protein level, and the mechanism of mitochondrial transition pore have been deemed responsible for neuronal cell death in vivo and in vitro models for neurodegenerative diseases. In this review, we will explore ATP synthase assembly and function in physiological and pathological conditions by referring to the recent cryo-EM studies and by exploring human disease models.