Tubular Mitochondria Research Articles

Trehalose enhances mitochondria deficits in human NPC1 mutant fibroblasts but disrupts mouse Purkinje cell dendritic growth ex vivo.

Lysosomes play important roles in catabolism, nutrient sensing, metabolic signaling, and homeostasis. NPC1 deficiency disrupts lysosomal function by inducing cholesterol accumulation that leads to early neurodegeneration in Niemann-Pick type C (NPC) disease. Mitochondria pathology and deficits in NPC1 deficient cells are associated with impaired lysosomal proteolysis and metabolic signaling. It is thought that activation of the transcription factor TFEB, an inducer of lysosome biogenesis, restores lysosomal-autophagy activity in lysosomal storage disorders. Here, we investigated the effect of trehalose, a TFEB activator, in the mitochondria pathology of NPC1 mutant fibroblasts in vitro and in mouse developmental Purkinje cells ex vivo. We found that in NPC1 mutant fibroblasts, serum starvation or/and trehalose treatment, both activators of TFEB, reversed mitochondria fragmentation to a more tubular mitochondrion. Trehalose treatment also decreased the accumulation of Filipin+ cholesterol in NPC1 mutant fibroblasts. However, trehalose treatment in cerebellar organotypic slices (COSCs) from wild-type and Npc1nmf164 mice caused mitochondria fragmentation and lack of dendritic growth and degeneration in developmental Purkinje cells. Our data suggest, that although trehalose successfully restores mitochondria length and decreases cholesterol accumulation in NPC1 mutant fibroblasts, in COSCs, Purkinje cells mitochondria and dendritic growth are negatively affected possibly through the overactivation of the TFEB-lysosomal-autophagy pathway.

Crucial physicochemical factors mediating mitochondrial toxicity of nanoparticles at noncytotoxic concentration

Nanomaterials have been extensively applied in multiple industries, among which silver nanoparticles (AgNPs), silicon dioxide nanoparticles (SiNPs), and gold nanoparticles (AuNPs) have become representative of widely consumed NPs. Limited knowledge is available regarding the subcellular responses of NPs with different physicochemical properties, i.e. material type and size, under the noncytotoxic concentrations. Macrophages are important sensitive cells exposed to NPs, and mitochondria are sensitive organelles that respond at the subcellular level. Herein, we found that sublethal concentrations of AgNPs and SiNPs, not AuNPs, decreased the mitochondrial membrane potential (MMP) and tubular mitochondria, and further resulted in an increase of ROS level and a decrease of ATP generation. AgNPs and SiNPs can also disturb mitochondrial dynamics manifested as increasing Mfn2 expression and decreasing Drp1 expression. Further assessments for mitochondrial function showed that AgNPs and SiNPs exposure led to a decrease in the gene expressions related to complex I (Ndufa8 and Ndufs2), complex III (Uqcrc2 and Uqcrfs1), complex IV (Cox6b1), and activity of complex I, suggesting their potential roles in impairing cellular respiration. In terms of the effects of NPs with different sizes, stronger toxicity was observed in smaller-sized nanoparticles. Among the above mitochondrial changes, we identified that ROS, ATP, MMP, tubular mitochondria, and expression of Drp1 were relatively sensitive indicators in subcellular response to NPs. With the above sensitive indicators, the comparison of heterogeneity between material type and size of the NPs showed that material type occupied a main influence on subcellular mitochondrial effects. Our finding provided important data on the potential subcellular risks of NPs, and indicated the vital role of material type for a better understanding of the nanomaterial biological safety.

Red blood cell trapping in the kidney vasculature promotes mitochondrial dysfunction independent of ischemia time

Red blood cell (RBC) trapping presents as the expansion of- and obstruction, of the outer-medullary capillaries of the kidney with tightly packed RBCs. RBC trapping has been thought to promote hypoxic tubular injury by extending ischemia time to the outer-medulla. Recent evidence from our laboratory; however, indicates that RBC trapping may promote toxic tubular injury, secondary to the mitochondrial uptake of iron from hemoglobin released from damaged RBCs in nearby congested capillaries. In the current study, we tested the hypothesis that ‘RBC trapping promotes tubular mitochondrial dysfunction independent of ischemia’. We tested our hypothesis using 9–10-week-old male Wistar-Kyoto rats. In an initial study, rats underwent bilateral warm arterial ischemia for 45 minutes with 2 hours of reperfusion before harvesting the kidneys for analysis by transmission electron microscopy (n=3). In a second study, the left kidney from each of n=10 rats underwent either arterial, venous or sham control clamping for 45 minutes. An alternate procedure (either arterial, venous or sham clamping) was performed on the right kidney. To examine the effect of ischemia alone on mitochondrial function, renal arteries were clamped, and the kidney excised without releasing the clamp. To examine the effect of ischemia with RBC trapping on mitochondrial function, the renal vein was clamped. Renal venous clamping mimics the obstruction of the medullary venous vasculature that occurs during reperfusion from arterial clamping, resulting in both kidney ischemia and RBC trapping. In all animals body temperature was maintained using a servo-controlled heating table and heat lamp. At harvest the kidney medulla and cortex separated and mitochondria isolated from each region. Mitochondrial membrane potential (TMRM fluorescence) and hydrogen peroxide production (Amplex red) was then determined from n=6-7 kidneys for each intervention. Data were compared using ANOVA. Consistent with iron uptake into mitochondria, when viewed using transmission electron microscopy, following 2 hours of reperfusion from arterial clamping, tubular mitochondria near RBC congested vessels often appeared darkened. Both ischemia alone and ischemia with RBC trapping, resulted in a significant increase in mitochondrial H 2 0 2 production compared to control (7063±562 and 7519±803 vs 5688±389 A.U per mg of protein, respectively, p<0.001). In contrast, ischemia alone resulted in a lower mitochondrial membrane potential (228±18 A.U) compared to control (253±18), whereas, mitochondrial membrane potential following ischemia with RBC trapping (308±26) was significantly greater than both ischemia alone and control (p<0.001). Our data supports the hypothesis that congestion of the kidney vasculature with RBCs directly promotes mitochondrial dysfunction independent of ischemia. This likely occurs secondary to mitochondrial uptake of iron from hemoglobin released from damaged RBCs. NIH NIDDK ISAC 21AU4231, NIH NHLBI P01 HL134604 and an American Heart Association Transformational Project Award 970585 to P.O and NIH NIDDK F31 DK127683 to S.R.M. NIH R01 HL148114 D.I. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High-throughput single-molecule localization microscopy: Potential clinical applications.

High-throughput single-molecule localization microscopy: Potential clinical applications.

Erythrose inhibits the progression to invasiveness and reverts drug resistance of cancer stem cells of glioblastoma

Glioblastoma (GBM) is the most frequent brain cancer and more lethal than other cancers. Characteristics of this cancer are its high drug resistance, high recurrence rate and invasiveness. Invasiveness in GBM is related to overexpression of matrix metalloproteinases (MMPs) which are mediated by wnt/β-catenin and induced by the activation of signaling pathways extracellularly activated by the cytokine neuroleukin (NLK) in cancer stem cells (CSC). Therefore, in this work we evaluated the effect of the tetrose saccharide, erythrose (Ery), a NLK inhibitor of invasiveness and drug sensitization in glioblastoma stem cells (GSC). GSC were obtained from parental U373 cell line by a CSC phenotype enrichment protocol based on microenvironmental stress conditions such as hypoxia, hipoglycemia, drug exposition and serum starvation. Enriched fraction of GSC overexpressed the typical markers of brain CSC: low CD133+ and high CD44; in addition, epithelial to mesenchyme transition (EMT) markers and MMPs were increased several times in GSC vs. U373 correlating with higher invasiveness, elongated and tubular mitochondrion and temozolomide (TMZ) resistance. IC50 of Ery was found at nM concentration and at 24h induced a severe diminution of EMT markers, MMPs and invasiveness in GSC. Furthermore, the phosphorylation pattern of NLK after Ery exposition also was affected. In addition, when Ery was administered to GSC at subIC50, it was capable of reverting TMZ resistance at concentrations innocuous to non-tumor cancer cells. Moreover, Ery added daily induced the death of all GSC. Those findings indicated that the phytodrug Ery could be used as adjuvant therapy in GBM.

Mitochondria-localized lncRNA HITT inhibits fusion by attenuating formation of mitofusin-2 homotypic or heterotypic complexes

Long noncoding RNAs (lncRNAs) are emerging as essential players in multiple biological processes. Mitochondrial dynamics, comprising the continuous cycle of fission and fusion, are required for healthy mitochondria that function properly. Despite long-term recognition of its significance in cell-fate control, the mechanism underlying mitochondrial fusion is not completely understood, particularly regarding the involvement of lncRNAs. Here, we show that the lncRNA HITT (HIF-1α inhibitor at translation level) can specifically localize in mitochondria. Cells expressing higher levels of HITT contain fragmented mitochondria. Conversely, we show that HITT knockdown cells have more tubular mitochondria than is present in control cells. Mechanistically, we demonstrate HITT directly binds mitofusin-2 (MFN2), a core component that mediates mitochondrial outer membrane fusion, by the invitro RNA pull-down and UV-cross-linking RNA-IP assays. In doing so, we found HITT disturbs MFN2 homotypic or heterotypic complex formation, attenuating mitochondrial fusion. Under stress conditions, such as ultraviolet radiation, we in addition show HITT stability increases as a consequence of MiR-205 downregulation, inhibiting MFN2-mediated fusion and leading to apoptosis. Overall, our data provide significant insights into the roles of organelle (mitochondria)-specific resident lncRNAs in regulating mitochondrial fusion and also reveal how such a mechanism controls cellular sensitivity to UV radiation-induced apoptosis.

Targeting mitochondrial dynamics to overcome therapeutic resistance

Mitochondria are frequently described as the powerhouse of the cell for apparent reasons. However, these organelles are dynamic was not known until recently. Scientists have found that mitochondria must undergo well-organized cycles of fragmentation/fission and fusion to maintain structural integrity, size, and distribution. These fission and fusion events are collectively called “mitochondrial dynamics” and are considered crucial for regulating organelle function. Mitochondrial fission accounts for the division of one mitochondrion into two. It is regulated by GTPase dynamin-related protein 1 (DRP1) and its adaptor proteins such as mitochondrial fission protein 1 (FIS1), mitochondrial fission factor (MFF), and mitochondrial dynamics protein of 49 and 51 kDa (Mid49, Mid51). DRP1, a cytosolic protein, is recruited to mitochondria to cause fragmentation upon activation through upregulation of serine 616 and downregulation of serine 637 phosphorylation. In contrast, mitochondrial fusion involves the fusion of two separate small mitochondria into one large mitochondrion, thereby generating a network of elongated or tubular mitochondria. These fusion events are regulated by GTPase dynamin-like proteins located on the outer (Mitofusin 1, MFN1 and mitofusin 2, MFN2) and inner (optic atrophy protein 1, OPA1) mitochondrial membrane. Fission is generally coupled with apoptosis, while fusion is associated with pro-survival signals. However, cancer cells can utilize mitochondrial dynamics, depending on their cellular state; this is reflected in the current conflicting literature explaining mitochondrial fission or fusion influencing tumor progression. Nonetheless, alterations in mitochondrial dynamics have been implicated as one of the key factors in tumor progression and therapeutic resistance across a wide spectrum of cancers. As a result, targeting mitochondrial dynamics is emerging as a potential strategy for solid tumors.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

Dynamic changes in mitochondrial 3D structure during folliculogenesis and luteal formation in the goat large luteal cell lineage

In mammalian ovaries, mitochondria are integral sites of energy production and steroidogenesis. While shifts in cellular activities and steroidogenesis are well characterized during the differentiation of large luteal cells in folliculogenesis and luteal formation, mitochondrial dynamics during this process have not been previously evaluated. In this study, we collected ovaries containing primordial follicles, mature follicles, corpus hemorrhagicum, or corpus luteum from goats at specific times in the estrous cycle. Enzyme histochemistry, ultrastructural observations, and 3D structural analysis of serial sections of mitochondria revealed that branched mitochondrial networks were predominant in follicles, while spherical and tubular mitochondria were typical in large luteal cells. Furthermore, the average mitochondrial diameter and volume increased from folliculogenesis to luteal formation. In primordial follicles, the signals of cytochrome c oxidase and ATP synthase were undetectable in most cells, and the large luteal cells from the corpus hemorrhagicum also showed low enzyme signals and content when compared with granulosa cells in mature follicles or large luteal cells from the corpus luteum. Our findings suggest that the mitochondrial enlargement could be an event during folliculogenesis and luteal formation, while the modulation of mitochondrial morphology and respiratory enzyme expressions may be related to tissue remodeling during luteal formation.

Tubular mitochondrial AKT1 is activated during ischemia reperfusion injury and has a critical role in predisposition to chronic kidney disease

Kidney tubular dysfunction contributes to acute kidney injury and to the transition to chronic kidney disease. Although tubular mitochondria have been implicated in the pathophysiology of kidney failure, the mechanisms are not yet clear. Here, we demonstrated that ischemia-reperfusion injury induced acute translocation and activation of mitochondrial protein kinase B (also known as AKT1) in the kidney tubules. We hypothesized that mitochondrial AKT1 signaling protects against the development of acute kidney injury and subsequent chronic kidney disease. To test this prediction, we generated two novel kidney tubule-specific transgenic mouse strains with inducible expression of mitochondria-targeted dominant negative AKT1 or constitutively active AKT1, using a Cre-Lox strategy. Inhibition of mitochondrial AKT1 in mitochondria-targeted dominant negative AKT1 mice aggravated azotemia, tubular injuries, kidney fibrosis, glomerulosclerosis, and negatively impacted survival after ischemia-reperfusion injury. Conversely, enhancing tubular mitochondrial AKT1 signaling in mitochondria-targeted constitutively active AKT1 mice attenuated kidney injuries, protected kidney function, and significantly improved survival after ischemia-reperfusion injury (76.9% vs. 20.8%, respectively). Uncoupled mitochondrial respiration and increased oxidative stress was found in the kidney tubules when mitochondria AKT1 was inhibited, supporting the role of mitochondrial dysfunction in the pathophysiology of kidney failure. Thus, our studies suggest tubular mitochondrial AKT1 signaling could be a novel target to develop new strategies for better prevention and treatment of kidney injury.

Mitofission impairs the anti-tumor activity of NK cells in tumor microenvironment

Abstract The survival and functional performance of NK cells is a metabolically demanding process. However, there is largely unknown how inappropriate metabolic reprogramming underlies disease microenvironments leads to NK dysfunction. Here, through analysis of human liver cancer samples (n=102) and benign liver samples (n=22), we found tumor infiltrative NK (TINK) cells present small and fragmented mitochondria in the cytoplasm, while normal liver NK and peripheral NK cells have big and tubular mitochondria. We also observed hypoxic tumor microenvironment is the main factor to induce NK cell excessive mitochondrial fission into fragmentation and that dependent on increasing Drp1 activity. Consequently, the fragmentation causes the metabolic disturbance and losing of TINK cells and that predicts poor survival in liver cancer patients. Revising mitochondrial morphology by targeting Drp1 improves mitochondrial metabolism and anti-tumor capacity of NK cells. Thus, disease-related mitochondrial restructuring drives NK cell exhaust and that is manipulated to the benefit of temper or enhance immunity.

Mitochondrial fragmentation limits NK cell-based tumor immunosurveillance.

Natural killer (NK) cells have crucial roles in tumor surveillance. We found that tumor-infiltrating NK cells in human liver cancers had small, fragmented mitochondria in their cytoplasm, whereas liver NK cells outside tumors, as well as peripheral NK cells, had normal large, tubular mitochondria. This fragmentation was correlated with reduced cytotoxicity and NK cell loss, resulting in tumor evasion of NK cell-mediated surveillance, which predicted poor survival in patients with liver cancer. The hypoxic tumor microenvironment drove the sustained activation of mechanistic target of rapamycin-GTPase dynamin-related protein 1 (mTOR-Drp1) in NK cells, resulting in excessive mitochondrial fission into fragments. Inhibition of mitochondrial fragmentation improved mitochondrial metabolism, survival and the antitumor capacity of NK cells. These data reveal a mechanism of immune escape that might be targetable and could invigorate NK cell-based cancer treatments.

Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo.

Accumulating evidence suggests that changes in the metabolic signature of microglia underlie their response to inflammation. We sought to increase our knowledge of how pro-inflammatory stimuli induce metabolic changes. Primary microglia exposed to lipopolysaccharide (LPS)-expressed excessive fission leading to more fragmented mitochondria than tubular mitochondria. LPS-mediated Toll-like receptor 4 (TLR4) activation also resulted in metabolic reprogramming from oxidative phosphorylation to glycolysis. Blockade of mitochondrial fission by Mdivi-1, a putative mitochondrial division inhibitor led to the reversal of the metabolic shift. Mdivi-1 treatment also normalized the changes caused by LPS exposure, namely an increase in mitochondrial reactive oxygen species production and mitochondrial membrane potential as well as accumulation of key metabolic intermediate of TCA cycle succinate. Moreover, Mdivi-1 treatment substantially reduced LPS induced cytokine and chemokine production. Finally, we showed that Mdivi-1 treatment attenuated expression of genes related to cytotoxic, repair, and immunomodulatory microglia phenotypes in an in vivo neuroinflammation paradigm. Collectively, our data show that the activation of microglia to a classically pro-inflammatory state is associated with a switch to glycolysis that is mediated by mitochondrial fission, a process which may be a pharmacological target for immunomodulation.

Geometric instability catalyzes mitochondrial fission.

The mitochondrial membrane undergoes extreme remodeling during fission. While a few membrane-squeezing proteins are recognized as the key drivers of fission, there is a growing body of evidence that strongly suggests that conical lipids play a critical role in regulating mitochondrial morphology and fission. However, the mechanisms by which proteins and lipids cooperate to execute fission have not been quantitatively investigated. Here, we computationally model the squeezing of the largely tubular mitochondrion and show that proteins and conical lipids can act synergistically to trigger buckling instability and achieve extreme constriction. More remarkably, the study reveals that the conical lipids can act with different fission proteins to induce hierarchical instabilities and create increasingly narrow and stable constrictions. We reason that this geometric plasticity imparts significant robustness to the fission reaction by arresting the elastic tendency of the membrane to rebound during protein polymerization and depolymerization cycles. Our in vitro study validates protein–lipid cooperativity in constricting membrane tubules. Overall, our work presents a general mechanism for achieving drastic topological remodeling in cellular membranes.

Abstract 100: PRDM16 is a Novel Regulator of Cardiac Hypertrophy, Remodeling and Mitochondrial Dynamics

Elucidation of the mechanisms that cause cardiac hypertrophy is essential for developing novel therapeutic strategies to prevent or reverse these prevalent conditions. Prdm16/Mel1 (PRDM16) belongs to the Prdm family of transcription factors involved in cell fate determination and development. PRDM16 deletion, found in humans with 1p36 deletion syndrome, has been associated with two main cardiomyopathies: left ventricle non-compaction (LVNC) and dilated cardiomyopathy. Nonetheless, its role in the heart and the underlying mechanisms by which these mutations contribute to the development of cardiomyopathy are unknown. We sought to directly assess the role of Prdm16 in the developing and adult heart by generating both a germline (cPRDM16) and a tamoxifen-inducible (ciPRDM16) cardiomyocyte-specific Prdm16 knockout mice. cPRDM16KO mice died shortly after birth, showing grossly abnormal ventricles that were reduced in size and often exhibiting a cleft at their apex, consistent with LVCN. Both heterozygous mice (cPRDM16KOhet) and ciPRDM16KO are viable and displayed markedly increased heart size, cardiomyocyte cross sectional area, collagen deposition and expression of genes involved in cardiac hypertrophy and ECM production. We observed an increased in TGF-β receptors mRNA expression as well as activation of Smad signaling in the hearts of cPRDM16KO neonates, cPRDM16 heterozygous, and ciPRDM16 KO mice. Interestingly, we observed a systematic reduction in Drp1 phosphorylation on Ser616 (activation) in the hearts of cPRDM16KO and cPRDM16het mice. The reduction in Drp1 phosphorylation was associated with more tubular mitochondria in cPRDM16KO mice. Interestingly, by 25 weeks, cPRDM16het mice accumulated more mitochondrial proteins, despite exhibiting no change in PGC1α. Together, these data reveal previously unknown roles for Prdm16 in protection against cardiac hypertrophy and fibrosis through the suppression of TGF-β/Smad signaling in the heart. They also reveal roles for PRDM16 in the modulation of mitochondrial dynamics and clearance in the heart, a process that likely involves the fission protein Drp1.

Cold PSM, but not TRAIL, triggers autophagic cell death: A therapeutic advantage of PSM over TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) are promising novel anticancer tools due to their strong anticancer activities and high tumor-selectivity. The present study demonstrated that PSM and TRAIL may trigger autophagy in human malignant melanoma and osteosarcoma cells. Live-cell imaging revealed that even under nutritional and stress-free conditions, these cells possessed a substantial level of autophagosomes, which were localized in the cytoplasm separately from tubular mitochondria. In response to cytotoxic levels of PSM, the mitochondria became highly fragmented, and aggregated and colocalized with the autophagosomes. The cytotoxic effects of PSM were suppressed in response to various pharmacological autophagy inhibitors, including 3-methyladenine (3-MA) and bafilomycin A1, thus indicating the induction of autophagic cell death (ACD). Lethal levels of PSM also resulted in non-apoptotic, non-autophagic cell death in a reactive oxygen species-dependent manner under certain circumstances. Furthermore, TRAIL exhibited only a modest cytotoxicity toward these tumor cells, and did not induce ACD and mitochondrial aberration. The combined use of TRAIL and subtoxic concentrations of 3-MA resulted in decreased basal autophagy, increased mitochondrial aberration, colocalization with autophagosomes and apoptosis. These results indicated that PSM may induce ACD, whereas TRAIL may trigger cytoprotective autophagy that compromises apoptosis. To the best of our knowledge, the present study is the first to demonstrate that PSM can induce ACD in human cancer cells. These findings provide a rationale for the advantage of PSM over TRAIL in the destruction of apoptosis-resistant melanoma and osteosarcoma cells.

SIRT 3 , a modifier of mitochondrial function in Huntington's disease

SIRT 3 is the major regulator of the mitochondrial acetylome, mediating adaptive responses of neurons to metabolic challenges. Here, we speculated that targeting SIRT 3 might influence mitochondrial function and oxidative status that are abnormal in models of Huntington's disease (HD), a motor neurodegenerative disorder mainly affecting the striatum. Protein and enzymatic analysis revealed that SIRT 3 is increased in several HD models. However, SIRT 3 knockdown aggravated mitochondrial-driven H 2 O 2 production and ATP levels. SIRT 3 levels in HD cells were normalized after antioxidant treatment, suggesting that augmented SIRT 3 may be triggered by oxidative stress. SIRT 3 overexpression (SIRT 3 OE) in HD striatal cells reestablished mitochondrial biogenesis markers, and functional imaging revealed improved mitochondrial membrane potential. Remarkably, the unbalanced mitochondrial dynamics observed in HD cells was reduced after SIRT 3 OE, with higher prevalence of tubular mitochondria due to decreased Drp1 accumulation. Additionally, overexpression of the Drosophila SIRT 3 ortholog dSirt 2 in HD model fruit flies reduced neurodegeneration of photoreceptor neurons. Overall, enhanced SIRT 3 activity ameliorates mitochondrial phenotypes, culminating in decreased neuronal damage in HD models.

Two-photon deep-tissue spatially resolved mitochondrial imaging using membrane potential fluorescence fluctuations.

Cell metabolism and viability are directly reflected in their mitochondria. Imaging-based analysis of mitochondrial morphological structure, size and dynamic characteristics can therefore provide critical insight into cell function. However, mitochondria are often very abundant, and due to their close to diffraction-limit size, it is often non-trivial to distinguish a tubular or large mitochondrion from an ensemble of punctate mitochondria. In this paper, we use membrane potential dependent fluorescence fluctuations of individual mitochondria to resolve them using an approach similar to single molecule localization microscopy. We use 2-photon microscopy to image mitochondrial intensity fluctuations at 200 μm deep inside an intact in-vivo mouse soleus muscle. By analyzing the acquired images, we can reconstruct images with an extra layer of information about individual mitochondria, separated from their ensemble. Our analysis shows a factor of 14 improvement in detection of mitochondria.

MTORC2 Promotes Tumorigenesis via Lipid Synthesis

Dysregulated mammalian target of rapamycin (mTOR) promotes cancer, but underlying mechanisms are poorly understood. We describe an mTOR-driven mouse model that displays hepatosteatosis progressing to hepatocellular carcinoma (HCC). Longitudinal proteomic, lipidomics, and metabolomic analyses revealed that hepatic mTORC2 promotes de novo fatty acid and lipid synthesis, leading to steatosis and tumor development. In particular, mTORC2 stimulated sphingolipid (glucosylceramide) and glycerophospholipid (cardiolipin) synthesis. Inhibition of fatty acid or sphingolipid synthesis prevented tumor development, indicating a causal effect in tumorigenesis. Increased levels of cardiolipin were associated with tubular mitochondria and enhanced oxidative phosphorylation. Furthermore, increased lipogenesis correlated with elevated mTORC2 activity and HCC in human patients. Thus, mTORC2 promotes cancer via formation of lipids essential for growth and energy production.

Mdivi-1 Protects Human W8B2+ Cardiac Stem Cells from Oxidative Stress and Simulated Ischemia-Reperfusion Injury.

Cardiac stem cell (CSC) therapy is a promising approach to treat ischemic heart disease. However, the poor survival of transplanted stem cells in the ischemic myocardium has been a major impediment in achieving an effective cell-based therapy against myocardial infarction. Inhibiting mitochondrial fission has been shown to promote survival of several cell types. However, the role of mitochondrial morphology in survival of human CSC remains unknown. In this study, we investigated whether mitochondrial division inhibitor-1 (Mdivi-1), an inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), can improve survival of a novel population of human W8B2+ CSCs in hydrogen peroxide (H2O2)-induced oxidative stress and simulated ischemia-reperfusion injury models. Mdivi-1 significantly reduced H2O2-induced cell death in a dose-dependent manner. This cytoprotective effect was accompanied by an increased proportion of cells with tubular mitochondria, but independent of mitochondrial membrane potential recovery and reduction of mitochondrial superoxide production. In simulated ischemia-reperfusion injury model, Mdivi-1 given as a pretreatment or throughout ischemia-reperfusion injury significantly reduced cell death. However, the cytoprotective effect of Mdivi-1 was not observed when given at reperfusion. Moreover, the cytoprotective effect of Mdivi-1 in the simulated ischemia-reperfusion injury model was not accompanied by changes in mitochondrial morphology, mitochondrial membrane potential, or mitochondrial reactive oxygen species production. Mdivi-1 also did not affect mitochondrial bioenergetics of intact W8B2+ CSCs. Taken together, these experiments demonstrated that Mdivi-1 treatment of human W8B2+ CSCs enhances their survival and can be employed to improve therapeutic efficacy of CSCs for ischemic heart disease.