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Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the immunochemistry data shown in Figs. 4K and 7G were strikingly similar to data appearing in different form in other research articles written by different authors at different research institutes that had either already been published, or were submitted for publication at around the same time. Owing to the fact that contentious data in the above article had already been published elsewhere prior to its submission to International Journal of Molecular Medicine , the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 44: 89‑102, 2019; DOI: 10.3892/ijmm.2019.4185].

The molecular mechanisms of aging are unsolved fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding with the 3′ untranslated regions (3′ UTR) in the target mRNAs. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF-1, a mitochondrial fission factor, and subsequently regulated longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. Those mutations reduced the fertility and extended the lifespan of C. elegans. Deep sequencing of total mRNAs from wild-type and puf-8 mutant worms as well as in vivo RNA Crosslinking and Immunoprecipitation (CLIP) experiments identified six PUF-8 regulated genes, which contain at least one PUF-binding element (PBE) at the 3′ UTR. One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8 mutant animals. We conclude that PUF-8 regulate the lifespan of C. elegans may not only via MFF but also via modulating pqm-1-related pathways.

Chemotherapy based on cisplatin (CDDP) has been established as the treatment of choice for head and neck squamous cell carcinoma (HNSCC). Malignant tumors respond to microenvironmental alterations through a dynamic balance between mitochondrial fission and fusion. HNSCCs are known to exhibit hypoxic conditions, yet the respective effects and underlying mechanisms of hypoxia on chemosensitivity and mitochondrial dynamics remain to be resolved. The effect of hypoxia on the chemosensitivity of HNCC cells was determined by flow cytometry. Mitochondrial fission factor (Mff) expression was assessed by RT-PCR and Western blotting in hypoxic HNSCC cells, and further verified in primary CDDP-sensitive and CDDP-resistant HSNCC samples. The biological function of Mff was evaluated by loss of function and gain of function analyses, both in vitro and in vivo. We found that hypoxia promoted mitochondrial fission and CDDP sensitivity in HNSCC cells. Importantly, Mff was found to be correlated with chemosensitivity in primary clinical samples under hypoxic conditions. Hypoxia-inducible factor 1α (HIF-1α) was found to markedly increase Mff transcription and to directly bind to Mff. Hypoxia enhanced the release of reactive oxygen species (ROS) and upregulated the expression of Mff via HIF-1α in HNSCC cells. ROS depletion in HNSCC cells attenuated HIF-1α expression, Mff expression and mitochondrial fission. Moreover, Mff knockdown led to suppression of hypoxia-induced mitochondrial fission and to decreased CDDP chemosensitivity in vivo and in vitro. Our findings indicate that hypoxia-induced release of ROS can promote mitochondrial fission and CDDP chemosensitivity via HIF1α/Mff regulation in HNSCC cells, indicating that Mff may serve as a biomarker to predict neoadjuvant chemosensitivity in HNSCC patients and as a target for overcoming chemoresistance.

Graphical abstract Previous work has shown an association between vitamin D3 deficiency and an increased risk for acquiring various inflammatory diseases. Vitamin D3 can reduce morbidity and mortality in these patients via different mechanisms. Lung inflammation is an important event in the initiation and development of respiratory disorders. However, the anti-inflammatory effects of vitamin D3 and the underlying mechanisms remained to be determined. The purpose of this study was to examine the effects and mechanisms of action of vitamin D3 (Vit. D) on the expression of intercellular adhesion molecule-1 (ICAM-1) in vitro and in vivo with or without tumor necrosis factor α (TNF-α) treatment. Pretreatment with Vit. D reduced the expression of ICAM-1 and leukocyte adhesion in TNF-α-treated A549 cells. TNF-α increased the accumulation of mitochondrial reactive oxygen species (mtROS), while Vit. D reduced this effect. Pretreatment with Vit. D attenuated TNF-α-induced mitochondrial fission, as shown by the increased expression of mitochondrial fission factor (Mff), phosphorylated dynamin-related protein 1 (p-DRP1), and mitophagy-related proteins (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3, Bnip3) in A549 cells. Inhibition of DRP1 or Mff significantly decreased ICAM-1 expression. In addition, we found that Vit. D decreased TNF-α-induced ICAM-1 expression, mitochondrial fission, and mitophagy via the AKT and NF-κB pathways. Moreover, ICAM-1 expression, mitochondrial fission, and mitophagy were increased in the lung tissues of TNF-α-treated mice, while Vit. D supplementation reduced these effects. In this study, we elucidated the mechanisms by which Vit. D reduces the expression of adhesion molecules in models of airway inflammation. Vit. D might be served as a novel therapeutic agent for the targeting of epithelial activation in lung inflammation. Graphical Headlights: • The expression of DRP1 and Mff, mitochondrial fission-related proteins, was increased in TNF-α-treated A549 cells. • The expression of Bnip3 and LC3B, mitophagy-related proteins, was increased in TNF-α-treated A549 cells. • Vit. D pretreatment decreased TNF-α-induced inflammation through the reduction of mitochondrial fission and mitophagy in A549 cells. Supplementary Information The online version contains supplementary material available at 10.1007/s10565-021-09629-6.

Mitochondrial dysfunction in proximal tubular epithelial cells is a key event in acute kidney injury (AKI), which is a risk factor for the development of chronic kidney disease (CKD). Apelin is a bioactive peptide that protects against AKI by alleviating inflammation, inhibiting apoptosis, and preventing lipid oxidation, but its role in protecting against mitochondrial damage remains unknown. Herein, we examined the protective effects of apelin on mitochondria in cisplatin-stimulated human renal proximal tubular epithelial cells and evaluated its therapeutic efficacy in cisplatin-induced AKI mice. In vitro, apelin inhibited the cisplatin-induced mitochondrial fission factor (MFF) upregulation and the fusion-promoting protein optic atrophy 1 (OPA1) downregulation. Apelin co-treatment reversed the decreased levels of the deacetylase, Sirt3, and the increased levels of protein acetylation in mitochondria of cisplatin-stimulated cells. Overall, apelin improved the mitochondrial morphology and membrane potential in vitro. In the AKI model, apelin administration significantly attenuated mitochondrial damage, as evidenced by longer mitochondrial profiles and increased ATP levels in the renal cortex. Suppression of MFF expression, and maintenance of Sirt3 and OPA1 expression in apelin-treated AKI mice was also observed. Finally, exogenous administration of apelin normalized the serum level of creatinine and urea nitrogen and the urine levels of NGAL and Kim-1. We also confirmed a regulatory pathway that drives mitochondrial homeostasis including PGC-1α, ERRα and Sirt3. In conclusion, we demonstrated that apelin ameliorates renal functions by protecting tubular mitochondria through Sirt3 upregulation, which is a novel protective mechanism of apelin in AKI. These results suggest that apelin has potential renoprotective effects and may be an effective agent for AKI treatment to significantly retard CKD progression.

Mitophagy, a form of autophagy, plays a role in cancer development, progression and recurrence. Cancer stem cells (CSCs) also play a key role in these processes, although it not known whether mitophagy can regulate the stemness of CSCs. Here, we employed the A549‐SD human non‐small cell lung adenocarcinoma CSC model that we have developed and characterized to investigate the effect of mitophagy on the stemness of CSCs. We observed a positive relationship between mitophagic activity and the stemness of lung CSCs. At the mechanistic level, our results suggest that augmentation of mitophagy in lung CSCs can be induced by FIS1 through mitochondrial fission. In addition, we assessed the clinical relevance of FIS1 in lung adenocarcinoma using The Cancer Genome Atlas database. An elevation in FIS1, when observed together with other prognostic markers for lung cancer progression, was found to correlate with shorter overall survival., In the human non‐small cell lung adenocarcinoma cancer stem cell model A549‐SD, inhibiting the expression of FIS1 can inhibit mitochondrial division, thereby inhibiting mitophagy and consequently inhibiting the stemness of cancer stem cells.

Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

Mitochondrial dynamics play vital roles in the tumorigenicity and malignancy of various types of cancers by promoting the tumor-initiating potential of cancer cells, suggesting that targeting crucial factors that drive mitochondrial dynamics may lead to promising anticancer therapies. In the current study, we report that overexpression of mitochondrial fission factor (MFF), which is upregulated significantly in liver cancer–initiating cells (LCIC), promotes mitochondrial fission and enhances stemness and tumor-initiating capability in non-LCICs. MFF-induced mitochondrial fission evoked mitophagy and asymmetric stem cell division and promoted a metabolic shift from oxidative phosphorylation to glycolysis that decreased mitochondrial reactive oxygen species (ROS) production, which prevented ROS-mediated degradation of the pluripotency transcription factor OCT4. CRISPR affinity purification in situ of regulatory elements showed that T-box transcription factor 19 (TBX19), which is overexpressed uniquely in LCICs compared with non-LCICs and liver progenitor cells, forms a complex with PRMT1 on the MFF promoter in LCICs, eliciting epigenetic histone H4R3me2a/H3K9ac-mediated transactivation of MFF. Targeting PRMT1 using furamidine, a selective pharmacologic inhibitor, suppressed TBX19-induced mitochondrial fission, leading to a profound loss of self-renewal potential and tumor-initiating capacity of LCICs. These findings unveil a novel mechanism underlying mitochondrial fission–mediated cancer stemness and suggest that regulation of mitochondrial fission via inhibition of PRMT1 may be an attractive therapeutic option for liver cancer treatment. Significance: These findings show that TBX19/PRMT1 complex–mediated upregulation of MFF promotes mitochondrial fission and tumor-initiating capacity in liver cancer cells, identifying PRMT1 as a viable therapeutic target in liver cancer.

A12-week experiment was conducted to evaluate the influences of thiamine ongrowth performance, and intestinal mitochondrial biogenesis and function of Megalobramaamblycephala fed a high-carbohydrate (HC) diet. Fish (24·73 (sem 0·45) g) were randomly assigned to one of four diets: two carbohydrate (CHO) levels (30 and 45 %) and two thiamine levels (0 and 1·5 mg/kg). HC diets significantly decreased DGC, GRMBW, FIMBW, intestinal activities of amylase, lipase, Na+, K+-ATPase, CK, complexes I, III and IV, intestinal ML, number of mitochondrial per field, ΔΨm, the P-AMPK: T-AMPK ratio, PGC-1β protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1β, mitochondrial transcription factor A, Opa-1, ND-1 and COX-1 and 2, while the opposite was true for ATP, AMP and reactive oxygen species, and the transcriptions of dynamin-related protein-1, fission-1 and mitochondrial fission factor. Dietarythiamine concentrations significantly increased DGC, GRMBW, intestinal activities of amylase, Na+, K+-ATPase, CK, complexes I and IV, intestinal ML, number of mitochondrial per field, ΔΨm, the P-AMPK:T-AMPK ratio, PGC-1β protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1β, Opa-1, ND-1, COX-1 and 2, SGLT-1 and GLUT-2. Furthermore, a significant interaction between dietary CHO and thiamine was observed in DGC, GRMBW, intestinal activities of amylase, CK, complexes I and IV, ΔΨm, the AMP:ATP ratio, the P-AMPK:T-AMPK ratio, PGC-1β protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1β, Opa-1, COX-1 and 2, SGLT-1 and GLUT-2. Overall, thiamine supplementation improved growth performance, and intestinal mitochondrial biogenesis and function of M. amblycephala fed HC diets.

Mitochondrial dysfunction is closely related to the occurrence of epilepsy. Homeostasis of mitochondrial fusion and division can alleviate mitochondrial dysfunction. The trafficking kinesin protein 1 (TRAK1) is a key regulator of mitochondrial movement and regulates mitochondrial fusion-fission balance. The pathogenic variants in TRAK1 result in the severe neurodevelopmental disorders. However, the role of TRAK1 in epilepsy remains unclear. In the present study, we report that TRAK1 has a crucial function in regulation of epileptogenesis in temporal lobe epilepsy (TLE). TRAK1 expression is decreased in the patient specimens and animal model of TLE. Knockdown of TRAK1 causes an increase in mitochondrial fission factor (MFF) in vitro and the susceptibility to seizures in vivo. Exogenous overexpression of TRAK1 can rescue the dysfunction caused by TRAK1 knockdown. These findings provide new insights into the fundamental mechanisms of TRAK1 in TLE and have important implications for understanding and treating TLE via targeting mitochondrion.

Mitochondrial fission factor (MFF) is a part of the protein complex that promotes mitochondrial and peroxisome fission. Mitochondria and peroxisome fission are complex processes of vital importance for cell growth and survival. Here, we are presenting a case of 5-year-old boy with spastic diplegic cerebral palsy, global developmental delay, and intellectual disability with a history of perinatal asphyxia and encephalopathy. MRI brain was suggestive of symmetrical hyper-intensities in bilateral thalami. Whole-genome sequencing indicates homozygous MFF gene mutation at exon 3 of c.19_20delAGinsTT; p.Ser7Phe variant.

Excessive mitochondrial fission has been identified as the central pathogenesis of diabetic kidney disease (DKD), but the precise mechanisms remain unclear. Disulfide-bond A oxidoreductase-like protein (DsbA-L) is highly expressed in mitochondria in tubular cells of the kidney, but its pathophysiological role in DKD is unknown. Our bioinformatics analysis showed that tubular DsbA-L mRNA levels were positively associated with eGFR but negatively associated with Scr and 24h-proteinuria in CKD patients. Furthermore, the genes that were coexpressed with DsbA-L were mainly enriched in mitochondria and were involved in oxidative phosphorylation. In vivo, knockout of DsbA-L exacerbated diabetic mice tubular cell mitochondrial fragmentation, oxidative stress and renal damage. In vitro, we found that DsbA-L was localized in the mitochondria of HK-2 cells. High glucose (HG, 30 mM) treatment decreased DsbA-L expression followed by increased mitochondrial ROS (mtROS) generation and mitochondrial fragmentation. In addition, DsbA-L knockdown exacerbated these abnormalities, but this effect was reversed by overexpression of DsbA-L. Mechanistically, under HG conditions, knockdown DsbA-L expression accentuated JNK phosphorylation in HK-2 cells. Furthermore, administration of a JNK inhibitor (SP600125) or the mtROS scavenger MitoQ significantly attenuated JNK activation and subsequent mitochondrial fragmentation in DsbA-L-knockdown HK-2 cells. Additionally, the down-regulation of DsbA-L also amplified the gene and protein expression of mitochondrial fission factor (MFF) via the JNK pathway, enhancing its ability to recruit DRP1 to mitochondria. Taken together, these results link DsbA-L to alterations in mitochondrial dynamics during tubular injury in the pathogenesis of DKD and unveil a novel mechanism by which DsbA-L modifies mtROS/JNK/MFF-related mitochondrial fission.

Encephalopathy due to defective mitochondrial and peroxisomal fission 2 caused by mitochondrial fission factor (MFF) gene mutation is a rare neurogenetic disorder. Pathogenic MFF mutations have been described in three reports in literature so far. We report a young child of Indian descent who presented to us with global developmental followed by regression of acquired milestones, spasticity, visual and auditory impairment, and was found to harbor a novel pathogenic homozygous MFF truncating variant c.433C>T; p.Arg145Ter. Cellular imaging of patient lymphoblastoid cell line had shown abnormal shapes of mitochondria due to fission defects. The patient has been started on mitochondrial cocktail with some improvement.

Myeloid cell leukemia-1 (Mcl-1) is involved in the regulation of mitochondrial fission and fusion. This report aims to explore whether Mcl-1 can interact with mitochondrial fission factor (Mff) and regulate Mff-mediated mitochondrial fragmentation and apoptosis. Fluorescence images of living cells coexpressing YFP-Mff and CFP-Mcl-1 showed that Mcl-1 markedly inhibited Mff-mediated mitochondrial fragmentation and apoptosis, suggesting that Mcl-1 played a key role in inhibiting mitochondrial fission. The cells coexpressing YFP-Mff and CFP-Mcl-1 exhibited consistent fluorescence resonance energy transfer (FRET) efficiency with that of the cells coexpressing CFP-Mcl-1 and YFP, demonstrating that Mcl-1 did not directly bind to Mff on mitochondria. Collectively, Mcl-1 inhibits Mff-mediated mitochondrial fission and apoptosis not via directly binding to Mff on mitochondria.

Aims Lipotoxic cardiomyopathy in diabetic and obese patients typically encompasses increased cardiac fatty acid (FA) uptake eventually surpassing the mitochondrial oxidative capacity. Lowering FA utilization via inhibition of lipolysis represents a strategy to counteract the development of lipotoxic heart dysfunction. However, defective cardiac triacylglycerol (TAG) catabolism and FA oxidation in humans (and mice) carrying mutated ATGL alleles provokes lipotoxic heart dysfunction questioning a therapeutic approach to decrease cardiac lipolysis. Interestingly, decreased lipolysis via cardiac overexpression of Perilipin 5 (Plin5), a binding partner of ATGL, is compatible with normal heart function and lifespan despite massive cardiac lipid accumulation. Herein, we decipher mechanisms that protect Plin5 transgenic mice from the development of heart dysfunction. Methods and results We generated mice with cardiac-specific overexpression of Plin5 encoding a serine-155 to alanine exchange (Plin5-S155A) of the protein kinase A phosphorylation site, which has been suggested as a prerequisite to stimulate lipolysis and may play a crucial role in the preservation of heart function. Plin5-S155A mice showed a substantial increase in cardiac TAG and ceramide levels, which was comparable to mice overexpressing non-mutated Plin5. Lipid accumulation was compatible with normal heart function even under mild stress. Plin5-S155A mice showed reduced cardiac FA oxidation but normal ATP production and changes in the Plin5-S155A phosphoproteome compared to Plin5 transgenic mice. Interestingly, mitochondrial recruitment of dynamin-related protein 1 (Drp1) was markedly reduced in cardiac muscle of Plin5-S155A and Plin5 transgenic mice accompanied by decreased phosphorylation of mitochondrial fission factor, a mitochondrial receptor of Drp1. Conclusions This study suggests that low cardiac lipolysis is associated with reduced mitochondrial fission and may represent a strategy to combat the development of lipotoxic heart dysfunction.

Mitochondrial fission factor (MFF) gene mutations are rare mitochondrial fission disorders, resulting in autosomal recessive neurological disorders. We here report a rare case of MFF gene mutation running in a family which ultimately turned out to be a variant of unknown significance. A 29-year-old multigravida visited at 18-week gestation for prenatal genetic testing as her previous baby had cerebral palsy and global developmental delay. The exome sequencing of the affected baby revealed defective mitochondrial and peroxisomal fission 2 (AR-617086). On Sanger sequencing, the mother was homozygous and the father heterozygous for the same variant. In the current pregnancy, amniocentesis was done and the fetus was also homozygous for a similar mutation. The couple continued the pregnancy and delivered a healthy baby who had normal milestones at 11 months of age. As far as prenatal diagnostic testing is considered, our case is a real-world scenario, where patient expectations befuddle appropriate decision-making.

Mitochondrial remodeling is dysregulated in metabolic diseases but the underlying mechanism is not fully understood. We report here that BDNF (brain derived neurotrophic factor) provokes mitochondrial fission and clearance in skeletal muscle via the PRKAA/AMPK-PINK1-PRKN/Parkin and PRKAA-DNM1L/DRP1-MFF pathways. Depleting Bdnf expression in myotubes reduced fatty acid-induced mitofission and mitophagy, which was associated with mitochondrial elongation and impaired lipid handling. Muscle-specific bdnf knockout (MBKO) mice displayed defective mitofission and mitophagy, and accumulation of dysfunctional mitochondria in the muscle when they were fed with a high-fat diet (HFD). These animals also have exacerbated body weight gain, increased intramyocellular lipid deposition, reduced energy expenditure, poor metabolic flexibility, and more insulin resistance. In contrast, consuming a BDNF mimetic (7,8-dihydroxyflavone) increased mitochondrial content, and enhanced mitofission and mitophagy in the skeletal muscles. Hence, BDNF is an essential myokine to maintain mitochondrial quality and function, and its repression in obesity might contribute to impaired metabolism. Abbreviations: 7,8-DHF: 7,8-dihydroxyflavone; ACACA/ACC: acetyl Coenzyme A carboxylase alpha; ACAD: acyl-Coenzyme A dehydrogenase family; ACADVL: acyl-Coenzyme A dehydrogenase, very long chain; ACOT: acyl-CoA thioesterase; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; BDNF: brain derived neurotrophic factor; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CCL2/MCP-1: chemokine (C-C motif) ligand 2; CCL5: chemokine (C-C motif) ligand 5; CNS: central nervous system; CPT1B: carnitine palmitoyltransferase 1b, muscle; Cpt2: carnitine palmitoyltransferase 2; CREB: cAMP responsive element binding protein; DNM1L/DRP1: dynamin 1-like; E2: estrogen; EHHADH: enoyl-CoenzymeA hydratase/3-hydroxyacyl CoenzymeA dehydrogenase; ESR1/ER-alpha: estrogen receptor 1 (alpha); FA: fatty acid; FAO: fatty acid oxidation; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FFA: free fatty acids; FGF21: fibroblast growth factor 21; FUNDC1: FUN14 domain containing 1; HADHA: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha; HFD: high-fat diet; iWAT: inguinal white adipose tissues; MAP1LC3A/LC3A: microtubule-associated protein 1 light chain 3 alpha; MBKO; muscle-specific bdnf knockout; IL6/IL-6: interleukin 6; MCEE: methylmalonyl CoA epimerase; MFF: mitochondrial fission factor; NTRK2/TRKB: neurotrophic tyrosine kinase, receptor, type 2; OPTN: optineurin; PA: palmitic acid; PARL: presenilin associated, rhomboid-like; PDH: pyruvate dehydrogenase; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PRKAA/AMPK: protein kinase, AMP-activated, alpha 2 catalytic subunit; ROS: reactive oxygen species; TBK1: TANK-binding kinase 1; TG: triacylglycerides; TNF/TNFα: tumor necrosis factor; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1

Proteins controlling mitochondrial fission have been recognized as essential regulators of mitochondrial functions, mitochondrial quality control and cell apoptosis. In the present study, we identified the critical B cell survival regulator TRAF3 as a novel binding partner of the key mitochondrial fission factor, MFF, in B lymphocytes. Elicited by our unexpected finding that the majority of cytoplasmic TRAF3 proteins were localized at the mitochondria in resting splenic B cells after ex vivo culture for 2 days, we found that TRAF3 specifically interacted with MFF as demonstrated by co-immunoprecipitation and GST pull-down assays. We further found that in the absence of stimulation, increased protein levels of mitochondrial TRAF3 were associated with altered mitochondrial morphology, decreased mitochondrial respiration, increased mitochondrial ROS production and membrane permeabilization, which eventually culminated in mitochondria-dependent apoptosis in resting B cells. Loss of TRAF3 had the opposite effects on the morphology and function of mitochondria as well as mitochondria-dependent apoptosis in resting B cells. Interestingly, co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF as well as decreased ubiquitination of TRAF3. Moreover, lentivirus-mediated overexpression of MFF restored mitochondria-dependent apoptosis in TRAF3-deficient malignant B cells. Taken together, our findings provide novel insights into the apoptosis-inducing mechanisms of TRAF3 in B cells: as a result of survival factor deprivation or under other types of stress, TRAF3 is mobilized to the mitochondria through its interaction with MFF, where it triggers mitochondria-dependent apoptosis. This new role of TRAF3 in controlling mitochondrial homeostasis might have key implications in TRAF3-mediated regulation of B cell transformation in different cellular contexts. Our findings also suggest that mitochondrial fission is an actionable therapeutic target in human B cell malignancies, including those with TRAF3 deletion or relevant mutations.

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-xL, an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-xL interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-xL interaction. Our data suggest that Mff primes Drp1 binding to Bcl-xL at the mitochondria and that Mff and Bcl-xL can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-xL interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-xL interaction. Expressing a Bcl-xL mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-xL interaction that contributes to cell death following ischemia.

Mitochondria are important organelles in eukaryotes. Turnover and quality control of mitochondria are regulated at the transcriptional and posttranslational level by several cellular mechanisms. Removal of defective mitochondrial proteins is mediated by mitochondria resident proteases or by proteasomal degradation of individual proteins. Clearance of bulk mitochondria occurs via a selective form of autophagy termed mitophagy. In yeast and some developing metazoan cells (e.g., oocytes and reticulocytes), mitochondria are largely removed by ubiquitin-independent mechanisms. In such cases, the regulation of mitophagy is mediated via phosphorylation of mitochondria-anchored autophagy receptors. On the other hand, ubiquitin-dependent recruitment of cytosolic autophagy receptors occurs in situations of cellular stress or disease, where dysfunctional mitochondria would cause oxidative damage. In mammalian cells, a well-studied ubiquitin-dependent mitophagy pathway induced by mitochondrial depolarization is regulated by the mitochondrial protein kinase PINK1, which upon activation recruits the ubiquitin ligase parkin. Here, we review mechanisms of mitophagy with an emphasis on posttranslational modifications that regulate various mitophagy pathways. We describe the autophagy components involved with particular emphasis on posttranslational modifications. We detail the phosphorylations mediated by PINK1 and parkin-mediated ubiquitylations of mitochondrial proteins that can be modulated by deubiquitylating enzymes. We also discuss the role of accessory factors regulating mitochondrial fission/fusion and the interplay with pro- and antiapoptotic Bcl-2 family members. Comprehensive knowledge of the processes of mitophagy is essential for the understanding of vital mitochondrial turnover in health and disease.

While psoriasis is known as a T cell- and dendritic cell-driven skin inflammation disease, macrophages are also reported to play some roles in its development. However, the signaling pathway of activated macrophages contributing to psoriasis is not entirely understood. Thus, we aimed to explore the possible mechanisms of how macrophages initiate and sustain psoriasis. The differentiated THP1 cells, stimulated by imiquimod (IMQ), were utilized as the activated macrophage model. IMQ was also employed to produce psoriasis-like lesions in mice. A transcriptomic assay of macrophages revealed that the expressions of pro-inflammatory mediators and GDAP1L1 were largely increased after an IMQ intervention. The depletion of GDAP1L1 by short hairpin (sh)RNA could inhibit cytokine release by macrophages. GDAP1L1 modulated cytokine production by activating the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways. Besides GDAP1L1, another mitochondrial fission factor, Drp1, translocated from the cytosol to mitochondria after IMQ stimulation, followed by the mitochondrial fragmentation according to the immunofluorescence imaging. Clodronate liposomes were injected into the mice to deplete native macrophages for examining the latter’s capacity on IMQ-induced inflammation. The THP1 cells, with or without GDAP1L1 silencing, were then transplanted into the mice to monitor the deposition of macrophages. We found a significant THP1 accumulation in the skin and lymph nodes. The silencing of GDAP1L1 in IMQ-treated animals reduced the psoriasiform severity score from 8 to 2. After depleting GDAP1L1, the THP1 recruitment in the lymph nodes was decreased by 3-fold. The skin histology showed that the GDAP1L1-mediated macrophage activation induced neutrophil chemotaxis and keratinocyte hyperproliferation. Thus, mitochondrial fission can be a target for fighting against psoriatic inflammation.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail‐anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl‐xl, an antiapoptotic protein, on the action of Mff by using live‐cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl‐xl. Microscopic imaging and live‐cell fluorescence resonance energy transfer analysis demonstrated that Bcl‐xl reconstructs the Mff network from punctate distribution of higher‐order oligomers to filamentous distribution of lower‐order oligomers. Live‐cell fluorescence resonance energy transfer two‐hybrid assay showed that Bcl‐xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl‐xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria., Mitochondrial fission factor (Mff)‐mediated mitochondrial fission is positively correlated with the self‐oligomerization of Mff. Bcl‐xl directly interacts with Mff to prevent Mff‐mediated mitochondrial fission and apoptosis. Bcl‐xl interacts with Mff to form heterogenous hexamers with 1 : 2 stoichiometry in cytoplasm and heterogenous tetramers with 1 : 1 stoichiometry on the mitochondrial membrane, respectively.

Aims Previous studies showed that abnormal mitochondrial structure and function were involved in the pathological process of diabetic nephropathy (DN). The dynamic mitochondrial processes, including fusion and fission, maintain the mass and quantity of mitochondria. Podocyte injury is a critical factor in the development and progression of DN. The present study evaluated the mitochondrial fission of podocytes in patients with DN. Methods We recruited 31 patients with biopsy-confirmed DN. A quantitative analysis of the mitochondrial morphology was conducted with electron microscopy using a computer-assisted morphometric analysis application to calculate the aspect ratio values. Immunofluorescence assays were used to evaluate protein colocalization in the glomeruli of patients. Results The urine protein level was significantly increased in DN patients compared to non-DN patients (P r = −0.574, P = 0.01), and multiple regression analysis verified that the mitochondrial aspect ratio was significantly and independently associated with the urine protein level (β = −0.519, P = 0.007). In addition, Drp1, a mitochondrial fission factor, preferentially combines with AKAP1, which is located in the mitochondrial membrane. Conclusions In the podocytes of DN patients, mitochondrial fragmentation was increased, and mitochondrial aspect ratio values were correlated with the proteinuria levels. The AKAP1-Drp1 pathway may contribute to mitochondrial fission in the pathogenesis of DN.

E5 protein, the major oncoprotein of the bovine Deltapapillomavirus genus, has been detected in 17 of the 19 urothelial cancers by molecular and morphological procedures. In 10 urothelial cancers, the oxygen sensitive subunit HIF-1α, which is upregulated by hypoxia, was overexpressed. Mitophagy, the selective autophagic removal of dysfunctional mitochondria, was upregulated in hypoxic neoplastic cells infected by BPVs which was mediated by FUNDC1, a mitochondrial outer-membrane protein. The FUNDC1 receptor was amplified by PCR, and amplicon sequencing showed a 100% homology with bovine FUNDC1 sequences deposited in GenBank (accession number: NM_001104982). Both transcripts and protein levels of FUNDC1 were significantly decreased in hypoxic neoplastic cells relative to healthy, non-neoplastic cells. FUNDC1 interacted with the LC3 protein, a marker of autophagosome (mitophagosome) membrane, the Hsc70/Hsp70 chaperone, and Bag3 co-chaperone. Bag3 may play a role in mitophagosome formation together with the Synpo2 protein, and may be involved in the degradation of Hsc70/Hsp70-bound CHIP-ubiquitinated cargoes, in association with its chaperone. Ultrastructural findings revealed the presence of mitochondria exhibiting severe fragmentation and loss of cristae, as well as numerous mitochondria-containing autophagosomes. Total and phosphorylated GTPase dynamin-related protein 1 (DRP1), which plays a crucial role in mitochondrial fission, a pre-requisite for mitophagy, was overexpressed at the mitochondrial level. Total and phosphorylated mitochondrial fission factor (Mff), mitochondrial fission protein 1 (Fis1), mitochondrial dynamics 51 (MiD51), and MiD49, which are DRP1 receptors responsible and/or co-responsible for its mitochondrial recruitment were overexpressed.

Mitochondrial dynamics play a critical role in deciding the fate of a cell under normal and diseased condition. Recent surge of studies indicate their regulatory role in meeting energy demands in renal cells making them critical entities in the progression of diabetic nephropathy. Diabetes is remarkably associated with abnormal fuel metabolism, a basis for free radical generation, which if left unchecked may devastate the mitochondria structurally and functionally. Impaired mitochondrial function and their aberrant accumulation have been known to be involved in the manifestation of diabetic nephropathy, indicating perturbed balance of mitochondrial dynamics, and mitochondrial turnover. Mitochondrial dynamics emphasize the critical role of mitochondrial fission proteins such as mitochondrial fission 1, dynamin-related protein 1 and mitochondrial fission factor and fusion proteins including mitofusin-1, mitofusin-2 and optic atrophy 1. Clearance of dysfunctional mitochondria is aided by translocation of autophagy machinery to the impaired mitochondria and subsequent activation of mitophagy regulating proteins PTEN-induced putative kinase 1 and Parkin, for which mitochondrial fission is a prior event. In this review, we discuss recent progression in our understanding of the molecular mechanisms targeting reactive oxygen species mediated alterations in mitochondrial energetics, mitophagy related disorders, impaired glucose transport, tubular atrophy, and renal cell death. The molecular cross talks linking autophagy and renoprotection through an intervention of 5'-AMP-activated protein kinase, mammalian target of rapamycin, and SIRT1 factors are also highlighted here, as in-depth exploration of these pathways may help in deriving therapeutic strategies for managing diabetes provoked end-stage renal disease.

AMPK, a master regulator of metabolic homeostasis, is activated by both AMP-dependent and AMP-independent mechanisms. The conditions under which these different mechanisms operate, and their biological implications are unclear. Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.

Arsenic-based drugs as food additive were used in poultry. However, excessive arsenic exposure can disturb myocardial cell metabolism, which results in the inhibition of growth and development of chickens. Since disordered mitochondria influences cardiac physiology and pathology, a better understanding of the mechanisms modulating cardiomyocyte mitochondria process is critical for identifying the potent detoxication targets under arsenic exposure in chickens. Male Hy-line chickens (1-day-old) were fed either a basal diet or an arsenic trioxide (As2O3)-supplemented diet containing 7.5, 15, and 30 mg/kg As2O3 for 90 d. The concentrations of ions ([Na, Mg, Al, Si, K, Ca, As, Mn, Fe, Zn] and [Cr, Ni, Cu, Ba]) significantly increased and decreased in the heart of chicken under As2O3 exposure, respectively. Moreover, we observed that As2O3 decreased high-density lipoprotein cholesterol concentrations and increased total cholesterol concentrations in the serum. We also observed arterial wall degeneration, biochemical character of mitochondria undergoing either fission or fusion, typical apoptotic cells, typical DNA fragments and TdT-mediated dUTP nick end labeling positive nuclei under As2O3 exposure in the heart. Further quantitative real-time PCR demonstrated that B cell lymphoma/leukemia 2 (Bcl2) were significantly decreased and dynamin-related protein 1 (Drp1), Optic atrophy 1 (Opa1), mitochondrial fission factor 1 (Mfn1), Mfn2, p53, caspase-8, Bcl-2 associated X protein (Bax), caspase-3, caspase-9 and cytochrome C were significantly increased in all As2O3 group. In conclusion, As2O3 can disturb the trace elements homeostasis, which might favor the development of mitochondrial damage. Moreover, we suspected that As2O3-increased mitochondrial dynamics might trigger the apoptosis to limit cell metabolism. These features might identify the role of the mitochondrial dynamics under arsenic-induced cardiovascular disease in the chickens.

BackgroundMitochondria are highly dynamic organelles which remain in a continuous state of fission/ fusion dynamics to meet the metabolic needs of a cell. However, this fission/fusion dynamism has been reported to be dysregulated in most cancers. Such enhanced mitochondrial fission is demonstrated to be positively regulated by some activating oncogenic mutations; such as those of KRAS (Kristen rat sarcoma viral oncogene homologue) or BRAF (B- rapidly accelerated fibrosarcoma), thereby increasing tumor progression/ chemotherapeutic resistance and metabolic deregulation. However, the underlying mechanism(s) are still not clear, thus highlighting the need to further explore possible mechanism(s) of intervention. We sought to investigate how BRAFV600E driven CRC (colorectal cancer) progression is linked to mitochondrial fission/fusion dynamics and whether this window could be exploited to target CRC progression.MethodsWestern blotting was employed to study the differences in expression levels of key proteins regulating mitochondrial dynamics, which was further confirmed by confocal microscopy imaging of mitochondria in endogenously expressing BRAFWT and BRAFV600E CRC cells. Proliferation assays, soft agar clonogenic assays, glucose uptake/lactate production, ATP/ NADPH measurement assays were employed to study the extent of carcinogenesis and metabolic reprograming in BRAFV600E CRC cells. Genetic knockdown (shRNA/ siRNA) and/or pharmacologic inhibition of Dynamin related protein1/Pyruvate dehydrogenase kinase1 (DRP1/PDK1) and/or BRAFV600E were employed to study the involvement and possible mechanism of these proteins in BRAFV600E driven CRC. Statistical analyses were carried out using Graph Pad Prism v 5.0, data was analyzed by unpaired t-test and two-way ANOVA with appropriate post hoc tests.ResultsOur results demonstrate that BRAFV600E CRC cells have higher protein levels of mitochondrial fission factor- DRP1/pDRP1S616 leading to a more fragmented mitochondrial state compared to those harboring BRAFWT. This fragmented mitochondrial state was found to confer glycolytic phenotype, clonogenic potential and metastatic advantage to cells harboring BRAFV600E. Interestingly, such fragmented mitochondrial state seemed positively regulated by mitochondrial PDK1 as observed through pharmacologic as well as genetic inhibition of PDK1.ConclusionIn conclusion, our data suggest that BRAFV600E driven colorectal cancers have fragmented mitochondria which confers glycolytic phenotype and growth advantage to these tumors, and such phenotype is dependent at least in part on PDK1- thus highlighting a potential therapeutic target.

Mitochondria are highly dynamic organelles subjected to fission and fusion events. During mitosis, mitochondrial fission ensures equal distribution of mitochondria to daughter cells. If and how this process can actively drive mitotic progression remains largely unknown. Here, we discover a pathway linking mitochondrial fission to mitotic progression in mammalian cells. The mitochondrial fission factor (MFF), the main mitochondrial receptor for the Dynamin-related protein 1 (DRP1), is directly phosphorylated by Protein Kinase D (PKD) specifically during mitosis. PKD-dependent MFF phosphorylation is required and sufficient for mitochondrial fission in mitotic but not in interphasic cells. Phosphorylation of MFF is crucial for chromosome segregation and promotes cell survival by inhibiting adaptation of the mitotic checkpoint. Thus, PKD/MFF-dependent mitochondrial fission is critical for the maintenance of genome integrity during cell division., Cell Reports, 35 (7), ISSN:2666-3864, ISSN:2211-1247

Myocardial infarction is the leading cause of morbidity and mortality worldwide. Although myocardial reperfusion after ischemia (I/R) is an effective method to save ischemic myocardium, it can cause adverse reactions, including increased oxidative stress and cardiomyocyte apoptosis. Mitochondrial fission and mitophagy are essential factors for mitochondrial quality control, but whether they play key roles in cardiac I/R injury remains unknown. New pharmacological or molecular interventions to alleviate reperfusion injury are currently considered desirable therapies. Vitamin D3 (Vit D3) regulates cardiovascular function, but its physiological role in I/R-exposed hearts, especially its effects on mitochondrial homeostasis, remains unclear. An in vitro hypoxia/reoxygenation (H/R) model was established in H9c2 cells to simulate myocardial I/R injury. H/R treatment significantly reduced H9c2 cell viability, increased apoptosis, and activated caspase 3. In addition, H/R treatment increased mitochondrial fission, as manifested by increased expression of phosphorylated dynein-related protein 1 (p-Drp1) and mitochondrial fission factor (Mff) as well as increased mitochondrial translocation of Drp1. Treatment with the mitochondrial reactive oxygen species scavenger MitoTEMPO increased cell viability and decreased mitochondrial fission. H/R conditions elicited excessive mitophagy, as indicated by increased expression of BCL2-interacting protein 3 (BNIP3) and light chain (LC3BII/I) and increased formation of autolysosomes. In contrast, Vit D3 reversed these effects. In a mouse model of I/R, apoptosis, mitochondrial fission, and mitophagy were induced. Vit D3 treatment mitigated apoptosis, mitochondrial fission, mitophagy, and myocardial ultrastructural abnormalities. The results indicate that Vit D3 exerts cardioprotective effects against I/R cardiac injury by protecting mitochondrial structural and functional integrity and reducing mitophagy.

Mitochondria are multifunctional organelles that produce energy and are critical for various signaling pathways. Mitochondrial antiviral signaling (MAVS) is a mitochondrial outer membrane protein essential for the anti-RNA viral immune response, which is regulated by mitochondrial dynamics and energetics; however, the molecular link between mitochondrial metabolism and immunity is unclear. Here we show in cultured mammalian cells that MAVS is activated by mitochondrial fission factor (Mff), which senses mitochondrial energy status. Mff mediates the formation of active MAVS clusters on mitochondria, independent of mitochondrial fission and dynamin-related protein 1. Under mitochondrial dysfunction, Mff is phosphorylated by the cellular energy sensor AMP-activated protein kinase (AMPK), leading to the disorganization of MAVS clusters and repression of the acute antiviral response. Mff also contributes to immune tolerance during chronic infection by disrupting the mitochondrial MAVS clusters. Taken together, Mff has a critical function in MAVS-mediated innate immunity, by sensing mitochondrial energy metabolism via AMPK signaling., Mitochondrial antiviral signalling protein (MAVS) is an adaptor of RIG-I like receptor. Here the authors show that mitochondrial fission factor (Mff) is required for clustering of MAVS on the outer mitochondrial membrane during antiviral response.

Background CAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF). Materials and Methods Overexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells. Results Overexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells. Conclusions Overexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells. Disclosure Information A. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.

Renal fibrosis is the common pathological process of various chronic kidney diseases (CKD). Recent studies indicate that mitochondrial fragmentation is closely associated with renal fibrosis in CKD. However, the molecular mechanisms leading to mitochondrial fragmentation remain to be elucidated. The present study investigated the role of regulators of calcineurin 1 (RCAN1) in mitochondrial fission and renal interstitial fibrosis using conditional knockout mice in which RCAN1 was genetically deleted in tubular epithelial cells (TECs). TEC-specific deletion of RCAN1 attenuated tubulointerstitial fibrosis and epithelial to mesenchymal transition (EMT)-like phenotype change after unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI) through suppressing TGF-β1/Smad3 signaling pathway. TEC-specific deletion of RCAN1 also reduced the tubular apoptosis after UUO by inhibiting cytochrome c/caspase-9 pathway. Ultrastructure analysis revealed a marked decrease in mitochondrial fragmentation in TECs of RCAN1-deficient mice in experimental CKD models. The expression of mitochondrial profission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff) was also downregulated in obstructed kidney of TEC-specific RCAN1-deficient mice. Furthermore, TEC-specific deletion of RCAN1 attenuated the dysfunctional tubular autophagy by regulating PINK1/Parkin-induced mitophagy in CKD. RCAN1 knockdown and knockout similarly improved the mitochondrial quality control in HK-2 cells and primary cultured mouse tubular cells stimulated by TGF-β1. Put together, our data indicated that RCAN1 plays an important role in the progression of tubulointerstitial fibrosis through regulating the mitochondrial quality. Therefore, targeting RCAN1 may provide a potential therapeutic approach in CKD.

Harderian gland (HG) plays an important role in the physiological adaptation to terrestrial life, however, the mechanisms underlying the changes in the structure and function of the HG during aging remain unclear. This study investigated autophagy and apoptosis in the HG of striped dwarf hamsters (Cricetulus barabensis) of different ages (sub-adult, adult and aged groups) in both males and females. The results showed that LC3II/LC3I and puncta of LC3 were significantly higher in adult and aged individuals than sub-adults, whereas P62 decreased with age. Bax/bcl2was the highest in sub-adults of male and female individuals. Caspase3 activity was the highest in sub-adults of male and female individuals, and the citrate synthase activity was highest in sub-adults of females. ATP synthase, citrate synthase, dynamin-related protein 1 and mitochondrial fission factor (Mff) were the highest in sub-adults of females. Peptidylglycine α-amidating monooxygenase were the highest in the aged group, and those of gonadotropin-releasing hormone was the highest in the adult group. LC3II/LC3I, P62, Drp1, Fis, and bax/bcl2 were higher in males than that in females. These results suggest that apoptosis mainly affects growth and development in the HG, whereas autophagy affects aging. The difference of the HG weight and mitochondrial function between sexes is mainly related to the apoptosis.

Background Mitochondrial fission counterbalances fusion to maintain organelle morphology, but its role during development remains poorly characterized. Mammalian spermatogenesis is a complex developmental process involving several drastic changes to mitochondrial shape and organization. Mitochondria are generally small and spherical in spermatogonia, elongate during meiosis, and fragment in haploid round spermatids. Near the end of spermatid maturation, small mitochondrial spheres line the axoneme, elongate, and tightly wrap around the midpiece to form the mitochondrial sheath, which is critical for fueling flagellar movements. It remains unclear how these changes in mitochondrial morphology are regulated and how they affect sperm development. Methods We used genetic ablation of Mff (mitochondrial fission factor) in mice to investigate the role of mitochondrial fission during mammalian spermatogenesis. Results Our analysis indicates that Mff is required for mitochondrial fragmentation in haploid round spermatids and for organizing mitochondria in the midpiece in elongating spermatids. In Mff mutant mice, round spermatids have aberrantly elongated mitochondria that often show central constrictions, suggestive of failed fission events. In elongating spermatids and spermatozoa, mitochondrial sheaths are disjointed, containing swollen mitochondria with large gaps between organelles. These mitochondrial abnormalities in Mff mutant sperm are associated with reduced respiratory chain Complex IV activity, aberrant sperm morphology and motility, and reduced fertility. Conclusions Mff is required for organization of the mitochondrial sheath in mouse sperm. General Significance Mitochondrial fission plays an important role in regulating mitochondrial organization during a complex developmental process.