Developmental dynamics of mitochondrial fission and fusion proteins in functionally divergent skeletal muscles of goat.

During skeletal muscle development, the intricate mitochondrial network formation relies on continuous fission and fusion. This process in larger mammals differs from rodents, the most used animal models. However, the expression pattern of proteins regulating mitochondrial dynamics in developing skeletal muscle remains unexplored in larger mammals. Therefore, we characterized the cellular expression and tissue-level distribution of these proteins during development taking goat as a model. We have performed histological and immunohistochemical analyses to study metabolic features in various muscles. Neonatal muscles display uniform distribution of mitochondrial activity. In contrast, adult muscles exhibit clear distinctions based on their function, whether dedicated for posture maintenance or facilitating locomotion. Mitochondrial fission proteins like DRP-1, MFF, and fusion proteins like MFN-1 and 2 are abundantly expressed in neonatal muscles. Fission proteins exhibit drastic downregulation with limited peripheral expression, whereas fusion proteins continue to express in a fiber-specific manner during adulthood. Locomotory muscles exhibit different fibers based on mitochondrial activity and peripheralization with high SDH activity. The proximity ligation assay between MFN1 and MFN2 demonstrates that their interaction is restricted to subsarcolemmal mitochondria in adult fibers while distributed evenly in neonatal fibers. These differences between postural and locomotory muscles suggest their physiological and metabolic properties are different.

Dynamics of membrane tubulation coupled with fission by a two-component module.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

Imbalanced mitochondrial dynamics contributes to the pathogenesis of X-linked adrenoleukodystrophy.

The peroxisomal disease adrenoleukodystrophy (X-ALD) is caused by loss of the transporter of very-long-chain fatty acids (VLCFAs), ABCD1. An excess of VLCFAs disrupts essential homeostatic functions crucial for axonal maintenance, including redox metabolism, glycolysis and mitochondrial respiration. As mitochondrial function and morphology are intertwined, we set out to investigate the role of mitochondrial dynamics in X-ALD models. Using quantitative 3D transmission electron microscopy, we revealed mitochondrial fragmentation in corticospinal axons in Abcd1- mice. In patient fibroblasts, an excess of VLCFAs triggers mitochondrial fragmentation through the redox-dependent phosphorylation of DRP1 (DRP1S616). The blockade of DRP1-driven fission by the peptide P110 effectively preserved mitochondrial morphology. Furthermore, mRNA inhibition of DRP1 not only prevented mitochondrial fragmentation but also protected axonal health in a Caenorhabditis elegans model of X-ALD, underscoring DRP1 as a potential therapeutic target. Elevated levels of circulating cell-free mtDNA in patients' CSF align this leukodystrophy with primary mitochondrial disorders. Our findings underscore the intricate interplay between peroxisomal dysfunction, mitochondrial dynamics and axonal integrity in X-ALD, shedding light on potential avenues for therapeutic intervention.

Mitochondrial quality control in human health and disease.

Mitochondria, the most crucial energy-generating organelles in eukaryotic cells, play a pivotal role in regulating energy metabolism. However, their significance extends beyond this, as they are also indispensable in vital life processes such as cell proliferation, differentiation, immune responses, and redox balance. In response to various physiological signals or external stimuli, a sophisticated mitochondrial quality control (MQC) mechanism has evolved, encompassing key processes like mitochondrial biogenesis, mitochondrial dynamics, and mitophagy, which have garnered increasing attention from researchers to unveil their specific molecular mechanisms. In this review, we present a comprehensive summary of the primary mechanisms and functions of key regulators involved in major components of MQC. Furthermore, the critical physiological functions regulated by MQC and its diverse roles in the progression of various systemic diseases have been described in detail. We also discuss agonists or antagonists targeting MQC, aiming to explore potential therapeutic and research prospects by enhancing MQC to stabilize mitochondrial function.

mTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions.

BACKGROUND: The larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated. MATERIALS AND RESULTS: This study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration. CONCLUSION: In summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

USP7 cardiomyocyte specific knockout causes disordered mitochondrial biogenesis and dynamics and early neonatal lethality in mice.

BACKGROUND: Ubiquitination is an enzymatic modification involving ubiquitin chains, that can be reversed by deubiquitination (DUB) enzymes. Ubiquitin-specific protease 7 (USP7), which is also known as herpes virus-associated ubiquitin-specific protease (HAUSP), has been shown to play a vital role in cardiovascular diseases. However, the underlying molecular mechanism by which USP7 regulates cardiomyocyte function has not been reported. METHODS: To understand the physiological function of USP7 in the heart, we constructed cardiomyocyte-specific USP7 conditional knockout mice. RESULTS: We found that homozygous knockout mice died approximately three weeks after birth, while heterozygous knockout mice grew normally into adulthood. Severe cardiac dysfunction, hypertrophy, fibrosis, and cell apoptosis were observed in cardiomyocyte-specific USP7 knockout mice, and these effects were accompanied by disordered mitochondrial dynamics and cardiometabolic-related proteins. CONCLUSIONS: In summary, we investigated changes in the growth status and cardiac function of cardiomyocyte-specific USP7 knockout mice, and preliminarily explored the underlying mechanism.

Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Regulatory roles of microRNAs in modulating mitochondrial dynamics, amyloid beta fibrillation, microglial activation, and cholinergic signaling: Implications for alzheimer's disease pathogenesis.

Alzheimer's Disease (AD) remains a formidable challenge due to its complex pathology, notably involving mitochondrial dysfunction and dysregulated microRNA (miRNA) signaling. This study delves into the underexplored realm of miRNAs' impact on mitochondrial dynamics and their interplay with amyloid-beta (Aß) aggregation and tau pathology in AD. Addressing identified gaps, our research utilizes advanced molecular techniques and AD models, alongside patient miRNA profiles, to uncover miRNAs pivotal in mitochondrial regulation. We illuminate novel miRNAs influencing mitochondrial dynamics, Aß, and tau, offering insights into their mechanistic roles in AD progression. Our findings not only enhance understanding of AD's molecular underpinnings but also spotlight miRNAs as promising therapeutic targets. By elucidating miRNAs' roles in mitochondrial dysfunction and their interactions with hallmark AD pathologies, our work proposes innovative strategies for AD therapy, aiming to mitigate disease progression through targeted miRNA modulation. This contribution marks a significant step toward novel AD treatments, emphasizing the potential of miRNAs in addressing this complex disease.

Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory.

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. In vitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted in vivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and in vivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

Nrf2 expression, mitochondrial fission, and neuronal apoptosis in the prefrontal cortex of methamphetamine abusers and rats.

Methamphetamine (MA), a representative amphetamine-type stimulant, is one of the most abused drugs worldwide. Studies have shown that MA-induced neurotoxicity is strongly associated with oxidative stress and apoptosis. While nuclear factor E2-related factor 2 (Nrf2), an antioxidant transcription factor, is known to exert neuroprotective effects, its role in MA-induced dopaminergic neuronal apoptosis remains incompletely understood. In the present study, we explored the effects of MA on the expression levels of Nrf2, dynamin-related protein 1 (Drp1), mitofusin 1 (Mfn1), cytochrome c oxidase (Cyt-c), and cysteine aspartate-specific protease 3 (Caspase 3), as well as the correlations between Nrf2 and mitochondrial dynamics and apoptosis. Brain tissue from MA abusers was collected during autopsy procedures. An MA-dependent rat model was also established by intraperitoneal administration of MA (10 mg/kg daily) for 28 consecutive days, followed by conditioned place preference (CPP) testing. Based on immunohistochemical staining and western blot analysis, the protein expression levels of Nrf2 and Mfn1 showed a decreasing trend, while levels of Drp1, Cyt-c, and Caspase 3 showed an increasing trend in the cerebral prefrontal cortex of both MA abusers and MA-dependent rats. Notably, the expression of Nrf2 was positively associated with the expression of Mfn1, but negatively associated with the expression levels of Drp1, Cyt-c, and Caspase 3. These findings suggest that oxidative stress and mitochondrial fission contribute to neuronal apoptosis, with Nrf2 potentially playing a critical role in MA-induced neurotoxicity.

ISGylation of DRP1 closely balances other post-translational modifications to mediate mitochondrial fission.

Dynamin related protein 1 (DRP1), a pivotal mitochondrial fission protein, is post-translationally modified by multiple mechanisms. Here we identify a new post-translational modification of DRP1 by the ubiquitin-like protein, interferon-stimulated gene 15 (ISG15). DRP1 ISGylation is mediated by ISG15 E3 ligase, HERC5; this promotes mitochondrial fission. DeISGylation of DRP1 however leads to hyperfusion. Heterologous expression of SARS-CoV2 PLpro, a deISGylating enzyme, results in similar mitochondrial filamentation, significant decrease in total DRP1 protein levels and efflux of mtDNA. We report that deISGylated DRP1 gets ubiquitylated and degraded by TRIM25, instead of PARKIN and MITOL. While the cytosolic pool of DRP1 is primarily ISGylated, both mitochondrial and cytosolic fractions may be ubiquitylated. It is known that phosphorylation of DRP1 at S616 residue regulates its mitochondrial localisation; we show that ISGylation of phospho-DRP1 (S616) renders fission competence at mitochondria. This is significant because DRP1 ISGylation affects its functionality and mitochondrial dynamics in Alzheimer's disease pathophysiology.

IGF2BP3-stabilized CAMK1 regulates the mitochondrial dynamics of renal tubule to alleviate diabetic nephropathy.

BACKGROUND: CAMK1 has been shown to be involved in human disease progression via regulating mitochondrial dynamics. However, whether CAMK1 mediates mitochondrial dynamics to regulate diabetic nephropathy (DN) process remains unclear. METHODS: Mice were injected with streptozotocin (STZ) to mimic diabetic mice models in vivo, and mice with proximal tubule-specific knockout of CAMK1 (CAMK1-KO) were generated. HK-2 cells were treated with high-glucose (HG) to mimic DN cell model in vitro. Histopathological analysis was performed to confirm kidney injury in mice. ROS production and apoptosis were assessed by DHE staining and TUNEL staining. Mitochondria morphology was observed and analyzed by electron microscopy. Mitochondrial membrane potential was detected by JC-1 staining, and cell proliferation was measured by EdU assay. The mRNA and protein expression were examined by qRT-PCR, western blot and immunostaining. RNA interaction was confirmed by RIP assay and dual-luciferase reporter assay. The mRNA stability was tested by actinomycin D treatment, and m6A level was examined by MeRIP assay. RESULTS: CAMK1 was reduced in DN patients and STZ-induced diabetic mice. Conditional deletion of CAMK1 aggravated kidney injury and promoted mitochondrial fission in diabetic mice. CAMK1 overexpression inhibited mitochondrial fission to alleviate HG-induced HK-2 cell apoptosis. IGF2BP3 promoted the stability of CAMK1 mRNA by m6A modification. IGF2BP3 inhibited mitochondrial fission to repress cell apoptosis in vitro and kidney injury in vivo by increasing CAMK1 expression. CONCLUSION: IGF2BP3-mediated CAMK1 mRNA stability alleviated DN progression by inhibiting mitochondria fission.

Recent insights into the control of mitochondrial fission.

Mitochondria are the powerhouse of the cell. They undergo fission and fusion to maintain cellular homeostasis. In this review, we explore the intricate regulation of mitochondrial fission at various levels, including the protein level, the post-translational modification level, and the organelle level. Malfunctions in mitochondrial fission can have detrimental effects on cells. Therefore, we also examine the association between mitochondrial fission with diseases such as breast cancer and cardiovascular disorders. We anticipate that a comprehensive investigation into the control of mitochondrial fission will pave the way for the development of innovative therapeutic strategies.

Significance of quantitative analyses of the impact of heterogeneity in mitochondrial content and shape on cell differentiation.

Mitochondria, classically known as the powerhouse of cells, are unique double membrane-bound multifaceted organelles carrying a genome. Mitochondrial content varies between cell types and precisely doubles within cells during each proliferating cycle. Mitochondrial content also increases to a variable degree during cell differentiation triggered after exit from the proliferating cycle. The mitochondrial content is primarily maintained by the regulation of mitochondrial biogenesis, while damaged mitochondria are eliminated from the cells by mitophagy. In any cell with a given mitochondrial content, the steady-state mitochondrial number and shape are determined by a balance between mitochondrial fission and fusion processes. The increase in mitochondrial content and alteration in mitochondrial fission and fusion are causatively linked with the process of differentiation. Here, we critically review the quantitative aspects in the detection methods of mitochondrial content and shape. Thereafter, we quantitatively link these mitochondrial properties in differentiating cells and highlight the implications of such quantitative link on stem cell functionality. Finally, we discuss an example of cell size regulation predicted from quantitative analysis of mitochondrial shape and content. To highlight the significance of quantitative analyses of these mitochondrial properties, we propose three independent rationale based hypotheses and the relevant experimental designs to test them.

ER-mitochondria contact sites in mitochondrial DNA dynamics, maintenance, and distribution.

Mitochondria are central cellular metabolic hubs. Their function requires proteins encoded by nuclear DNA, but also mitochondrial DNA (mtDNA) whose maintenance is essential for the proper function of the organelle. Defective mtDNA maintenance and distribution are associated with mitochondrial diseases. mtDNA is organized into nucleo-protein complexes called nucleoids that dynamically move along the mitochondrial network and interact with each other. mtDNA replication and nucleoid distribution is an active process regulated by the complex interplay of mitochondrial dynamics, endoplasmic reticulum (ER)-mitochondria contact sites, and cytoskeletal networks. For example, defects in mitochondrial fusion and fission or ER-mitochondria contact sites affect nucleoid maintenance and distribution. In this review, we discuss the process of nucleoid dynamics and the factors regulating nucleoid maintenance and distribution.

Celastrol Alleviates Mitochondrial Oxidative Stress and Brain Injury After Intracerebral Hemorrhage by Promoting OPA1-Dependent Mitochondrial Fusion.

Mitochondrial oxidative stress is one of the characteristics of secondary brain injury (SBI) after intracerebral hemorrhage (ICH), contributing largely to the apoptosis of neurons. Celastrol, a quinone methide triterpene that possesses antioxidant and mitochondrial protective properties, has emerged as a neuroprotective agent. However, the activity of celastrol has not been tested in ICH-induced SBI. In this study, we found that celastrol could effectively alleviate neurological function deficits and reduce brain oedema and neuronal apoptosis caused by ICH. Through electron microscopy, we found that celastrol could significantly attenuate mitochondrial morphology impairment. Therefore, we tested the regulatory proteins of mitochondrial dynamics and found that celastrol could reverse the downwards trend of OPA1 expression after ICH. In view of this, by culturing OPA1-deficient primary neurons and constructing neuron-specific OPA1 conditional knockout mice, we found that the protective effects of celastrol on mitochondrial morphology and function after ICH were counteracted in the absence of OPA1. Further experiments also showed that OPA1 is indispensable for the protective effects of celastrol on ICH-induced secondary brain injury. In summary, we have demonstrated that celastrol is a potential drug for the treatment of ICH and have revealed a novel mechanism by which celastrol exerts its antioxidant effects by promoting OPA1-mediated mitochondrial fusion.

Recombinant bovine FGF1 promotes muscle satellite cells mitochondrial fission and proliferation in serum-free conditions.

Cell cultured meat is a novel and promising technology, but developing specific culture medium for muscle cells remains one of the main technical obstacles. FGF1 signaling is reported to promote proliferation and maintain proliferative capacity of satellite cells. However, the effect of FGF1 as a supplement to serum-free medium on satellite cells in vitro culture is still unclear. In this study, an efficient method for the production of soluble and biologically active recombinant bovine FGF1 (rbFGF1) protein in Escherichia coli was established. The soluble expression level of TrxA-rbFGF1 fusion protein was 562 mg/L in shake flasks, resulting in 5.5 mg of pure rbFGF1 from 0.1 L of starting culture. In serum-free culture conditions, rbFGF1 effectively promoted the proliferation and regulated the mitochondrial morphology and function of C2C12 myoblasts.rbFGF1 activated extracellular signal-regulated kinases1/2 (ERK1/2) signaling in C2C12 myoblasts, which further stimulated dynamin related protein 1 (DRP1) Ser616 phosphorylation. These findings highlighted the potential application of rbFGF1 in developing effective serum-free medium for cultured meat production.

The interplay between sex, time of day, fasting status, and their impact on cardiac mitochondrial structure, function, and dynamics.

Mitochondria morphology and function, and their quality control by mitophagy, are essential for heart function. We investigated whether these are influenced by time of the day (TOD), sex, and fed or fasting status, using transmission electron microscopy (EM), mitochondrial electron transport chain (ETC) activity, and mito-QC reporter mice. We observed peak mitochondrial number at ZT8 in the fed state, which was dependent on the intrinsic cardiac circadian clock, as hearts from cardiomyocyte-specific BMAL1 knockout (CBK) mice exhibit different TOD responses. In contrast to mitochondrial number, mitochondrial ETC activities do not fluctuate across TOD, but decrease immediately and significantly in response to fasting. Concurrent with the loss of ETC activities, ETC proteins were decreased with fasting, simultaneous with significant increases of mitophagy, mitochondrial antioxidant protein SOD2, and the fission protein DRP1. Fasting-induced mitophagy was lost in CBK mice, indicating a direct role of BMAL1 in regulating mitophagy. This is the first of its kind report to demonstrate the interactions between sex, fasting, and TOD on cardiac mitochondrial structure, function and mitophagy. These studies provide a foundation for future investigations of mitochondrial functional perturbation in aging and heart diseases.

C-reactive protein accelerates DRP1-mediated mitochondrial fission by modulating ERK1/2-YAP signaling in cardiomyocytes.

C-reactive protein (CRP) is an inflammatory marker and risk factor for atherosclerosis and cardiovascular diseases. However, the mechanism through which CRP induces myocardial damage remains unclear. This study aimed to determine how CRP damages cardiomyocytes via the change of mitochondrial dynamics and whether survivin, an anti-apoptotic protein, exerts a cardioprotective effect in this process. We treated H9c2 cardiomyocytes with CRP and found increased intracellular ROS production and shortened mitochondrial length. CRP treatment phosphorylated ERK1/2 and promoted increased expression, phosphorylation, and translocation of DRP1, a mitochondrial fission-related protein, from the cytoplasm to the mitochondria. The expression of mitophagy proteins PINK1 and PARK2 was also increased by CRP. YAP, a transcriptional regulator of PINK1 and PARK2, was also increased by CRP. Knockdown of YAP prevented CRP-induced increases in DRP1, PINK1, and PARK2. Furthermore, CRP-induced changes in the expression of DRP1 and increases in YAP, PINK1, and PARK2 were inhibited by ERK1/2 inhibition, suggesting that ERK1/2 signaling is involved in CRP-induced mitochondrial fission. We treated H9c2 cardiomyocytes with a recombinant TAT-survivin protein before CRP treatment, which reduced CRP-induced ROS accumulation and reduced mitochondrial fission. CRP-induced activation of ERK1/2 and increases in the expression and activity of YAP and its downstream mitochondrial proteins were inhibited by TAT-survivin. This study shows that mitochondrial fission occurs during CRPinduced cardiomyocyte damage and that the ERK1/2-YAP axis is involved in this process, and identifies that survivin alters these mechanisms to prevent CRP-induced mitochondrial damage. [BMB Reports 2023; 56(12): 663-668].