Crista Junctions Research Articles

Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats

This study investigated whether chronic undernutrition alters the mitochondrial structure and function in renal proximal tubule cells, thus impairing fluid transport and homeostasis. We previously showed that chronic undernutrition downregulates the renal proximal tubules (Na++K+)ATPase, the main molecular machine responsible for fluid transport and ATP consumption. Male rats received a multifactorial deficient diet, the so-called Regional Basic Diet (RBD), mimicking those used in impoverished regions worldwide, from weaning to a juvenile age (3 months). The diet has a low content (8 %) of poor-quality proteins, low lipids, and no vitamins compared to control (CTR). We investigated citrate synthase activity, mitochondrial respiration (oxygraphy) in phosphorylating and non-phosphorylating conditions with different substrates/inhibitors, potential across the internal membrane (Δψ), and anion superoxide/H2O2 formation. The data were correlated with ultrastructural alterations evaluated using transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM). Citrate synthase activity decreased (∼50 %) in RBD rats, accompanied by a similar reduction in respiration in non-phosphorylating conditions, maximum respiratory capacity, and ATP synthesis. The Δψ generation and its dissipation after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone remained unmodified in the survival mitochondria. H2O2 production increased (∼100 %) after Complex II energization. TEM demonstrated intense matrix vacuolization and disruption of cristae junctions in a subpopulation of RBD mitochondria, which was also demonstrated in the 3D analysis of FIB-SEM tomography. In conclusion, chronic undernutrition impairs mitochondrial functions in renal proximal tubules, with profound alterations in the matrix and internal membrane ultrastructure that culminate with the compromise of ATP supply for transport processes.

Implications of mitochondrial membrane potential gradients on signaling and ATP production analyzed by correlative multi-parameter microscopy

The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨC) and inner boundary (ΔΨIBM) membranes. We have developed a robust and flexible approach to visualize the CJ permeability with super-resolution microscopy as a readout of local mitochondrial membrane potential (ΔΨmito) fluctuations. This method involves analyzing the distribution of TMRM fluorescence intensity in a model that is restricted to the mitochondrial geometry. We show that mitochondrial Ca2+ elevation hyperpolarizes the CM most likely caused by Ca2+ sensitive increase of mitochondrial tricarboxylic acid cycle (TCA) and subsequent oxidative phosphorylation (OXPHOS) activity in the cristae. Dynamic multi-parameter correlation measurements of spatial mitochondrial membrane potential gradients, ATP levels, and mitochondrial morphometrics revealed a CJ-based membrane potential overflow valve mechanism protecting the mitochondrial integrity during excessive cristae hyperpolarization.

Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

An interactive deep learning-based approach reveals mitochondrial cristae topologies.

The convolution of membranes called cristae is a critical structural and functional feature of mitochondria. Crista structure is highly diverse between different cell types, reflecting their role in metabolic adaptation. However, their precise three-dimensional (3D) arrangement requires volumetric analysis of serial electron microscopy and has therefore been limiting for unbiased quantitative assessment. Here, we developed a novel, publicly available, deep learning (DL)-based image analysis platform called Python-based human-in-the-loop workflow (PHILOW) implemented with a human-in-the-loop (HITL) algorithm. Analysis of dense, large, and isotropic volumes of focused ion beam-scanning electron microscopy (FIB-SEM) using PHILOW reveals the complex 3D nanostructure of both inner and outer mitochondrial membranes and provides deep, quantitative, structural features of cristae in a large number of individual mitochondria. This nanometer-scale analysis in micrometer-scale cellular contexts uncovers fundamental parameters of cristae, such as total surface area, orientation, tubular/lamellar cristae ratio, and crista junction density in individual mitochondria. Unbiased clustering analysis of our structural data unraveled a new function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in regulating the balance between lamellar versus tubular cristae subdomains.

Abnormal expression of fission and fusion genes and the morphology of mitochondria in eutopic and ectopic endometrium

Mitochondria play a pivotal role in physiological and metabolic function of the cell. Mitochondrial dynamics orchestrate mitochondrial function and morphology, involving fission and fusion as well as ultrastructural remodeling. Mounting evidence unravels the close link between mitochondria and endometriosis. However, how mitochondrial architecture changes through fission and fusion in eutopic and ectopic tissues of women with ovarian endometriosis remains unknown. We detected the expression of fission and fusion genes and the morphology of mitochondria in eutopic and ectopic endometrium in ovarian endometriosis. The results showed that the expression of DRP1 and LCLAT1 was upregulated in eutopic endometrial stromal cells (ESCs), and the expression of DRP1, OPA1, MFN1, MFN2, and LCLAT1 was significantly downregulated in ectopic ESCs, and reduced number of mitochondria, wider cristae width and narrower cristae junction width was observed, but there was no difference in cell survival rate. The altered mitochondrial dynamics and morphology might, respectively, provide an advantage for migration and adhesion in eutopic ESCs and be the adaptive response in ectopic endometrial cells to survive under hypoxic and oxidative stress environment.

MICU1 deficiency alters mitochondrial morphology and cytochrome C release

The mitochondrial inner boundary membrane harbors a protein called MICU1, which is sensitive to Ca2+ and binds to the MICOS components Mic60 and CHCHD2. Changes in the mitochondrial cristae junction structure and organization in MICU1−/− cells lead to increased cytochrome c release, membrane potential rearrangement, and changes in mitochondrial Ca2+ uptake dynamics. These findings shed new light on the multifaceted role of MICU1, highlighting its involvement not only as an interaction partner and regulator of the MCU complex but also as a crucial determinant of mitochondrial ultrastructure and, thus, an essential player in processes initiating apoptosis.

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

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

Surviving without oxygen involves major tissue specific changes in the proteome of crucian carp (Carassius carassius).

The crucian carp (Carassius carassius) can survive complete oxygen depletion (anoxia) for several months at low temperatures, making it an excellent model for studying molecular adaptations to anoxia. Still, little is known about how its global proteome responds to anoxia and reoxygenation. By applying mass spectrometry-based proteome analyses on brain, heart and liver tissue from crucian carp exposed to normoxia, five days anoxia, and reoxygenation, we found major changes in particularly cardiac and hepatic protein levels in response to anoxia and reoxygenation. These included tissue-specific differences in mitochondrial proteins involved in aerobic respiration and mitochondrial membrane integrity. Enzymes in the electron transport system (ETS) decreased in heart and increased massively in liver during anoxia and reoxygenation but did not change in the brain. Importantly, the data support a special role for the liver in succinate handling upon reoxygenation, as suggested by a drastic increase of components of the ETS and uncoupling protein 2, which could allow for succinate metabolism without excessive formation of reactive oxygen species (ROS). Also during reoxygenation, the levels of proteins involved in the cristae junction organization of the mitochondria changed in the heart, possibly functioning to suppress ROS formation. Furthermore, proteins involved in immune (complement) system activation changed in the anoxic heart compared to normoxic controls. The results emphasize that responses to anoxia are highly tissue-specific and related to organ function.

A Cristae-Like Microcompartment in Desulfobacterota.

Some Alphaproteobacteria contain intracytoplasmic membranes (ICMs) and proteins homologous to those responsible for the mitochondrial cristae, an observation which has given rise to the hypothesis that the Alphaproteobacteria endosymbiont had already evolved cristae-like structures and functions. However, our knowledge of microbial fine structure is still limited, leaving open the possibility of structurally homologous ICMs outside the Alphaproteobacteria. Here, we report on the detailed characterization of lamellar cristae-like ICMs in environmental sulfate-reducing Desulfobacterota that form syntrophic partnerships with anaerobic methane-oxidizing (ANME) archaea. These structures are junction-bound to the cytoplasmic membrane and resemble the form seen in the lamellar cristae of opisthokont mitochondria. Extending these observations, we also characterized similar structures in Desulfovibrio carbinolicus, a close relative of the magnetotactic D. magneticus, which does not contain magnetosomes. Despite a remarkable structural similarity, the key proteins involved in cristae formation have not yet been identified in Desulfobacterota, suggesting that an analogous, but not a homologous, protein organization system developed during the evolution of some members of Desulfobacterota. IMPORTANCE Working with anaerobic consortia of methane oxidizing ANME archaea and their sulfate-reducing bacterial partners recovered from deep sea sediments and with the related sulfate-reducing bacterial isolate D. carbinolicus, we discovered that their intracytoplasmic membranes (ICMs) appear remarkably similar to lamellar cristae. Three-dimensional electron microscopy allowed for the novel analysis of the nanoscale attachment of ICMs to the cytoplasmic membrane, and these ICMs are structurally nearly identical to the crista junction architecture seen in metazoan mitochondria. However, the core junction-forming proteins must be different. The outer membrane vesicles were observed to bud from syntrophic Desulfobacterota, and darkly stained granules were prominent in both Desulfobacterota and D. carbinolicus. These findings expand the taxonomic breadth of ICM-producing microorganisms and add to our understanding of three-dimensional microbial fine structure in environmental microorganisms.

Does Disruption of Optic Atrophy-1 (OPA1) Contribute to Cell Death in HL-1 Cardiomyocytes Subjected to Lethal Ischemia-Reperfusion Injury?

Disruption of mitochondrial structure/function is well-recognized to be a determinant of cell death in cardiomyocytes subjected to lethal episodes of ischemia-reperfusion (IR). However, the precise mitochondrial event(s) that precipitate lethal IR injury remain incompletely resolved. Using the in vitro HL-1 cardiomyocyte model, our aims were to establish whether: (1) proteolytic processing of optic atrophy protein-1 (OPA1), the inner mitochondrial membrane protein responsible for maintaining cristae junction integrity, plays a causal, mechanistic role in determining cardiomyocyte fate in cells subjected to lethal IR injury; and (2) preservation of OPA1 may contribute to the well-documented cardioprotection achieved with ischemic preconditioning (IPC) and remote ischemic conditioning. We report that HL-1 cells subjected to 2.5 h of simulated ischemia displayed increased activity of OMA1 (the metalloprotease responsible for proteolytic processing of OPA1) during the initial 45 min following reoxygenation. This was accompanied by processing of mitochondrial OPA1 (i.e., cleavage to yield short-OPA1 peptides) and release of short-OPA1 into the cytosol. However, siRNA-mediated knockdown of OPA1 content did not exacerbate lethal IR injury, and did not attenuate the cardioprotection seen with IPC and a remote preconditioning stimulus, achieved by transfer of ‘reperfusate’ medium (TRM-IPC) in this cell culture model. Taken together, our results do not support the concept that maintenance of OPA1 integrity plays a mechanistic role in determining cell fate in the HL-1 cardiomyocyte model of lethal IR injury, or that preservation of OPA1 underlies the cardioprotection seen with ischemic conditioning.

Structural insights into crista junction formation by the Mic60-Mic19 complex.

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function.

ORP5/8 and MIB/MICOS link ER-mitochondria and intra-mitochondrial contacts for non-vesicular transport of phosphatidylserine.

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes.

MICU1 controls spatial membrane potential gradients and guides Ca2+ fluxes within mitochondrial substructures

Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.

The role of IR‐induced swelling in OPA1‐MICOS interaction in cardiac mitochondria

Mitochondria are membrane‐bound organelles composed of two membranes, the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM), with the intermembrane space (IMS) localized between them. The IMM is divided into two subcompartments, the inner boundary membrane (IBM) that lies parallel to the OMM, and the cristae membrane (CM), which are IMM invaginations folded into cristae. The IBM and CM are connected at terminals forming circular‐like openings known as cristae junctions (CJs). The structural integrity of the mitochondrial CJs is maintained by the mitochondrial contact site and cristae organizing system (MICOS), a specific protein complex containing several structural and regulatory proteins. The MICOS proteins control the IMM architecture through direct membrane shaping, formation of contact sites, and biogenesis of proteins and lipids. In addition, the optic atrophy 1 (OPA1), a mitochondrial fusion protein, localized also in the CM, has been shown to participate in IMM remodeling which mediates the fusion of the IMM. The structural organization of the IMM is regulated by changes in the matrix volume of mitochondria; excessive matrix swelling in response to energetic and oxidative stress induced by pathological stimuli such as cardiac ischemia‐reperfusion (IR) impairs the integrity of CJs and thereby, alters mitochondrial function. The main goal of this study is to investigate the effects of cardiac IR‐induced swelling on OPA1 and MICOS proteins. Hearts were isolated from male Sprague Dawley rats and perfused with Krebs‐Henseleit solution (KHS) using the Langendorff‐mode technique at a constant flow rate (10‐12 ml/min). The animals were randomly assigned to the following groups: i) perfusion (no ischemia) for 55 min (C‐55 group), ii) ischemia (I group) for 25 min, iii) ischemia in the presence of sanglifehrin A (SfA), an inhibitor of the permeability transition pore (IS), iv) perfusion (no ischemia) for 95 min (C‐95), v) ischemia for 25‐min followed by 40‐min reperfusion (IR group), and vi) IR in the presence of SfA (IRS). SfA (0.5 µM) was present 10 min before ischemia (IS group) and throughout the entire period of reperfusion (IRS group). LDH activity was determined in the coronary effluent as a marker of cell death. At the end of reperfusion, mitochondria were isolated from the hearts by differential centrifugation for analysis of oxygen consumption rates, mitochondrial swelling, and protein levels of long and short forms of OPA1, and MICOS proteins. Our results show that ischemia diminished post‐ischemic recovery of the hearts as evidenced by impaired cardiac contractility, increased LDH activity in the coronary effluent, and reduced mitochondrial respiration. Ischemia alone and IR differently affected the expression of both, OPA1 and mitofilin (Mic‐60, a core MICOS protein) as well as other MICOS components. In conclusion, our data suggest that OPA1 and MICOS proteins are affected differently by ischemia alone and IR.

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome

So‐called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in‐depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.

Progressive Mitochondrial SOD1G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis.

In recent years, the “non-autonomous motor neuron death” hypothesis has become more consolidated behind amyotrophic lateral sclerosis (ALS). It postulates that cells other than motor neurons participate in the pathology. In fact, the involvement of the autonomic nervous system is fundamental since patients die of sudden death when they become unable to compensate for cardiorespiratory arrest. Mitochondria are thought to play a fundamental role in the physiopathology of ALS, as they are compromised in multiple ALS models in different cell types, and it also occurs in other neurodegenerative diseases. Our study aimed to uncover mitochondrial alterations in the sympathoadrenal system of a mouse model of ALS, from a structural, bioenergetic and functional perspective during disease instauration. We studied the adrenal chromaffin cell from mutant SOD1G93A mouse at pre-symptomatic and symptomatic stages. The mitochondrial accumulation of the mutated SOD1G93A protein and the down-regulation of optic atrophy protein-1 (OPA1) provoke mitochondrial ultrastructure alterations prior to the onset of clinical symptoms. These changes affect mitochondrial fusion dynamics, triggering mitochondrial maturation impairment and cristae swelling, with increased size of cristae junctions. The functional consequences are a loss of mitochondrial membrane potential and changes in the bioenergetics profile, with reduced maximal respiration and spare respiratory capacity of mitochondria, as well as enhanced production of reactive oxygen species. This study identifies mitochondrial dynamics regulator OPA1 as an interesting therapeutic target in ALS. Additionally, our findings in the adrenal medulla gland from presymptomatic stages highlight the relevance of sympathetic impairment in this disease. Specifically, we show new SOD1G93A toxicity pathways affecting cellular energy metabolism in non-motor neurons, which offer a possible link between cell specific metabolic phenotype and the progression of ALS.

Conserved GxxxG and WN motifs of MIC13 are essential for bridging two MICOS subcomplexes

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.

Loss of MIC60 Aggravates Neuronal Death by Inducing Mitochondrial Dysfunction in a Rat Model of Intracerebral Hemorrhage.

Mitochondrial damage has been reported to be a critical factor for secondary brain injury (SBI) induced by intracerebral hemorrhage (ICH). MIC60 is a key element of the mitochondrial contact site and cristae junction organizing system (MICOS), which takes a principal part in maintaining mitochondrial structure and function. The role of MIC60 and its underlying mechanisms in ICH-induced SBI are not clear, which will be investigated in this present study. To establish and emulate ICH model in vivo and in vitro, autologous blood was injected into the right basal ganglia of Sprague-Dawley (SD) rats; and primary-cultured cortical neurons were treated by oxygen hemoglobin (OxyHb). First, after ICH induction, mitochondria were damaged and exhibited mitochondrial crista-structure remodeling, and MIC60 protein levels were reduced. Furthermore, MIC60 overexpression reduced ICH-induced neuronal death both in vivo and in vitro. In addition, MIC60 upregulation reduced ICH-induced cerebral edema, neurobehavioral impairment, and cognitive dysfunction; by contrast, MIC60 knockdown had the opposite effect. Additionally, in primary-cultured neurons, MIC60 overexpression could reverse ICH-induced neuronal cell death and apoptosis, mitochondrial membrane potential collapse, and decrease of mitophagy, indicating that MIC60 overexpression can maintain the integrity of mitochondrial structures. Moreover, loss of MIC60 is after ICH-induced reduction in PINK1 levels and mislocalization of Parkin in primary-cultured neurons. Taken together, our findings suggest that MIC60 plays an important role in ICH-induced SBI and may represent a promising target for ICH therapy.

Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.

ABSTRACTMitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Animals homozygous for a CRISPR-Cas9-generated knockout allele of nduf-11 arrested at the second larval (L2) development stage. Reducing (but not eliminating) expression using RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its supercomplexes and perturbation of respiratory function. The loss of NADH dehydrogenase activity was compensated by enhanced complex II activity, with the potential for detrimental reactive oxygen species (ROS) production. Cryo-electron tomography highlighted aberrant morphology of cristae and widening of both cristae junctions and the intermembrane space. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I and supercomplex maintenance. This highlights the importance of respiratory complex integrity for health and the potential for its perturbation to cause mitochondrial disease.This article has an associated First Person interview with Amber Knapp-Wilson, joint first author of the paper.

Emerging Roles of the MICOS Complex in Cristae Dynamics and Biogenesis.

Simple SummaryMitochondria possess an outer and inner membrane. The part of the inner membrane parallel to the outer membrane is termed the inner boundary membrane, while the cristae membrane folds towards the mitochondrial matrix and houses the respiratory chain complexes. Crista junctions are located at the interface of the inner boundary membrane and the cristae membrane and contain the important ‘mitochondrial contact site and cristae organizing system’ complex. Despite the growing evidence that the mitochondrial inner membrane could remodel, cristae membranes were largely considered static for nearly seventy years, as the observations were mostly based on electron microscopy and tomography. Recently, using fluorescence super-resolution techniques, several studies showed that cristae membranes undergo dynamic remodeling in living cells, and probably even fission and fusion of the inner membrane. In this review, we discuss the important recent literature conveying the emerging role of the MICOS complex in cristae dynamics and its relation to cristae biogenesis. As the aberrant inner membrane architecture is connected to various pathologies such as cardiomyopathies, neurodegeneration and diabetes, understanding the roles of various molecules connected with cristae biogenesis and dynamics would shed light on the pathophysiology, probably leading to therapeutics in the near future.Mitochondria are double membrane-enclosed organelles performing important cellular and metabolic functions such as ATP generation, heme biogenesis, apoptosis, ROS production and calcium buffering. The mitochondrial inner membrane (IM) is folded into cristae membranes (CMs) of variable shapes using molecular players including the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex, the dynamin-like GTPase OPA1, the F1FO ATP synthase and cardiolipin. Aberrant cristae structures are associated with different disorders such as diabetes, neurodegeneration, cancer and hepato-encephalopathy. In this review, we provide an updated view on cristae biogenesis by focusing on novel roles of the MICOS complex in cristae dynamics and shaping of cristae. For over seven decades, cristae were considered as static structures. It was recently shown that cristae constantly undergo rapid dynamic remodeling events. Several studies have re-oriented our perception on the dynamic internal ambience of mitochondrial compartments. In addition, we discuss the recent literature which sheds light on the still poorly understood aspect of cristae biogenesis, focusing on the role of MICOS and its subunits. Overall, we provide an integrated and updated view on the relation between the biogenesis of cristae and the novel aspect of cristae dynamics.