Inner Membrane Fusion Research Articles

Severe mitochondrial encephalomyopathy caused by de novo variants in OPA1 gene.

Mitochondria adjust their shape in response to the different energetic and metabolic requirements of the cell, through extremely dynamic fusion and fission events. Several highly conserved dynamin-like GTPases are involved in these processes and, among those, the OPA1 protein is a key player in the fusion of inner mitochondrial membranes. Hundreds of monoallelic or biallelic pathogenic gene variants have been described in OPA1, all associated with a plethora of clinical phenotypes without a straightforward genotype-phenotype correlation. Here we report two patients harboring novel de novo variants in OPA1. DNA of two patients was analyzed using NGS technology and the pathogenicity has been evaluated through biochemical and morphological studies in patient's derived fibroblasts and in yeast model. The two patients here reported manifest with neurological signs resembling Leigh syndrome, thus further expanding the clinical spectrum associated with variants in OPA1. In cultured skin fibroblasts we observed a reduced amount of mitochondrial DNA (mtDNA) and altered mitochondrial network characterized by more fragmented mitochondria. Modeling in yeast allowed to define the deleterious mechanism and the pathogenicity of the identified gene mutations. We have described two novel-single OPA1 mutations in two patients characterized by early-onset neurological signs, never documented, thus expanding the clinical spectrum of this complex syndrome. Moreover, both yeast model and patients derived fibroblasts showed mitochondrial defects, including decreased mtDNA maintenance, correlating with patients' clinical phenotypes.

A single chemotherapy administration induces muscle atrophy, mitochondrial alterations and apoptosis in breast cancer patients.

Breast cancer patients are commonly treated with sequential administrations of epirubicin-cyclophosphamide (EC) and paclitaxel (TAX). The chronic effect of this treatment induces skeletal muscle alterations, but the specific effect of each chemotherapy agent is unknown. This study aimed to investigate the effect of EC or TAX administration on skeletal muscle homeostasis in breast cancer patients. Twenty early breast cancer patients undergoing EC followed by TAX chemotherapies were included. Two groups of 10 women were established and performed vastus lateralis skeletal muscle biopsies either before the first administration (pre) of EC (50±14years) or TAX (50±16years) and 4days later (post). Mitochondrial respiratory capacity recording, reactive oxygen species production, western blotting and histological analyses were performed. Decrease in muscle fibres cross-sectional area was only observed post-EC (-25%; P<0.001), associated with a reduction in mitochondrial respiratory capacity for the complex I (CI)-linked substrate state (-32%; P=0.001), oxidative phosphorylation (OXPHOS) by CI (-35%; P=0.002), CI&CII (-26%; P=0.022) and CII (-24%; P=0.027). If H2 O2 production was unchanged post-EC, an increase was observed post-TAX for OXPHOS by CII (+25%; P=0.022). We found a decrease in makers of mitochondrial content, as shown post-EC by a decrease in the protein levels of citrate synthase (-53%; P<0.001) and VDAC (-39%; P<0.001). Despite no changes in markers of mitochondrial fission, a decrease in the expression of a marker of mitochondrial inner-membrane fusion was found post-EC (OPA1; -60%; P<0.001). We explored markers of mitophagy and found reductions post-EC in the protein levels of PINK1 (-63%; P<0.001) and Parkin (-56%; P=0.005), without changes post-TAX. An increasing trend in Bax protein level was found post-EC (+96%; P=0.068) and post-TAX (+77%; P=0.073), while the Bcl-2 level was decreased only post-EC (-52%; P=0.007). If an increasing trend in TUNEL-positive signal was observed post-EC (+68%; P=0.082), upregulation was highlighted post-TAX (+86%; P<0.001), suggesting activation of the apoptosis process. We demonstrated that a single administration of EC induced, in only 4days, skeletal muscle atrophy and mitochondrial alterations in breast cancer patients. These alterations were characterized by reductions in mitochondrial function and content as well as impairment of mitochondrial dynamics and an increase in apoptosis. TAX administration did not worsen these alterations as this group had already received EC during the preceding weeks. However, it resulted in an increased apoptosis, likely in response to the increased H2 O2 production.

Loss of UCP2 causes mitochondrial fragmentation by OMA1-dependent proteolytic processing of OPA1 in podocytes.

Mitochondrial dysfunction plays an important role in the onset and progression of podocyte injury and proteinuria. However, the process by which the change in the podocyte mitochondria occurs is not well understood. Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which is located in the mitochondrial inner membrane. Here, we reported that mice with podocyte-specific Ucp2 deficiency developed podocytopathy with proteinuria with aging. Furthermore, those mice exhibited increased proteinuria in experimental models evoked by Adriamycin. Our findings suggest that UCP2 mediates mitochondrial dysfunction by regulating mitochondrial dynamic balance. Ucp2-deleted podocytes exhibited increased mitochondrial fission and deficient in ATP production. Mechanistically, opacity protein 1 (OPA1), a key protein in fusion of mitochondrial inner membrane, was regulated by UCP2. Ucp2 deficiency promoted proteolysis of OPA1 by activation OMA1 which belongs to mitochondrial inner membrane zinc metalloprotease. Those finding demonstrate the role of UCP2 in mitochondrial dynamics in podocytes and provide new insights into pathogenesis associated with podocyte injury and proteinuria.

CryoEM of OPA1 helical structures gives perspective to mitochondrial dysfunction.

The leading cause of childhood blindness, Dominant Optic Atrophy (DOA), is primarily caused by mutation of the gene encoding the Optic Atrophy 1 (OPA1) protein. These OPA1 mutations putatively dysregulate the inner-membrane (IM) fusion processes of the mitochondria, leading to mitochondrial network disruption and DOA pathology. OPA1 exists in two forms in vivo: the long-form which is tethered to the inner mitochondrial membrane through a single transmembrane domain, and the short form that results from proteolytic cleavage in the inner membrane space as a response to mitochondrial membrane depolarization. Using cryo-electron microscopy, we solved helical structures of the short form of human OPA1 isoform-1 (sOPA1) with and without nucleotide. These helical assemblies formed around a lipid core, with densely packed protein rungs marked by minimal inter-rung connectivity. Notably, we also observed nucleotide-dependent dimerization of the GTPase domains, a hallmark of dynamin superfamily proteins. The sOPA1 assemblies contained several unique secondary structures to strengthen membrane association, pointing to the importance of sOPA1 membrane interaction to its function. The novel structural features and interfaces also shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly, and membrane interactions. Further, the biological relevance of the sOPA1 helical assemblies was supported by in-cell assays, where disruption of the structurally identified interfaces and membrane binding regions caused fragmentation of the mitochondrial network.

Mitochondrial OMA1 and OPA1 as Gatekeepers of Organellar Structure/Function and Cellular Stress Response.

Mammalian mitochondria are emerging as a critical stress-responsive contributor to cellular life/death and developmental outcomes. Maintained as an organellar network distributed throughout the cell, mitochondria respond to cellular stimuli and stresses through highly sensitive structural dynamics, particularly in energetically demanding cell settings such as cardiac and muscle tissues. Fusion allows individual mitochondria to form an interconnected reticular network, while fission divides the network into a collection of vesicular organelles. Crucially, optic atrophy-1 (OPA1) directly links mitochondrial structure and bioenergetic function: when the transmembrane potential across the inner membrane (ΔΨm) is intact, long L-OPA1 isoforms carry out fusion of the mitochondrial inner membrane. When ΔΨm is lost, L-OPA1 is cleaved to short, fusion-inactive S-OPA1 isoforms by the stress-sensitive OMA1 metalloprotease, causing the mitochondrial network to collapse to a fragmented population of organelles. This proteolytic mechanism provides sensitive regulation of organellar structure/function but also engages directly with apoptotic factors as a major mechanism of mitochondrial participation in cellular stress response. Furthermore, emerging evidence suggests that this proteolytic mechanism may have critical importance for cell developmental programs, particularly in cardiac, neuronal, and stem cell settings. OMA1’s role as a key mitochondrial stress-sensitive protease motivates exciting new questions regarding its mechanistic regulation and interactions, as well as its broader importance through involvement in apoptotic, stress response, and developmental pathways.

Homeostatic p62 levels and inclusion body formation in CHCHD2 knockout mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics.

Mitochondrial dynamics is essential for the organelle's diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Structural insights into G domain dimerization and pathogenic mutation of OPA1.

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF3-, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

Long OPA1 isoforms mediate a membrane potential‐dependent threshold for mitochondrial fusion in AC16 cardiomyocytes

The bioenergetic function of mitochondria is linked to its organellar structural dynamics by optic atrophy‐1 (OPA1), a nuclear‐encoded protein that mediates fusion of the mitochondrial inner membrane. OPA1‐mediated inner membrane fusion is dependent on the transmembrane potential across the mitochondrial inner membrane (Δψm): long (L‐OPA1) isoforms accomplish fusion for an interconnected reticular morphology when Δψm is intact. Upon loss of Δψm, L‐OPA1 is cleaved by the OMA1 metalloprotease, causing accumulation of short S‐OPA1 and collapse of mitochondrial fusion to a fragmented morphology. We examined Δψm‐sensitive mitochondrial fusion in human AC16 cardiomyocytes treated with carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), an uncoupler of Δψm. Upon increasing titration with CCCP, mitochondrial fusion collapses at [CCCP] &gt; 2 μM, as determined using blinded ImageJ quantification of mitochondrial morphology. Similarly, OPA1 Western blotting revealed that AC16s show a significant loss of fusion‐active L‐OPA1 at [CCCP] &gt; 2 μM. Taken together, these findings indicate that L‐OPA1 maintains mitochondrial fusion, but that OMA1 is activated at [CCCP] &gt; 2 μM, causing L‐OPA1 cleavage and collapse of mitochondrial fusion. Experiments in progress are examining OMA1 activation at this Δψm breakpoint, as well as impact of altered OPA1 and OMA1 expression.Support or Funding InformationNIGMS 5SC3GM116669 (to R.G.)NIGMS 1SC3GM132053 (to M.K.)UTRGV Presidential Graduate Research Assistantship (to P. D.)

Structural analysis of a trimeric assembly of the mitochondrial dynamin-like GTPase Mgm1

The fusion of inner mitochondrial membranes requires dynamin-like GTPases, Mgm1 in yeast and OPA1 in mammals, but how they mediate membrane fusion is poorly understood. Here, we determined the crystal structure of Saccharomyces cerevisiae short Mgm1 (s-Mgm1) in complex with GDP. It revealed an N-terminal GTPase (G) domain followed by two helix bundles (HB1 and HB2) and a unique C-terminal lipid-interacting stalk (LIS). Dimers can form through antiparallel HB interactions. Head-to-tail trimers are built by intermolecular interactions between the G domain and HB2-LIS. Biochemical and in vivo analyses support the idea that the assembly interfaces observed here are native and critical for Mgm1 function. We also found that s-Mgm1 interacts with negatively charged lipids via both the G domain and LIS. Based on these observations, we propose that membrane targeting via the G domain and LIS facilitates the in cis assembly of Mgm1, potentially generating a highly curved membrane tip to allow inner membrane fusion.

Two forms of Opa1 cooperate to complete fusion of the mitochondrial inner-membrane.

Mitochondrial membrane dynamics is a cellular rheostat that relates metabolic function and organelle morphology. Using an in vitro reconstitution system, we describe a mechanism for how mitochondrial inner-membrane fusion is regulated by the ratio of two forms of Opa1. We found that the long-form of Opa1 (l-Opa1) is sufficient for membrane docking, hemifusion and low levels of content release. However, stoichiometric levels of the processed, short form of Opa1 (s-Opa1) work together with l-Opa1 to mediate efficient and fast membrane pore opening. Additionally, we found that excess levels of s-Opa1 inhibit fusion activity, as seen under conditions of altered proteostasis. These observations describe a mechanism for gating membrane fusion.

A Molecular Perspective on Mitochondrial Membrane Fusion: From the Key Players to Oligomerization and Tethering of Mitofusin.

Mitochondria are dynamic organelles characterized by an ultrastructural organization which is essential in maintaining their quality control and ensuring functional efficiency. The complex mitochondrial network is the result of the two ongoing forces of fusion and fission of inner and outer membranes. Understanding the functional details of mitochondrial dynamics is physiologically relevant as perturbations of this delicate equilibrium have critical consequences and involved in several neurological disorders. Molecular actors involved in this process are large GTPases from the dynamin-related protein family. They catalyze nucleotide-dependent membrane remodeling and are widely conserved from bacteria to higher eukaryotes. Although structural characterization of different family members has contributed in understanding molecular mechanisms of mitochondrial dynamics in more detail, the complete structure of some members as well as the precise assembly of functional oligomers remains largely unknown. As increasing structural data become available, the domain modularity across the dynamin superfamily emerged as a foundation for transfering the knowledge towards less characterized members. In this review, we will first provide an overview of the main actors involved in mitochondrial dynamics. We then discuss recent example of computational methodologies for the study of mitofusin oligomers, and present how the usage of integrative modeling in conjunction with biochemical data can be an asset in progressing the still challenging field of membrane dynamics.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

Structure and assembly of the mitochondrial membrane remodelling GTPase Mgm1.

Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G)domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.

Advances in the Structural and Biochemical Determination of Several Dynamin-Like GTPases

Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of lipid membranes throughout the cell. The dynamin-like protein Optic Atrophy 1 (Opa1) is responsible for the fusion of the mitochondrial inner membrane and plays a role in maintaining cristae shape. Atlastin mediates fusion of homotypic three-way junctions in the endoplasmic reticulum (ER). Mutations in these, and other dynamin-like proteins, can lead to neuropathies including Dominant Optic Atrophy, Hereditary Spastic Paraplegia, and Charcot-Marie-Tooth, among others. Currently, structural and biochemical data is limiting for Opa1. In addition, crystal structures of ATL with nucleotide do not entirely explain the GTPase cycle. We have developed a protocol for expressing and purifying biologically relevant and biochemically active shortened isoforms of Opa1 (Opa1GG) in sufficient quantity to begin crystallography and biochemical assays. Furthermore, we have optimized sample prep and begun reconstructions of aproteolytically processed short form, Opa1S, by cryo-EM on a lipid surface and are examining the role nucleotide plays in structural rearrangements. In addition, to verify work performed by x-ray crystallography, we have begun probing the GTPase cycle of Atlastin using single particle cryo-EM methods.

Astaxanthin but not quercetin preserves mitochondrial integrity and function, ameliorates oxidative stress, and reduces heat-induced skeletal muscle injury.

Heat stress causes mitochondrial dysfunction and increases mitochondrial production of reactive oxygen species (ROS), both of which contribute to heat-induced skeletal muscle injury. In this study, we tested whether either astaxanthin or quercetin, two dietary antioxidants, could ameliorate heat-induced skeletal muscle oxidative injury. In mouse C2C12 myoblasts exposed to 43°C heat stress, astaxanthin inhibited heat-induced ROS production in a concentration-dependent manner (1-20 μM), whereas the ROS levels remained high in cells treated with quercetin over a range of concentrations (2-100 µM). Because mitochondria are both the main source and a primary target of heat-induced ROS, we then tested the effects of astaxanthin and quercetin on mitochondrial integrity and function, under both normal temperature (37°C) and heat stress conditions. Quercetin treatment at 37°C induced mitochondrial fragmentation and decreased membrane potential (ΔΨ m ), accompanied by reduced protein expression of the master regulator of mitochondrial biogenesis peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). It also induced cleavage of mitochondrial inner-membrane fusion protein OPA1. In contrast, astaxanthin at 37°C increased protein expression of PGC-1α and mitochondrial transcription factor A (TFAM), and maintained tubular structure and normal ΔΨm . Under 43°C heat stress conditions, whereas quercetin failed to rescue C2C12 cells from injury, astaxanthin treatment prevented heat-induced mitochondrial fragmentation and depolarization, and apoptotic cell death. We also isolated rat flexor digitorum brevis myofibers and confirmed the data from C2C12 myoblasts that astaxanthin but not quercetin preserves mitochondrial integrity and function and ameliorates heat-induced skeletal muscle injury. These results confirm that mitochondria may be a potential therapeutic target for heat-related illness and suggest that astaxanthin may potentially be an effective preventive strategy.

Relationship between OPA1 and cardiolipin in mitochondrial inner-membrane fusion

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. The large GTPase optic atrophy 1 (OPA1) is identified as a core component of inner membrane (IM) fusion. OPA1 exists as the membrane-anchored L-OPA1 and the proteolytically cleavage soluble S-OPA1. Recently, we showed that OPA1 and mitochondria-localized lipid cardiolipin (CL) cooperate in heterotypic IM fusion [Ban et al., Nat. Cell Biol. 19 (2017) 856–863]. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 and S-OPA1 expressed in silkworm and found that L-OPA1 on one side of the membrane and CL on the other side were sufficient for mitochondrial fusion. L-OPA1 is the major fusion-prone factor in heterotypic fusion. However, the role of S-OPA1 remains unknown as S-OPA1 promoted L-OPA1-dependent heterotypic membrane fusion and homotypic CL-containing membrane fusion, but S-OPA1 alone was not sufficient for heterotypic membrane fusion. L-OPA1- and CL-mediated heterotypic mitochondrial fusion was confirmed in living cells, but tafazzin (Taz1), the causal gene product of Barth syndrome, was not essential for mitochondrial fusion. Taz1-dependent CL maturation might have other roles in the remodeling of mitochondrial DNA nucleoids.

ROS as Regulators of Mitochondrial Dynamics in Neurons.

Mitochondrial dynamics is a complex process, which involves the fission and fusion of mitochondrial outer and inner membranes. These processes organize the mitochondrial size and morphology, as well as their localization throughout the cells. In the last two decades, it has become a spotlight due to their importance in the pathophysiological processes, particularly in neurological diseases. It is known that Drp1, mitofusin 1 and 2, and Opa1 constitute the core of proteins that coordinate this intricate and dynamic process. Likewise, changes in the levels of reactive oxygen species (ROS) lead to modifications in the expression and/or activity of the proteins implicated in the mitochondrial dynamics, suggesting an involvement of these molecules in the process. In this review, we discuss the role of ROS in the regulation of fusion/fission in the nervous system, as well as the involvement of mitochondrial dynamics proteins in neurodegenerative diseases.

Mitochondrial Fusion Proteins: A Tale of Two Membranes

Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of cellular lipid membranes throughout the cell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin (Mfn) 1 and 2 are responsible for the fusion of the mitochondrial inner and outer membranes, respectively. Mutations in any of these proteins can lead to neuropathies including blindness and Charcot-Marie-Tooth, a disease characterized by progressive loss of distal muscle tissue. Currently, little is known structurally or biochemically about any of these proteins. We have developed a protocol for expressing and purifying biologically relevant and biochemically active shortened isoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallographic studies. Both have comparable GTPase activity compared to full-length, unstimulated Dynamin 1 when assayed at room temperature and interestingly OpaGG exists as a tetramer when assayed by size exclusion chromatography. In addition, the long, membrane-bound isoforms of Opa1 and Mfn1 have been expressed and purified in large quantities. To date, we have shown full length Mfn1 can be incorporated into proteoliposomes and in the presence of GTP forms dense tethers as seen by cryo-EM. This tethering is reversible as shown by confocal microscopy. Currently we are developing tethering assays for Opa1 and fusion assays for both Opa1 and Mfn1.

Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model.

BackgroundRecent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain.ObjectivesTo investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries.Methods and ResultsWe examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p<0.01) and ex vivo (71.1±3.2% vs. 59.6±8.5%, respectively; p<0.05). No difference was observed in the expression of other main fission/fusion protein, oxidative phosphorylation, apoptotic markers, or mitochondrial permeability transition pore (mPTP) function. Analysis of calcium transients in isolated ventricular cardiomyocytes demonstrated a lower sarcoplasmic reticulum Ca2+ uptake, whereas cytosolic Ca2+ removal from the Na+/Ca2+ exchanger (NCX) was increased, whilst SERCA2a, phospholamban, and NCX protein expression levels were unaffected in Opa1+/- compared to WT mice. Simultaneous whole-cell patch-clamp recordings of mitochondrial Ca2+ movements and ventricular action potential (AP) showed impairment of dynamic mitochondrial Ca2+ uptake and a marked increase in the AP late repolarization phase in conjunction with greater occurrence of arrhythmia in Opa1+/- mice.ConclusionOpa1 deficiency was associated with increased sensitivity to I/R, imbalance in dynamic mitochondrial Ca2+ uptake, and subsequent increase in NCX activity.