Long Forms Of OPA1 Research Articles

Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

Absence of Cardiolipin From the Outer Leaflet of a Mitochondrial Inner Membrane Mimic Restricts Opa1-Mediated Fusion.

Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.

Loss of CHCHD2 and CHCHD10 activates OMA1 peptidase to disrupt mitochondrial cristae phenocopying patient mutations.

Dominant mutations in the mitochondrial paralogs coiled-helix-coiled-helix (CHCHD) domain 2 (C2) and CHCHD10 (C10) were recently identified as causing Parkinson's disease and amyotrophic lateral sclerosis/frontotemporal dementia/myopathy, respectively. The mechanism by which they disrupt mitochondrial cristae, however, has been uncertain. Using the first C2/C10 double knockout (DKO) mice, we report that C10 pathogenesis and the normal function of C2/C10 are intimately linked. Similar to patients with C10 mutations, we found that C2/C10 DKO mice have disrupted mitochondrial cristae, because of cleavage of the mitochondrial-shaping protein long form of OPA1 (L-OPA1) by the stress-induced peptidase OMA1. OMA1 was found to be activated similarly in affected tissues of mutant C10 knock-in (KI) mice, demonstrating that L-OPA1 cleavage is a novel mechanism for cristae abnormalities because of both C10 mutation and C2/C10 loss. Using OMA1 activation as a functional assay, we found that C2 and C10 are partially functionally redundant, and some but not all disease-causing mutations have retained activity. Finally, C2/C10 DKO mice partially phenocopied mutant C10 KI mice with the development of cardiomyopathy and activation of the integrated mitochondrial integrated stress response in affected tissues, tying mutant C10 pathogenesis to C2/C10 function.

Mitochondrial serine protease Omi/HtrA2 accentuates brain ischemia/reperfusion injury in rats and oxidative stress injury in vitro by modulating mitochondrial stress proteins CHOP and ClpP and physically interacting with mitochondrial fusion protein OPA1

ABSTRACT Serine protease Omi/HtrA2, a member of the HtrA family, is closely related to the maintenance of mitochondrial integrity and participates in apoptosis but its role in cerebral ischemia/reperfusion (I/R) injury and cellular oxidative stress response remains unclear. In this study, we found that I/R injury resulted in a time-dependent increase in Omi/HtrA2 expression in rat brain tissue. Inhibition of Omi/HtrA2 significantly inhibited XIAP cleavage in H2O2-induced PC12 cells. In addition, inhibition of Omi/HtrA2 significantly inhibited the up-regulation of mitochondrial stress proteins CHOP and ClpP, significantly reduced mitochondrial aggregation, and attenuated the decline of mitochondrial ΔΨm in PC12 cells. Studies show that there is a physical interaction between Omi/HtrA2 and OPA1. We found that Omi/HtrA2 and OPA1 are closely related to the oxidative stress mitochondrial response in PC12 cells. The current study has demonstrated that Omi/HtrA2 is upregulated in brain I/R injury in vivo and is implicated in mitochondrial response to oxidative stress in vitro by regulating mitochondrial stress proteins CHOP and CLpP and by interacting with mitochondrial cristae remodeling protein OPA1. These findings suggest that Omi/HtrA2 could be a candidate molecular target in diseases that involve oxidative stress such as in I/R injury. Abbreviation: ATP: Adenosine tripHospHate; Bax: BCL2-Associated X; Bcl-2: B-cell lympHoma-2; BSA: Albumin from bovine serum; DMEM: Dulbecco’s Minimum Essential Medium; DMSO: Dimethyl sulfoxide; HSP60: Heat shock protein60, 70; L-OPA1: Long forms of OPA1; Omi/HtrA2: high-temperature-regulated A2; MCAO: Middle cerebral artery occlusion; OPA1: Optic AtropHy; PBS: PHospHate buffered saline; PMSF: pHenylmethyl sulfonylfluoride; ROS: reactive oxygen species; SDS: Sodium dodecyl sulfate; S-OPA1: Short forms of OPA1; TTC: TripHenyltetrazalium chloride; XIAP: X-linked inhibitor apoptosis protein

In vivo stabilization of OPA1 in hepatocytes potentiates mitochondrial respiration and gluconeogenesis in a prohibitin-dependent way

Patients with fatty liver diseases present altered mitochondrial morphology and impaired metabolic function. Mitochondrial dynamics and related cell function require the uncleaved form of the dynamin-like GTPase OPA1. Stabilization of OPA1 might then confer a protective mechanism against stress-induced tissue damages. To study the putative role of hepatic mitochondrial morphology in a sick liver, we expressed a cleavage-resistant long form of OPA1 (L-OPA1Δ) in the liver of a mouse model with mitochondrial liver dysfunction (i.e. the hepatocyte-specific prohibitin-2 knockout (Hep-Phb2-/-) mice). Liver prohibitin-2 deficiency caused excessive proteolytic cleavage of L-OPA1, mitochondrial fragmentation, and increased apoptosis. These molecular alterations were associated with lipid accumulation, abolished gluconeogenesis, and extensive liver damage. Such liver dysfunction was associated with severe hypoglycemia. In prohibitin-2 knockout mice, expression of L-OPA1Δ by in vivo adenovirus delivery restored the morphology but not the function of mitochondria in hepatocytes. In prohibitin-competent mice, elongation of liver mitochondria by expression of L-OPA1Δ resulted in excessive glucose production associated with increased mitochondrial respiration. In conclusion, mitochondrial dynamics participates in the control of hepatic glucose production.

Tumor suppressor KIF1Bβ regulates mitochondrial apoptosis in collaboration with YME1L1.

KIF1Bβ, a member of the kinesin superfamily of motor proteins, is a haploinsufficient tumor suppressor mapped to chromosome 1p36.2, which is frequently deleted in neural crest–derived tumors, including neuroblastoma and pheochromocytoma. While KIF1Bβ acts downstream of the nerve growth factor (NGF) pathway to induce apoptosis, further molecular functions of this gene product have largely been unexplored. In this study, we report that KIF1Bβ destabilizes the morphological structure of mitochondria, which is critical for cell survival and apoptosis. We identified YME1L1, a mitochondrial metalloprotease responsible for the cleavage of the mitochondrial GTPase OPA1, as a physical interacting partner of KIF1Bβ. KIF1Bβ interacted with YME1L1 through its death‐inducing region, as initiated the protease activity of YME1L1 to cleave the long forms of OPA1, resulting in mitochondrial fragmentation. Overexpression of YME1L1 promoted apoptosis, while knockdown of YME1L1 promoted cell growth. High YME1L1 expression was significantly associated with a better prognosis in neuroblastoma. Furthermore, in NGF‐deprived PC12 cells, KIF1Bβ and YME1L1 were upregulated, accompanied by mitochondrial fragmentation and apoptotic cell death. Small interfering RNA–mediated knockdown of either protein alone, however, remarkably inhibited the NGF depletion–induced apoptosis. Our findings indicate that tumor suppressor KIF1Bβ plays an important role in intrinsic mitochondria–mediated apoptosis through the regulation of structural and functional dynamics of mitochondria in collaboration with YME1L1. Dysfunction of the KIF1Bβ/YME1L1/OPA1 mechanism may be involved in malignant biological features of neural crest–derived tumors as well as the initiation and progression of neurodegenerative diseases.

Dietary supplementation with acetyl-l-carnitine counteracts age-related alterations of mitochondrial biogenesis, dynamics and antioxidant defenses in brain of old rats

We previously reported the ability of dietary supplementation with acetyl-l-carnitine (ALCAR) to prevent age-related decreases of mitochondrial biogenesis in skeletal muscle and liver of old rats. Here, we investigate the effects of ALCAR supplementation in cerebral hemispheres and cerebellum of old rats by analyzing several parameters linked to mitochondrial biogenesis, mitochondrial dynamics and antioxidant defenses.We measured the level of the coactivators PGC-1α and PGC-1β and of the factors regulating mitochondrial biogenesis, finding an age-related decrease of PGC-1β, whereas PGC-1α level was unvaried. Twenty eight-month old rats supplemented with ALCAR for one and two months showed increased levels of both factors. Accordingly, the expression of the two transcription factors NRF-1 and TFAM followed the same trend of PGC-1β. The level of mtDNA, ND1 and the activity of citrate synthase, were decreased with aging and increased following ALCAR treatment. Furthermore, ALCAR counteracted the age-related increase of deleted mtDNA. We also analyzed the content of proteins involved in mitochondrial dynamics (Drp1, Fis1, OPA1 and MNF2) and found an age-dependent increase of MFN2 and of the long form of OPA1. ALCAR treatment restored the content of the two proteins to the level of the young rats. No changes with aging and ALCAR were observed for Drp1 and Fis1. ALCAR reduced total cellular levels of oxidized PRXs and counteracted the age-related decrease of PRX3 and SOD2.Overall, our findings indicate a systemic positive effect of ALCAR dietary treatment and a tissue specific regulation of mitochondrial homeostasis in brain of old rats. Moreover, it appears that ALCAR acts as a nutrient since in most cases its effects were almost completely abolished one month after treatment suspension. Dietary supplementation of old rats with this compound seems a valuable approach to prevent age-related mitochondrial dysfunction and might ultimately represent a strategy to delay age-associated negative consequences in mitochondrial homeostasis.

Distinct types of protease systems are involved in homeostasis regulation of mitochondrial morphology via balanced fusion and fission.

Mitochondrial morphology is dynamically regulated by fusion and fission. Several GTPase proteins control fusion and fission, and posttranslational modifications of these proteins are important for the regulation. However, it has not been clarified how the fusion and fission is balanced. Here, we report the molecular mechanism to regulate mitochondrial morphology in mammalian cells. Ablation of the mitochondrial fission, by repression of Drp1 or Mff, or by over-expression of MiD49 or MiD51, results in a reduction in the fusion GTPase mitofusins (Mfn1 and Mfn2) in outer membrane and long form of OPA1 (L-OPA1) in inner membrane. RNAi- or CRISPR-induced ablation of Drp1 in HeLa cells enhanced the degradation of Mfns via the ubiquitin-proteasome system (UPS). We further found that UPS-related protein BAT3/BAG6, here we identified as Mfn2-interacting protein, was implicated in the turnover of Mfns in the absence of mitochondrial fission. Ablation of the mitochondrial fission also enhanced the proteolytic cleavage of L-OPA1 to soluble S-OPA1, and the OPA1 processing was reversed by inhibition of the inner membrane protease OMA1 independent on the mitochondrial membrane potential. Our findings showed that the distinct degradation systems of the mitochondrial fusion proteins in different locations are enhanced in response to the mitochondrial morphology.

Mitochondrial dynamics regulate brown adiopcyte energy expenditure

Brown adipose tissue has gained much attention as a potential therapeutic approach for obesity and insulin resistance. This tissue is unique in its ability to go through hormonal mediated fast transition from efficient, ATP‐generating metabolism to inefficient, heat producing metabolism. As such, the brown adipocyte is a unique system in which the relationship between mitochondrial architecture and bioenergetics can be studied.Brown pre‐adipocyes were harvested from C57BL6/J mice and differentiated in culture. Oxygen consumption (using Seahorse XF24) and mitochondrial membrane potential (using TMRE and Zeiss LSM 710 confocal microscope) were measured before and after activation with NE (1 uM) and FFA (Plamitate or Oleate 0.4 mM).Immediately upon stimulation, mitochondrial networks underwent complete fragmentation and arrest of fusion. Fragmentation could only be achieved by beta3‐adrenergic stimulation and could not be mimicked by treatment with free fatty acids. Examination of the mitochondrial fusion proteins revealed that the long form of Opa1 was cleaved during the activation. Remarkably, within each cell, mitochondrial membrane depolarization and mitochondrial fragmentation propagated in a wave‐like fashion. To evaluate the role of mitochondrial fragmentation in energy expenditure, brown adipocytes were infected with a DRP1 dominant negative adenovirus. Inhibition of mitochondrial fission resulted in reduced levels of hormone stimulated oxygen consumption and membrane potential depolarization by 50%.Together, these data reveals that mitochondrial fission is essential for the activation of uncoupled respiration in brown adipocytes and its transition from bioenergetic efficient to inefficient metabolism.

Mitochondrial shape changes: orchestrating cell pathophysiology

Mitochondria are highly dynamic organelles, the location, size and distribution of which are controlled by a family of proteins that modulate mitochondrial fusion and fission. Recent evidence indicates that mitochondrial morphology is crucial for cell physiology, as changes in mitochondrial shape have been linked to neurodegeneration, calcium signalling, lifespan and cell death. Because immune cells contain few mitochondria, these organelles have been considered to have only a marginal role in this physiological context-which is conversely well characterized from the point of view of signalling. Nevertheless, accumulating evidence shows that mitochondrial dynamics have an impact on the migration and activation of immune cells and on the innate immune response. Here, we discuss the roles of mitochondrial dynamics in cell pathophysiology and consider how studying dynamics in the context of the immune system could increase our knowledge about the role of dynamics in key signalling cascades.

Fussy mitochondria fuse in response to stress

Fussy mitochondria fuse in response to stress