Mitochondrial dynamics in cell death and neurodegeneration

Mitochondria are highly dynamic organelles that continuously undergo two opposite processes, fission and fusion. Mitochondrial dynamics influence not only mitochondrial morphology, but also mitochondrial biogenesis, mitochondrial distribution within the cell, cell bioenergetics, and cell injury or death. Drp1 mediates mitochondrial fission, whereas Mfn1/2 and Opa1 control mitochondrial fusion. Neurons require large amounts of energy to carry out their highly specialized functions. Thus, mitochondrial dysfunction is a prominent feature in a variety of neurodegenerative diseases. Mutations of Mfn2 and Opa1 lead to neuropathies such as Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. Moreover, both Aβ peptide and mutant huntingtin protein induce mitochondrial fragmentation and neuronal cell death. In addition, mutants of Parkinson’s disease-related genes also show abnormal mitochondrial morphology. This review highlights our current understanding of abnormal mitochondrial dynamics relevant to neuronal synaptic loss and cell death in neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.     

Regulation of mitochondrial dynamics: convergences and divergences between yeast and vertebrates

In eukaryotic cells, the shape of mitochondria can be tuned to various physiological conditions by a balance of fusion and fission processes termed mitochondrial dynamics. Mitochondrial dynamics controls not only the morphology but also the function of mitochondria, and therefore is crucial in many aspects of a cell’s life. Consequently, dysfunction of mitochondrial dynamics has been implicated in a variety of human diseases including cancer. Several proteins important for mitochondrial fusion and fission have been discovered over the past decade. However, there is emerging evidence that there are as yet unidentified proteins important for these processes and that the fusion/fission machinery is not completely conserved between yeast and vertebrates. The recent characterization of several mammalian proteins important for the process that were not conserved in yeast, may indicate that the molecular mechanisms regulating and controlling the morphology and function of mitochondria are more elaborate and complex in vertebrates. This difference could possibly be a consequence of different needs in the different cell types of multicellular organisms. Here, we review recent advances in the field of mitochondrial dynamics. We highlight and discuss the mechanisms regulating recruitment of cytosolic Drp1 to the mitochondrial outer membrane by Fis1, Mff, and MIEF1 in mammals and the divergences in regulation of mitochondrial dynamics between yeast and vertebrates.     

A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Δψ _m) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Δψ _m required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Δψ _m were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted ρ⁰) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Δψ _m, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Δψ _m with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Δψ _m below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events.     

Bcl-2 family proteins and mitochondrial fission/fusion dynamics

Mitochondria are dynamic organelles and can undergo regulated fission/fragmentation to produce smaller organelles or, alternatively, can undergo fusion to produce tubular or net-like mitochondrial structures. Although some of the molecules that control mitochondrial fission and fusion are known, new molecules and pathways that control this process continue to be discovered, suggesting that this process is more complex than previously appreciated. In addition to their crucial role in the regulation of apoptosis, recent studies have implicated members of the Bcl-2 family in maintenance of the mitochondrial network. Here, we discuss the mechanisms governing mitochondrial fission/fusion and summarize current knowledge concerning the role of Bcl-2 family members in regulating mitochondrial fission/fusion dynamics.     

Mitochondrial dynamics as regulators of cancer biology

Mitochondria are dynamic organelles that supply energy required to drive key cellular processes, such as survival, proliferation, and migration. Critical to all of these processes are changes in mitochondrial architecture, a mechanical mechanism encompassing both fusion and fragmentation (fission) of the mitochondrial network. Changes to mitochondrial shape, size, and localization occur in a regulated manner to maintain energy and metabolic homeostasis, while deregulation of mitochondrial dynamics is associated with the onset of metabolic dysfunction and disease. In cancers, oncogenic signals that drive excessive proliferation, increase intracellular stress, and limit nutrient supply are all able to alter the bioenergetic and biosynthetic requirements of cancer cells. Consequently, mitochondrial function and shape rapidly adapt to these hostile conditions to support cancer cell proliferation and evade activation of cell death programs. In this review, we will discuss the molecular mechanisms governing mitochondrial dynamics and integrate recent insights into how changes in mitochondrial shape affect cellular migration, differentiation, apoptosis, and opportunities for the development of novel targeted cancer therapies.     

Biosynthesis and roles of phospholipids in mitochondrial fusion,division and mitophagy

Mitochondria move, fuse and divide in cells. The dynamic behavior of mitochondria is central to the control of their structure and function. Three conserved mitochondrial dynamin-related GTPases (i.e., mitofusin, Opa1 and Drp1 in mammals and Fzo1, Mgm1 and Dnm1 in yeast) mediate mitochondrial fusion and division. In addition to dynamins, recent studies demonstrated that phospholipids in mitochondria also play key roles in mitochondrial dynamics by interacting with dynamin GTPases and by directly changing the biophysical properties of the mitochondrial membranes. Changes in phospholipid composition also promote mitophagy, which is a selective mitochondrial degradation process that is mechanistically coupled to mitochondrial division. In this review, we will discuss the biogenesis and function of mitochondrial phospholipids.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells

Summary The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells.

The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Mitochondrial network remodeling: an important feature of myogenesis and skeletal muscle regeneration

The remodeling of the mitochondrial network is a critical process in maintaining cellular homeostasis and is intimately related to mitochondrial function. The interplay between the formation of new mitochondria (biogenesis) and the removal of damaged mitochondria (mitophagy) provide a means for the repopulation of the mitochondrial network. Additionally, mitochondrial fission and fusion serve as a bridge between biogenesis and mitophagy. In recent years, the importance of these processes has been characterised in multiple tissue- and cell-types, and under various conditions. In skeletal muscle, the robust remodeling of the mitochondrial network is observed, particularly after injury where large portions of the tissue/cell structures are damaged. The significance of mitochondrial remodeling in regulating skeletal muscle regeneration has been widely studied, with alterations in mitochondrial remodeling processes leading to incomplete regeneration and impaired skeletal muscle function. Needless to say, important questions related to mitochondrial remodeling and skeletal muscle regeneration still remain unanswered and require further investigation. Therefore, this review will discuss the known molecular mechanisms of mitochondrial network remodeling, as well as integrate these mechanisms and discuss their relevance in myogenesis and regenerating skeletal muscle.

     

Parvalbumin alters mitochondrial dynamics and affects cell morphology

The Ca²⁺-binding protein parvalbumin (PV) and mitochondria play important roles in Ca²⁺ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.     

Morphine but not fentanyl and methadone affects mitochondrial membrane potential by inducing nitric oxide release in glioma cells

We have observed that treatment of human glioma cells with morphine in the nanomolar range of concentration affects the mitochondrial membrane potential. The effect is specific to morphine and is mediated by naloxone-sensitive receptors, and is thus better observed on glioma cells treated with desipramine; moreover, the mitochondrial impairment is not inducible by fentanyl or methadone treatment and is prevented by the nitric oxide (NO) synthase inhibitor L-NAME. We conclude that in cultured glioma cells, the morphine-induced NO release decreases the mitochondrial membrane potential, as one might expect based on the rapid inhibition of the respiratory chain by NO. The identification of new intra-cellular pathways involved in the mechanism of action of morphine opens additional hypotheses, providing a novel rationale relevant to the therapy and toxicology of opioids.Received 19 August 2004; received after revision 16 September 2004; accepted 7 October 2004     

The biogenesis and function of eukaryotic porins.

Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial protein import with a few aberrations: a) porin contains an uncleaved NH2-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential delta psi, although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involves at least one receptor protein. Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.     

Host defense peptides as new weapons in cancer treatment

In the last decade intensive research has been conducted to determine the role of innate immunity host defense peptides (also termed antimicrobial peptides) in the killing of prokaryotic and eukaryotic cells. Many antimicrobial peptides damage the cellular membrane as part of their killing mechanism. However, it is not clear what makes cancer cells more susceptible to some of these peptides, and what the molecular mechanisms underlying these activities are. Two general mechanisms were suggested: (i) plasma membrane disruption via micellization or pore formation, and (ii) induction of apoptosis via mitochondrial membrane disruption. To be clinically used, these peptides need to combine high and specific anticancer activity with stability in serum. Although so far very limited, new studies have paved the way for promising anticancer host defense peptides with a new mode of action and with a broad spectrum of anticancer activity.     

Control of cell growth: Rag GTPases in activation of TORC1

The target of rapamycin (TOR) is a central regulator controlling cell growth. TOR is highly conserved from yeast to mammals, and is deregulated in human cancers and diabetes. TOR complex 1 (TORC1) integrates signals from growth factors, cellular energy status, stress, and amino acids to control cell growth, mitochondrial metabolism, and lipid biosynthesis. The mechanisms of growth factors and cellular energy status in regulating TORC1 have been well established, whereas the mechanism by which amino acid induces TORC1 remains largely unknown. Recent studies revealed that Rag GTPases play a central role in the regulation of TORC1 activation in response to amino acids. In this review, we will discuss the recent progress in our understanding of Rag GTPase-regulated TORC1 activation in response to amino acids. Particular focus will be given to the function of Rag GTPases in TORC1 activation and how Rag GTPases are regulated by amino acids.     

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action

A large variety of snake toxins evolved from PLA₂ digestive enzymes through a process of ‘accelerated evolution’. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA₂s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca²⁺ concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells. Received 05 March 2008; received after revision 08 April 2008; accepted 29 April 2008     

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.     

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.     

The biogenesis and function of eukaryotic porins

Summary Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial, protein import with a few aberrations: a) porin contains an,uncleaved NH₂-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential , although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involved at least one receptor protein.Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.