Giovanni Benard

Mitochondrial bioenergetics and structural network organization

Mitochondria form a dynamic network, and it remains unclear how the alternate configurations interact with bioenergetics properties. The metabolic signals that link mitochondrial structure to its functional states have not been fully characterized. In this report, we analyze the bidirectional relationships between mitochondrial morphology and function in living human cells. First, we determined the effect of mitochondrial fission on energy production by using small interfering RNA (siRNA) targeting DRP1, which revealed the importance of membrane fluidity on the control of bioenergetics. Second, we followed the effect of rotenone, a specific inhibitor of respiratory chain complex I, which causes large structural perturbations, once a threshold was reached. Last, we followed changes in the mitochondrial network configuration in human cells that had been treated with modulators of oxidative phosphorylation, and in fibroblasts from two patients with mitochondrial disease where the respiratory rate, ΔΨ and the generation of reactive oxygen species (ROS) were measured. Our data demonstrate that the relationship between mitochondrial network organization and bioenergetics is bidirectional, and we provide a model for analyzing the metabolic signals involved in this crosstalk.

Mitochondrial CB1 receptors regulate neuronal energy metabolism

The mammalian brain is one of the organs with the highest energy demands, and mitochondria are key determinants of its functions. Here we show that the type-1 cannabinoid receptor (CB1) is present at the membranes of mouse neuronal mitochondria (mtCB1), where it directly controls cellular respiration and energy production. Through activation of mtCB1 receptors, exogenous cannabinoids and in situ endocannabinoids decreased cyclic AMP concentration, protein kinase A activity, complex I enzymatic activity and respiration in neuronal mitochondria. In addition, intracellular CB1 receptors and mitochondrial mechanisms contributed to endocannabinoid-dependent depolarization-induced suppression of inhibition in the hippocampus. Thus, mtCB1 receptors directly modulate neuronal energy metabolism, revealing a new mechanism of action of G protein–coupled receptor signaling in the brain.

Ultrastructure of the Mitochondrion and Its Bearing on Function and Bioenergetics

The recently ascertained network and dynamic organization of the mitochondrion, as well as the demonstration of energy proteins and metabolites subcompartmentalization, have led to a reconsideration of the relationships between organellar form and function. In particular, the impact of mitochondrial morphological changes on bioenergetics is inseparable. Several observations indicate that mitochondrial energy production may be controlled by structural rearrangements of the organelle both interiorly and globally, including the remodeling of cristae morphology and elongation or fragmentation of the tubular network organization, respectively. These changes are mediated by fusion or fission reactions in response to physiological signals that remain unidentified. They lead to important changes in the internal diffusion of energy metabolites, the sequestration and conduction of the electric membrane potential (Delta Psi), and possibly the delivery of newly synthesized ATP to various cellular areas. Moreover, the physiological or even pathological context also determines the morphology of the mitochondrion, suggesting a tight and mutual control between mitochondrial form and bioenergetics. In this review, we delve into the link between mitochondrial structure and energy metabolism.

Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by different patterns of airway remodeling, which all include an increased mass of bronchial smooth muscle (BSM). A remaining major question concerns the mechanisms underlying such a remodeling of BSM. Because mitochondria play a major role in both cell proliferation and apoptosis, we hypothesized that mitochondrial activation in BSM could play a role in this remodeling. We describe that both the mitochondrial mass and oxygen consumption were higher in the BSM from asthmatic subjects than in that from both COPD and controls. This feature, which is specific to asthma, was related to an enhanced mitochondrial biogenesis through up-regulation of peroxisome proliferator-activated receptor γ coactivator (PGC)–1α, nuclear respiratory factor-1, and mitochondrial transcription factor A. The priming event of such activation was an alteration in BSM calcium homeostasis. BSM cell apoptosis was not different in the three groups of subjects. Asthmatic BSM was, however, characterized by increased cell growth and proliferation. Both characteristics were completely abrogated in mitochondria-deficient asthmatic BSM cells. Conversely, in both COPD and control BSM cells, induction of mitochondrial biogenesis reproduced these characteristics. Thus, BSM in asthmatic patients is characterized by an altered calcium homeostasis that increases mitochondrial biogenesis, which, in turn, enhances cell proliferation, leading to airway remodeling.

Rheb Regulates Mitophagy Induced by Mitochondrial Energetic Status

Mitophagy has been recently described as a mechanism of elimination of damaged organelles. Although the regulation of the amount of mitochondria is a core issue concerning cellular energy homeostasis, the relationship between mitochondrial degradation and energetic activity has not yet been considered. Here, we report that the stimulation of mitochondrial oxidative phosphorylation enhances mitochondrial renewal by increasing its degradation rate. Upon high oxidative phosphorylation activity, we found that the small GTPase Rheb is recruited to the mitochondrial outer membrane. This mitochondrial localization of Rheb promotes mitophagy through a physical interaction with the mitochondrial autophagic receptor Nix and the autophagosomal protein LC3-II. Thus, Rheb-dependent mitophagy contributes to the maintenance of optimal mitochondrial energy production. Our data suggest that mitochondrial degradation contributes to a bulk renewal of the organelle in order to prevent mitochondrial aging and to maintain the efficiency of oxidative phosphorylation.

Multi-site control and regulation of mitochondrial energy production

With the extraordinary progress of mitochondrial science and cell biology, novel biochemical pathways have emerged as strategic points of bioenergetic regulation and control. They include mitochondrial fusion, fission and organellar motility along microtubules and microfilaments (mitochondrial dynamics), mitochondrial turnover (biogenesis and degradation), and mitochondrial phospholipids synthesis. Yet, much is still unknown about the mutual interaction between mitochondrial energy state, biogenesis, dynamics and degradation. Meanwhile, clinical research into metabolic abnormalities in tumors as diverse as renal carcinoma, glioblastomas, paragangliomas or skin leiomyomata, has designated new genes, oncogenes and oncometabolites involved in the regulation of cellular and mitochondrial energy production. Furthermore, the examination of rare neurological diseases such as Charcot-Marie Tooth type 2a, Autosomal Dominant Optic Atrophy, Lethal Defect of Mitochondrial and Peroxisomal Fission, or Spastic Paraplegia suggested involvement of MFN2, OPA1/3, DRP1 or Paraplegin, in the auxiliary control of mitochondrial energy production. Lastly, advances in the understanding of mitochondrial apoptosis have suggested a supplementary role for Bcl2 or Bax in the regulation of mitochondrial respiration and dynamics, which has fostered the investigation of alternative mechanisms of energy regulation. In this review, we discuss the regulatory mechanisms of cellular and mitochondrial energy production, and we emphasize the importance of the study of rare neurological diseases in addition to more common disorders such as cancer, for the fundamental understanding of cellular and mitochondrial energy production.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio).

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13 microg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

A cannabinoid link between mitochondria and memory.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

REEP1 mutations in SPG31: Frequency, mutational spectrum, and potential association with mitochondrial morpho-functional dysfunction†

Hereditary spastic paraplegias (HSP) constitute a heterogeneous group of neurodegenerative disorders characterized at least by slowly progressive spasticity of the lower limbs. Mutations in REEP1 were recently associated with a pure dominant HSP, SPG31. We sequenced all exons of REEP1 and searched for rearrangements by multiplex ligation‐dependent probe amplification (MLPA) in a large panel of 175 unrelated HSP index patients from kindreds with dominant inheritance (AD‐HSP), with either pure (n = 102) or complicated (n = 73) forms of the disease, after exclusion of other known HSP genes. We identified 12 different heterozygous mutations, including two exon deletions, associated with either a pure or a complex phenotype. The overall mutation rate in our clinically heterogeneous sample was 4.5% in French families with AD‐HSP. The phenotype was restricted to pyramidal signs in the lower limbs in most patients but nine had a complex phenotype associating axonal peripheral neuropathy (= 5/11 patients) including a Silver‐like syndrome in one patient, and less frequently cerebellar ataxia, tremor, dementia. Interestingly, we evidenced abnormal mitochondrial network organization in fibroblasts of one patient in addition to defective mitochondrial energy production in both fibroblasts and muscle, but whether these anomalies are directly or indirectly related to the mutations remains uncertain. Hum Mutat 32:1118–1127, 2011. ©2011 Wiley‐Liss, Inc.

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases.

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.