Publications by authors named "Etienne Hebert-Chatelain"

35 Publications

Evaluating the mitochondrial activity and inflammatory state of dimethyl sulfoxide differentiated PLB-985 cells.

Mol Immunol 2021 Apr 7;135:1-11. Epub 2021 Apr 7.

Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada; New Brunswick Center for Precision Medicine, Moncton, NB, Canada. Electronic address:

Neutrophils play a key role in the innate immunity with their ability to generate and release inflammatory mediators that promote the inflammatory response and consequently restore the hemostasis. As active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis, and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been suggested as an important therapeutic approach by numerous researchers. The neutrophils have a relatively short lifespan, which can be problematic for some in vitro experiments. To address this issue, researchers have used the human monomyelocyte cell line PLB-985 as an in vitro model for exploratory experiments addressing neutrophil-related physiological functions. PLB-985 cells can be differentiated into a neutrophil-like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB-985 affects important features related to the neutrophil's normal functions (i.e., mitochondrial activity, eicosanoid production) remains elusive, and characterizing these changes will be the focal point of this study. Our results indicate that the differentiation affected the proliferation of PLB-985 cells, without inducing apoptosis. A significant decrease in mitochondrial respiration was observed in differentiated PLB-985 cells. However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A, superoxide dismutase 2, ubiquinol-cytochrome c reductase core protein 2 and ATP synthase subunit α in differentiated PLB-985 cells. Finally, eicosanoids (leukotriene B, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells. In summary, our data demonstrate that the differentiation process of PLB-985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulation of the inflammatory state of the cell. Of importance, our data suggest that PLB-985 cells could be suitable in vitro candidates to study mitochondrial-related dysfunctions in inflammatory diseases.
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http://dx.doi.org/10.1016/j.molimm.2021.03.026DOI Listing
April 2021

Subcellular specificity of cannabinoid effects in striatonigral circuits.

Neuron 2021 May 25;109(9):1513-1526.e11. Epub 2021 Mar 25.

INSERM, U1215 NeuroCentre Magendie, Endocannabinoids and Neuroadaptation, Bordeaux, France; University of Bordeaux, Bordeaux, France.

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB receptors (pmCB), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB receptors (mtCB) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB receptor signaling within striatonigral circuits determines multimodal control of behavior.
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http://dx.doi.org/10.1016/j.neuron.2021.03.007DOI Listing
May 2021

Characterization of the interactome of c-Src within the mitochondrial matrix by proximity-dependent biotin identification.

Mitochondrion 2021 03 4;57:257-269. Epub 2021 Jan 4.

Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada; University of Moncton, Dept. of Biology, Moncton, NB, Canada. Electronic address:

C-Src kinase is localized in several subcellular compartments, including mitochondria where it is involved in the regulation of organelle functions and overall metabolism. Surprisingly, the characterization of the intramitochondrial Src interactome has never been fully determined. Using in vitro proximity-dependent biotin identification (BioID) coupled to mass spectrometry, we identified 51 candidate proteins that may interact directly or indirectly with c-Src within the mitochondrial matrix. Pathway analysis suggests that these proteins are involved in a large array of mitochondrial functions such as protein folding and import, mitochondrial organization and transport, oxidative phosphorylation, tricarboxylic acid cycle and metabolism of amino and fatty acids. Among these proteins, we identified 24 tyrosine phosphorylation sites in 17 mitochondrial proteins (AKAP1, VDAC1, VDAC2, VDAC3, LonP1, Hsp90, SLP2, PHB2, MIC60, UBA1, EF-Tu, LRPPRC, ACO2, OAT, ACAT1, ETFβ and ATP5β) as potential substrates for intramitochondrial Src using in silico prediction of tyrosine phospho-sites. Interaction of c-Src with SLP2 and ATP5β was confirmed using coimmunoprecipitation. This study suggests that the intramitochondrial Src could target several proteins and regulate different mitochondrial functions.
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http://dx.doi.org/10.1016/j.mito.2020.12.012DOI Listing
March 2021

Insight into the Interactome of Intramitochondrial PKA Using Biotinylation-Proximity Labeling.

Int J Mol Sci 2020 Nov 5;21(21). Epub 2020 Nov 5.

Department of Biology, University of Moncton, Moncton, NB E1A 3E9, Canada.

Mitochondria are fully integrated in cell signaling. Reversible phosphorylation is involved in adjusting mitochondrial physiology to the cellular needs. Protein kinase A (PKA) phosphorylates several substrates present at the external surface of mitochondria to maintain cellular homeostasis. However, few targets of PKA located inside the organelle are known. The aim of this work was to characterize the impact and the interactome of PKA located inside mitochondria. Our results show that the overexpression of intramitochondrial PKA decreases cellular respiration and increases superoxide levels. Using proximity-dependent biotinylation, followed by LC-MS/MS analysis and in silico phospho-site prediction, we identified 21 mitochondrial proteins potentially targeted by PKA. We confirmed the interaction of PKA with TIM44 using coimmunoprecipitation and observed that TIM44-S80 is a key residue for the interaction between the protein and the kinase. These findings provide insights into the interactome of intramitochondrial PKA and suggest new potential mechanisms in the regulation of mitochondrial functions.
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http://dx.doi.org/10.3390/ijms21218283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663848PMC
November 2020

Role of the Mitochondrial Pyruvate Carrier in the Occurrence of Metabolic Inflexibility in Exposed to Dietary Sucrose.

Metabolites 2020 Oct 14;10(10). Epub 2020 Oct 14.

Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB E1A 3E9, Canada.

Excess dietary carbohydrates are linked to dysregulation of metabolic pathways converging to mitochondria and metabolic inflexibility. Here, we determined the role of the mitochondrial pyruvate carrier (MPC) in the occurrence of this metabolic inflexibility in wild-type (WT) and MPC1-deficient (MPC1) flies that were exposed to diets with different sucrose concentrations for 15-25 days (Standard Diet: SD, Medium-Sucrose Diet: MSD, and High-Sucrose Diet: HSD). Our results showed that MPC1 flies had lower mitochondrial respiration rates than WT flies on the SD and MSD. However, when exposed to the HSD, WT flies displayed decreased mitochondrial respiration rates compared to MPC1 flies. WT flies exposed to the HSD also displayed increased proline contribution and slightly decreased expression. Surprisingly, when fed the MSD and the HSD, few metabolites were altered in WT flies whereas MPC1 flies display significant accumulation of glycogen, glucose, fructose, lactate, and glycerol. Overall, this suggests that metabolic inflexibility starts to occur in WT flies after 15-25 days of exposure to the HSD whereas the MPC1 flies display metabolic inflexibility independently of the diet provided. This study thus highlights the involvement of MPC as an essential protein in Drosophila to maintain proper metabolic homeostasis during changes in dietary resources.
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http://dx.doi.org/10.3390/metabo10100411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602203PMC
October 2020

Metabolic Characterization and Consequences of Mitochondrial Pyruvate Carrier Deficiency in .

Metabolites 2020 Sep 6;10(9). Epub 2020 Sep 6.

Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB E1A 3E9, Canada.

In insect, pyruvate is generally the predominant oxidative substrate for mitochondria. This metabolite is transported inside mitochondria via the mitochondrial pyruvate carrier (MPC), but whether and how this transporter controls mitochondrial oxidative capacities in insects is still relatively unknown. Here, we characterize the importance of pyruvate transport as a metabolic control point for mitochondrial substrate oxidation in two genotypes of an insect model, , differently expressing MPC1, an essential protein for the MPC function. We evaluated the kinetics of pyruvate oxidation, mitochondrial oxygen consumption, metabolic profile, activities of metabolic enzymes, and climbing abilities of wild-type (WT) flies and flies harboring a deficiency in MPC1 (MPC1). We hypothesized that MPC1 deficiency would cause a metabolic reprogramming that would favor the oxidation of alternative substrates. Our results show that the MPC1 flies display significantly reduced climbing capacity, pyruvate-induced oxygen consumption, and enzymatic activities of pyruvate kinase, alanine aminotransferase, and citrate synthase. Moreover, increased proline oxidation capacity was detected in MPC1 flies, which was associated with generally lower levels of several metabolites, and particularly those involved in amino acid catabolism such as ornithine, citrulline, and arginosuccinate. This study therefore reveals the flexibility of mitochondrial substrate oxidation allowing Drosophila to maintain cellular homeostasis.
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http://dx.doi.org/10.3390/metabo10090363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570025PMC
September 2020

Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Nature 2020 07 8;583(7817):603-608. Epub 2020 Jul 8.

INSERM, U1215 NeuroCentre Magendie, Bordeaux, France.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.
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http://dx.doi.org/10.1038/s41586-020-2470-yDOI Listing
July 2020

Multi-omics Reveal that c-Src Modulates the Mitochondrial Phosphotyrosine Proteome and Metabolism According to Nutrient Availability.

Cell Physiol Biochem 2020 May;54(4):517-537

Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada,

Background/aims: Src kinase family members, including c-Src, are involved in numerous signaling pathways and have been observed inside different cellular compartments. Notably, c-Src modulates carbohydrate and fatty acid metabolism and is involved in the metabolic rewiring of cancer cells. This kinase is found within mitochondria where it targets different proteins to impact on the organelle functions and overall metabolism. Surprisingly, no global metabolic characterization of Src has been performed although c-Src knock-out mice have been available for 30 years. Considering that c-Src is sensitive to various metabolites, c-Src might represent a crucial player in metabolic adjustments induced by nutrient stress. The aim of this work was to characterize the impact of c-Src on mitochondrial activity and overall metabolism using multi-omic characterization.

Methods: Src and Src mice were fed ad libitum or fasted during 24h and were then analyzed using multi-omics.

Results: We observed that deletion of c-Src is linked to lower phosphorylation of Y412-NDUFA8, inhibition of oxygen consumption and accumulation of metabolites involved in glycolysis, TCA cycle and amino acid metabolism in mice fed ad libitum. Finally, metabolomics and (phosphotyrosine) proteomics are differently impacted by Src according to nutrient availability.

Conclusion: The findings presented here highlight that c-Src reduces mitochondrial metabolism and impacts the metabolic adjustment induced by nutrient stress.
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http://dx.doi.org/10.33594/000000237DOI Listing
May 2020

Damage in Mitochondrial DNA Associated with Parkinson's Disease.

DNA Cell Biol 2020 Aug 12;39(8):1421-1430. Epub 2020 May 12.

Department of Biology and Université de Moncton, Moncton, Canada.

Mitochondria are the only organelles that contain their own genetic material (mtDNA). Mitochondria are involved in several key physiological functions, including ATP production, Ca homeostasis, and metabolism of neurotransmitters. Since these organelles perform crucial processes to maintain neuronal homeostasis, mitochondrial dysfunctions can lead to various neurodegenerative diseases. Several mitochondrial proteins involved in ATP production are encoded by mtDNA. Thus, any mtDNA alteration can ultimately lead to mitochondrial dysfunction and cell death. Accumulation of mutations, deletions, and rearrangements in mtDNA has been observed in animal models and patients suffering from Parkinson's disease (PD). Also, specific inherited variations associated with mtDNA genetic groups (known as mtDNA haplogroups) are associated with lower or higher risk of developing PD. Consequently, mtDNA alterations should now be considered important hallmarks of this neurodegenerative disease. This review provides an update about the role of mtDNA alterations in the physiopathology of PD.
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http://dx.doi.org/10.1089/dna.2020.5398DOI Listing
August 2020

Intramitochondrial Src kinase links mitochondrial dysfunctions and aggressiveness of breast cancer cells.

Cell Death Dis 2019 12 9;10(12):940. Epub 2019 Dec 9.

Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada.

High levels and activity of Src kinase are common among breast cancer subtypes, and several inhibitors of the kinase are currently tested in clinical trials. Alterations in mitochondrial activity is also observed among the different types of breast cancer. Src kinase is localized in several subcellular compartments, including mitochondria where it targets several proteins to modulate the activity of the organelle. Although the subcellular localization of other oncogenes modulates the potency of known treatments, nothing is known about the specific role of intra-mitochondrial Src (mtSrc) in breast cancer. The aim of this work was to determine whether mtSrc kinase has specific impact on breast cancer cells. We first observed that activity of mtSrc is higher in breast cancer cells of the triple negative subtype. Over-expression of Src specifically targeted to mitochondria reduced mtDNA levels, mitochondrial membrane potential and cellular respiration. These alterations of mitochondrial functions led to lower cellular viability, shorter cell cycle and increased invasive capacity. Proteomic analyses revealed that mtSrc targets the mitochondrial single-stranded DNA-binding protein, a regulator of mtDNA replication. Our findings suggest that mtSrc promotes aggressiveness of breast cancer cells via phosphorylation of mitochondrial single-stranded DNA-binding protein leading to reduced mtDNA levels and mitochondrial activity. This study highlights the importance of considering the subcellular localization of Src kinase in the development of potent therapy for breast cancer.
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http://dx.doi.org/10.1038/s41419-019-2134-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901437PMC
December 2019

Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle.

Cell Rep 2019 03;26(13):3784-3797.e8

Department of Biology, University of Padova, Via Ugo Bassi 58/b, 35131 Padova, Italy; CRIBI Biotechnology Centre, University of Padova, Via Ugo Bassi 58/b, 35131 Padova, Italy; CIR-Myo Myology Center, University of Padova, Via Ugo Bassi 58/b, 35131 Padova, Italy. Electronic address:

Skeletal muscle is composed of different myofiber types that preferentially use glucose or lipids for ATP production. How fuel preference is regulated in these post-mitotic cells is largely unknown, making this issue a key question in the fields of muscle and whole-body metabolism. Here, we show that microRNAs (miRNAs) play a role in defining myofiber metabolic profiles. mRNA and miRNA signatures of all myofiber types obtained at the single-cell level unveiled fiber-specific regulatory networks and identified two master miRNAs that coordinately control myofiber fuel preference and mitochondrial morphology. Our work provides a complete and integrated mouse myofiber type-specific catalog of gene and miRNA expression and establishes miR-27a-3p and miR-142-3p as regulators of lipid use in skeletal muscle.
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http://dx.doi.org/10.1016/j.celrep.2019.02.105DOI Listing
March 2019

Redox mechanism of levobupivacaine cytostatic effect on human prostate cancer cells.

Redox Biol 2018 09 31;18:33-42. Epub 2018 May 31.

Univ. Bordeaux, 146 rue Léo Saignat, F-33076 Bordeaux, France; INSERM U1211, F-33076 Bordeaux, France; Cellomet, CGFB, 146 Rue léo Saignat, F-33000 Bordeaux, France. Electronic address:

Anti-cancer effects of local anesthetics have been reported but the mode of action remains elusive. Here, we examined the bioenergetic and REDOX impact of levobupivacaine on human prostate cancer cells (DU145) and corresponding non-cancer primary human prostate cells (BHP). Levobupivacaine induced a combined inhibition of glycolysis and oxidative phosphorylation in cancer cells, resulting in a reduced cellular ATP production and consecutive bioenergetic crisis, along with reactive oxygen species generation. The dose-dependent inhibition of respiratory chain complex I activity by levobupivacaine explained the alteration of mitochondrial energy fluxes. Furthermore, the potency of levobupivacaine varied with glucose and oxygen availability as well as the cellular energy demand, in accordance with a bioenergetic anti-cancer mechanism. The levobupivacaine-induced bioenergetic crisis triggered cytostasis in prostate cancer cells as evidenced by a S-phase cell cycle arrest, without apoptosis induction. In DU145 cells, levobupivacaine also triggered the induction of autophagy and blockade of this process potentialized the anti-cancer effect of the local anesthetic. Therefore, our findings provide a better characterization of the REDOX mechanisms underpinning the anti-effect of levobupivacaine against human prostate cancer cells.
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http://dx.doi.org/10.1016/j.redox.2018.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019688PMC
September 2018

Mitochondrial cAMP-PKA signaling: What do we really know?

Biochim Biophys Acta Bioenerg 2018 09 23;1859(9):868-877. Epub 2018 Apr 23.

Université de Moncton, Department of Biology, 18 Av Antonine Maillet, Moncton, NB E1A2Y5, Canada. Electronic address:

Mitochondria are key organelles for cellular homeostasis. They generate the most part of ATP that is used by cells through oxidative phosphorylation. They also produce reactive oxygen species, neurotransmitters and other signaling molecules. They are important for calcium homeostasis and apoptosis. Considering the role of this organelle, it is not surprising that most mitochondrial dysfunctions are linked to the development of pathologies. Various mechanisms adjust mitochondrial activity according to physiological needs. The cAMP-PKA signaling emerged in recent years as a direct and powerful mean to regulate mitochondrial functions. Multiple evidence demonstrates that such pathway can be triggered from cytosol or directly within mitochondria. Notably, specific anchor proteins target PKA to mitochondria whereas enzymes necessary for generation and degradation of cAMP are found directly in these organelles. Mitochondrial PKA targets proteins localized in different compartments of mitochondria, and related to various functions. Alterations of mitochondrial cAMP-PKA signaling affect the development of several physiopathological conditions, including neurodegenerative diseases. It is however difficult to discriminate between the effects of cAMP-PKA signaling triggered from cytosol or directly in mitochondria. The specific roles of PKA localized in different mitochondrial compartments are also not completely understood. The aim of this work is to review the role of cAMP-PKA signaling in mitochondrial (patho)physiology.
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http://dx.doi.org/10.1016/j.bbabio.2018.04.005DOI Listing
September 2018

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue.

J Vis Exp 2018 04 7(134). Epub 2018 Apr 7.

Département de chimie et biochimie, Université de Moncton;

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.
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http://dx.doi.org/10.3791/57376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933415PMC
April 2018

Identification of proteins interacting with the mitochondrial small heat shock protein Hsp22 of Drosophila melanogaster: Implication in mitochondrial homeostasis.

PLoS One 2018 6;13(3):e0193771. Epub 2018 Mar 6.

Laboratoire de Génétique Cellulaire et Développementale, IBIS and PROTEO, Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Faculté de Médecine, Université Laval, Québec, Canada.

The small heat shock protein (sHsp) Hsp22 from Drosophila melanogaster (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient in vitro chaperone, its function within mitochondria in vivo remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839585PMC
June 2018

Linking Mitochondria and Synaptic Transmission: The CB1 Receptor.

Bioessays 2017 Dec 23;39(12). Epub 2017 Oct 23.

Department of Biology, Université de Moncton, 18 Av Antonine Maillet, Moncton, New Brunswick, Canada.

CB1 receptors are functionally present within brain mitochondria (mtCB1), although they are usually considered specifically targeted to plasma membrane. Acute activation of mtCB1 alters mitochondrial ATP generation, synaptic transmission, and memory performance. However, the detailed mechanism linking disrupted mitochondrial metabolism and synaptic transmission is still uncharacterized. CB1 receptors are among the most abundant G protein-coupled receptors in the brain and impact on several processes, including fear coping, anxiety, stress, learning, and memory. Mitochondria perform several key physiological processes for neuronal homeostasis, including production of ATP and reactive oxygen species, calcium buffering, metabolism of neurotransmitters, and apoptosis. It is therefore possible that acute activation of mtCB1 impacts on these different mitochondrial functions to modulate synaptic transmission. In reviewing and integrating across the literature in this area, we describe the possible mechanisms involved in the regulation of brain physiology by mtCB1 receptors.
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http://dx.doi.org/10.1002/bies.201700126DOI Listing
December 2017

[Mitochondria link between cannabinoid and memory].

Med Sci (Paris) 2017 Jun-Jul;33(6-7):579-581. Epub 2017 Jul 19.

Inserm U862, Neurocentre Magendie, université de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France.

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http://dx.doi.org/10.1051/medsci/20173306007DOI Listing
April 2019

Functional Analysis of Mitochondrial CB1 Cannabinoid Receptors (mtCB1) in the Brain.

Methods Enzymol 2017 14;593:143-174. Epub 2017 Jul 14.

Université de Moncton, Moncton, NB, Canada. Electronic address:

Recent evidence indicates that, besides its canonical localization at cell plasma membranes, the type-1 cannabinoid receptor, CB1 is functionally present at brain and muscle mitochondrial membranes (mtCB1). Through mtCB1 receptors, cannabinoids can directly regulate intramitochondrial signaling and respiration. This new and surprising discovery paves the way to new potential fields of research, dealing with the direct impact of G protein-coupled receptors on bioenergetic processes and its functional implications. In this chapter, we summarize some key experimental approaches established in our laboratories to identify anatomical, biochemical, and functional features of mtCB1 receptors in the brain. In particular, we describe the procedures to obtain reliable and controlled detection of mtCB1 receptors by immunogold electromicroscopy and by immunoblotting methods. Then, we address the study of direct cannabinoid effects on the electron transport system and oxidative phosphorylation. Finally, we present a functional example of the impact of mtCB1 receptors on mitochondrial mobility in cultured neurons. Considering the youth of the field, these methodological approaches will very likely be improved and refined in the future, but this chapter aims at presenting the methods that are currently used and, in particular, at underlining the need of rigorous controls to obtain reliable results. We hope that this chapter might help scientists becoming interested in this new and exciting field of research.
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http://dx.doi.org/10.1016/bs.mie.2017.06.023DOI Listing
April 2018

Sirtuin 5 protects mitochondria from fragmentation and degradation during starvation.

Biochim Biophys Acta Mol Cell Res 2017 Jan 15;1864(1):169-176. Epub 2016 Nov 15.

Department of Biology, University of Moncton, NB, Canada. Electronic address:

During starvation, intra-mitochondrial sirtuins, NAD sensitive deacylating enzymes that modulate metabolic homeostasis and survival, directly adjust mitochondrial function to nutrient availability; concomitantly, mitochondria elongate to escape autophagic degradation. However, whether sirtuins also impinge on mitochondrial dynamics is still uncharacterized. Here we show that the mitochondrial Sirtuin 5 (Sirt5) is essential for starvation induced mitochondrial elongation. Deletion of Sirt5 in mouse embryonic fibroblasts increased levels of mitochondrial dynamics of 51kDa protein and mitochondrial fission protein 1, leading to mitochondrial accumulation of the pro-fission dynamin related protein 1 and to mitochondrial fragmentation. During starvation, Sirt5 deletion blunted mitochondrial elongation, resulting in increased mitophagy. Our results indicate that starvation induced mitochondrial elongation and evasion from autophagic degradation requires the energy sensor Sirt5.
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http://dx.doi.org/10.1016/j.bbamcr.2016.10.015DOI Listing
January 2017

A cannabinoid link between mitochondria and memory.

Nature 2016 11 9;539(7630):555-559. Epub 2016 Nov 9.

INSERM U1215, NeuroCentre Magendie, Bordeaux 33077, France.

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 (mtCB) 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 mtCB receptors. Genetic exclusion of CB receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB receptors signal through intra-mitochondrial Gα 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 mtCB 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.
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http://dx.doi.org/10.1038/nature20127DOI Listing
November 2016

Cannabinoid control of brain bioenergetics: Exploring the subcellular localization of the CB1 receptor.

Mol Metab 2014 Jul 2;3(4):495-504. Epub 2014 Apr 2.

INSERM U862, NeuroCentre Magendie, 33077 Bordeaux, France ; University of Bordeaux, 33077 Bordeaux, France.

Brain mitochondrial activity is centrally involved in the central control of energy balance. When studying mitochondrial functions in the brain, however, discrepant results might be obtained, depending on the experimental approaches. For instance, immunostaining experiments and biochemical isolation of organelles expose investigators to risks of false positive and/or false negative results. As an example, the functional presence of cannabinoid type 1 (CB1) receptors on brain mitochondrial membranes (mtCB1) was recently reported and rapidly challenged, claiming that the original observation was likely due to artifact results. Here, we addressed this issue by directly comparing the procedures used in the two studies. Our results show that the use of appropriate controls and quantifications allows detecting mtCB1 receptor with CB1 receptor antibodies, and that, if mitochondrial fractions are enriched and purified, CB1 receptor agonists reliably decrease respiration in brain mitochondria. These data further underline the importance of adapted experimental procedures to study brain mitochondrial functions.
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http://dx.doi.org/10.1016/j.molmet.2014.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060213PMC
July 2014

Studying mitochondrial CB1 receptors: Yes we can.

Mol Metab 2014 Jul 12;3(4):339. Epub 2014 Apr 12.

INSERM U862, NeuroCentre Magendie, 33077 Bordeaux, France ; University of Bordeaux, 33077 Bordeaux, France.

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http://dx.doi.org/10.1016/j.molmet.2014.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060283PMC
July 2014

Pregnenolone can protect the brain from cannabis intoxication.

Science 2014 Jan;343(6166):94-8

INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U862, F-33000 Bordeaux, France.

Pregnenolone is considered the inactive precursor of all steroid hormones, and its potential functional effects have been largely uninvestigated. The administration of the main active principle of Cannabis sativa (marijuana), Δ(9)-tetrahydrocannabinol (THC), substantially increases the synthesis of pregnenolone in the brain via activation of the type-1 cannabinoid (CB1) receptor. Pregnenolone then, acting as a signaling-specific inhibitor of the CB1 receptor, reduces several effects of THC. This negative feedback mediated by pregnenolone reveals a previously unknown paracrine/autocrine loop protecting the brain from CB1 receptor overactivation that could open an unforeseen approach for the treatment of cannabis intoxication and addiction.
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http://dx.doi.org/10.1126/science.1243985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057431PMC
January 2014

Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes.

Mol Metab 2013 9;2(4):393-404. Epub 2013 Aug 9.

INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U862, F-33000 Bordeaux, France ; University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U862, F-33000 Bordeaux, France ; Neuropharmacology, Institute of Neurosciences, Université Catholique de Louvain, Av. Hippocrate 54, B1.54.10-10, 1200 Bruxelles, Belgium.

Type-1 cannabinoid (CB1) and leptin (ObR) receptors regulate metabolic and astroglial functions, but the potential links between the two systems in astrocytes were not investigated so far. Genetic and pharmacological manipulations of CB1 receptor expression and activity in cultured cortical and hypothalamic astrocytes demonstrated that cannabinoid signaling controls the levels of ObR expression. Lack of CB1 receptors also markedly impaired leptin-mediated activation of signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) in astrocytes. In particular, CB1 deletion determined a basal overactivation of STAT5, thereby leading to the downregulation of ObR expression, and leptin failed to regulate STAT5-dependent glycogen storage in the absence of CB1 receptors. These results show that CB1 receptors directly interfere with leptin signaling and its ability to regulate glycogen storage, thereby representing a novel mechanism linking endocannabinoid and leptin signaling in the regulation of brain energy storage and neuronal functions.
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http://dx.doi.org/10.1016/j.molmet.2013.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3854987PMC
December 2013

Rheb regulates mitophagy induced by mitochondrial energetic status.

Cell Metab 2013 May 18;17(5):719-30. Epub 2013 Apr 18.

EA4576, Maladies Rares: Génétique et Métabolisme, 33000 Bordeaux Cedex, France.

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.
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http://dx.doi.org/10.1016/j.cmet.2013.03.014DOI Listing
May 2013

Src kinases are important regulators of mitochondrial functions.

Int J Biochem Cell Biol 2013 Jan 20;45(1):90-8. Epub 2012 Aug 20.

INSERM-U688 Physiopathologie Mitochondriale, Université de Bordeaux, 146 rue Léo Saignat, Bordeaux 33076, France.

Mitochondria produce the most part of the energy used by the cells. This energetic production occurs through the oxidative phosphorylation (OXPHOS) process. Mitochondrial functions such as OXPHOS need to be tightly regulated to respect the needs of cells. Phosphorylation of mitochondrial proteins now appears as a major regulation pathway of mitochondrial functions. Several kinases and phosphatases are specifically targeted to mitochondria where they modulate mitochondrial functions. However, we still poorly understand the extent of tyrosine phosphorylation events on mitochondrial metabolism. Among the tyrosine-kinases observed in mitochondria, Src kinases emerge as key players. In the past years, several mitochondrial proteins were shown to be substrates of Src kinases. Notably, these kinases can impact greatly OXPHOS and apoptosis. Important regulators of Src kinases activity are also observed in mitochondria. The aim of this review is to summarize the recent findings on how overall mitochondrial tyrosine phosphorylation events and more specifically Src kinases can influence mitochondrial functions. The different mechanisms of Src kinases regulation and translocation into mitochondria will be also discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.
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http://dx.doi.org/10.1016/j.biocel.2012.08.014DOI Listing
January 2013

Mitochondrial CB₁ receptors regulate neuronal energy metabolism.

Nat Neurosci 2012 Mar 4;15(4):558-64. Epub 2012 Mar 4.

INSERM, Neurocentre Magendie, Physiopathologie de plasticité neuronale, Endocannabinoids and Neuroadaptation, U862, Bordeaux, France.

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 (CB(1)) is present at the membranes of mouse neuronal mitochondria (mtCB(1)), where it directly controls cellular respiration and energy production. Through activation of mtCB(1) 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 CB(1) receptors and mitochondrial mechanisms contributed to endocannabinoid-dependent depolarization-induced suppression of inhibition in the hippocampus. Thus, mtCB(1) receptors directly modulate neuronal energy metabolism, revealing a new mechanism of action of G protein-coupled receptor signaling in the brain.
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http://dx.doi.org/10.1038/nn.3053DOI Listing
March 2012

Preservation of NADH ubiquinone-oxidoreductase activity by Src kinase-mediated phosphorylation of NDUFB10.

Biochim Biophys Acta 2012 May 1;1817(5):718-25. Epub 2012 Feb 1.

INSERM-U688 Physiopathologie mitochondriale, Université de Bordeaux, 146 rue Léo Saignat, Bordeaux 33076, France.

The tyrosine kinase Src is upregulated in several cancer cells. In such cells, there is a metabolic reprogramming elevating aerobic glycolysis that seems partly dependent on Src activation. Src kinase was recently shown to be targeted to mitochondria where it modulates mitochondrial bioenergetics in non-proliferative tissues and cells. The main goal of our study was to determine if increased Src kinase activity could also influence mitochondrial metabolism in cancer cells (143B and DU145 cells). We have shown that 143B and DU145 cells produce most of the ATP through glycolysis but also that the inhibition of OXPHOS led to a significant decrease in proliferation which was not due to a decrease in the total ATP levels. These results indicate that a more important role for mitochondria in cancer cells could be ensuring mitochondrial functions other than ATP production. This study is the first to show a putative influence of intramitochondrial Src kinase on oxidative phosphorylation in cancer cells. Indeed, we have shown that Src kinase inhibition led to a decrease in mitochondrial respiration via a specific decrease in complex I activities (NADH-ubiquinone oxidoreductase). This decrease is associated with a lower phosphorylation of the complex I subunit NDUFB10. These results suggest that the preservation of complex I function by mitochondrial Src kinase could be important in the development of the overall phenotype of cancer.
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http://dx.doi.org/10.1016/j.bbabio.2012.01.014DOI Listing
May 2012

Novel mitochondrial DNA mutations responsible for maternally inherited nonsyndromic hearing loss.

Hum Mutat 2012 Apr 14;33(4):681-9. Epub 2012 Feb 14.

INSERM-U688 Physiopathologie Mitochondriale, Université Victor Segalen Bordeaux 2,146 rue Léo Saignat, Bordeaux, F-33076 France.

Some cases of maternally inherited isolated deafness are caused by mtDNA mutations, frequently following an exposure to aminoglycosides. Two mitochondrial genes have been clearly described as being affected by mutations responsible for this pathology: the ribosomal RNA 12S gene and the transfer RNA serine (UCN) gene. A previous study identified several candidate novel mtDNA mutations, localized in a variety of mitochondrial genes, found in patients with no previous treatment with aminoglycosides. Five of these candidate mutations are characterized in the present study. These mutations are localized in subunit ND1 of complex I of the respiratory chain (m.3388C>A [p.MT-ND1:Leu28Met]), the tRNA for Isoleucine (m.4295A>G), subunit COII of complex IV (m.8078G>A [p.MT-CO2:Val165Ile]), the tRNA of Serine 2 (AGU/C) (m.12236G>A), and Cytochrome B, subunit of complex III (m.15077G>A [p.MT-CYB:Glu111Lys]). Cybrid cell lines have been constructed for each of the studied mtDNA mutations and functional studies have been performed to assess the possible consequences of these mutations on mitochondrial bioenergetics. This study shows that a variety of mitochondrial genes, including protein-coding genes, can be responsible for nonsyndromic deafness, and that exposure to aminoglycosides is not required to develop the disease, giving new insights on the molecular bases of this pathology.
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http://dx.doi.org/10.1002/humu.22023DOI Listing
April 2012

AICAR inhibits cancer cell growth and triggers cell-type distinct effects on OXPHOS biogenesis, oxidative stress and Akt activation.

Biochim Biophys Acta 2011 Jun;1807(6):707-18

(MRGM) Maladies Rares: Génétique et Métabolisme, Université Victor Segalen, F-33076 Bordeaux, France.

The AMP-activated protein kinase agonist AICAR mimics a low intracellular energy state and inhibits the proliferation of cancer cells by different mechanisms, which may depend on the bioenergetic signature of these cells. AICAR can also stimulate mitochondrial biogenesis in myoblasts, neurons and HeLa cells. Yet, whether the reactivation of oxidative phosphorylation biogenesis by AICAR contributes to the growth arrest of cancer cells remains undetermined. To investigate this possibility, we looked at the impact of 24- and 48-hour treatments with 750 μM AICAR on human cancer cell lines (HeLa, DU145, and HEPG2), non-cancer cells (EM64, FM14, and HLF), embryonic cells (MRC5) and Rho(0) cells. We determined the bioenergetic profile of these cells and assessed the effect of AICAR on oxidative phosphorylation biogeneis, cell viability and cell proliferation, ROS generation, mitochondrial membrane potential and apoptosis induction. We also followed possible changes in metabolic regulators such as Akt and Hif1-α stabilization which might participate to the anti-proliferative effect of AICAR. Our results demonstrated a strong and cancer-specific anti-growth effect of AICAR that may be explained by three different modes according to cell type: the first mode included stimulation of the mitochondrial apoptotic pathway however with compensatory activation of Akt and upregulation of oxidative phosphorylation. In the second mode of action of AICAR Akt phosphorylation was reduced. In the third mode of action, apoptosis was activated by different pathways. The sensitivity to AICAR was higher in cells with a low steady-state ATP content and a high proliferation rate.
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http://dx.doi.org/10.1016/j.bbabio.2010.12.002DOI Listing
June 2011