Jean-Michel Franconi

Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of Multiple Sclerosis. Amelioration of clinical status by M2 activated monocyte administration

Objectives:We investigated proinflammatory M1 and immunomodulatory M2 activation profiles of circulating monocytes in relapsing experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, and tested whether altered M1/M2 equilibrium promotes CNS inflammation. Results:Approaches of MRI macrophage tracking with USPIO nanoparticles and expression patterns of M1/M2 macrophages and microglia in brain and M1/M2 monocytes in blood samples at various disease stages revealed that M1/M2 equilibrium in blood and CNS favors mild EAE, while imbalance towards M1 promotes relapsing EAE. We consequently investigated whether M2 activated monocyte restoration in peripheral blood could cure acute clinical EAE disease. Administration of ex vivo activated M2 monocytes both suppressed ongoing severe EAE and increased immunomodulatory expression pattern in lesions, confirming their role in the induction of recovery. Conclusion:We conclude that imbalance of monocyte activation profiles and impaired M2 expression, are key factors in development of relapses. Our study opens new perspectives for therapeutic applications in MS.

In Vivo Phage Display to Identify New Human Antibody Fragments Homing to Atherosclerotic Endothelial and Subendothelial Tissues

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.

Competition between glucose and lactate as oxidative energy substrates in both neurons and astrocytes: a comparative NMR study.

Competition between glucose and lactate as oxidative energy substrates was investigated in both primary cultures of astrocytes and neurons using physiological concentrations (1.1 mm for each). Glucose metabolism was distinguished from lactate metabolism by using alternatively labelled substrates in the medium ([1‐13C]glucose + lactate or glucose + [3‐13C]lactate). After 4 h of incubation, 1H and 13C‐NMR spectra were realized on perchloric acid extracts of both cells and culture media. For astrocytic cultures, spectra showed that amino acids (glutamine and alanine) were more labelled in the glucose‐labelled condition, indicating that glucose is a better substrate to support oxidative metabolism in these cells. The opposite was observed on spectra from neuronal cultures, glutamate being much more labelled in the lactate‐labelled condition, confirming that neurons consume lactate preferentially as an oxidative energy substrate. Analysis of glutamine and glutamate peaks (singlets or multiplets) also suggests that astrocytes have a less active oxidative metabolism than neurons. In contrast, they exhibit a stronger glycolytic metabolism than neurons as indicated by their high lactate production yield. Using a mathematical model, we have estimated the relative contribution of exogenous glucose and lactate to neuronal oxidative metabolism. Under the aforementioned conditions, it represents 25% for glucose and 75% for lactate. Altogether, these results obtained on separate astrocytic and neuronal cultures support the idea that lactate, predominantly produced by astrocytes, is used as a supplementary fuel by neurons in vivo already under resting physiological conditions.

A multicenter measurement of magnetization transfer ratio in normal white matter

To assess the importance of intercenter variations when measuring magnetization transfer ratio (MTR) in the brain, six European centers measured MTR in normal white matter. MTR ranged from 9 to 51 percent units (25 sequences). The effective flip angle of the saturating pulse divided by the pulse repetition time (ENRsat degrees/msec) was a good predictor of MTR (MTR = 3.25 ENRsat).J. Magn. Reson. Imaging 1999; 9:441–446.

Glucose-induced Remodeling of Intermediary and Energy Metabolism in Procyclic Trypanosoma brucei

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of 13C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F0/F1-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F0/F1-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.

A Mitochondrial NADH-dependent Fumarate Reductase Involved in the Production of Succinate Excreted by Procyclic Trypanosoma brucei

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.

Close coupling between astrocytic and neuronal metabolisms to fulfill anaplerotic and energy needs in the rat brain.

Carbon metabolism in the rat brain was studied in animals anesthetized with a light dose of pentobarbital and in awake animals under morphine, which were infused with either [1-13C]glucose+acetate or glucose+[2-13C]acetate for various periods of time. Brain amino-acid enrichments in tissue extracts were determined by nuclear magnetic resonance (NMR) spectroscopy and their time evolution was analyzed by automatic fitting. Acetyl-coenzyme A C2 enrichment and ratio between pyruvate carboxylase and pyruvate dehydrogenase activity (PC/PDH) were determined from glutamate and glutamine labeling. The following results were obtained: (i) amino-acid enrichment patterns implied metabolic compartmentation and occurrence of the glutamate—glutamine cycle; (ii) kinetics of aspartate, GABA, and glutamate labeling from [1-13C]glucose and of glutamine labeling from [2-13C]acetate indicated a twofold higher metabolic activity in awake than in anesthetized rat brain; (iii) evaluation of the contributions of the astrocytic and neuronal metabolisms to glutamine synthesis in both groups of rats indicated a coupling between neuronal tricarboxylic acid (TCA) cycle, glutamate—glutamine cycle and glial TCA cycle; and (iv) analyzing the extrapolations back to time zero and the steady-state values of PC/PDH indicated a close coupling between PC activity and both astrocytic and neuronal TCA cycles. All these results suggest a cooperative-like behavior of astrocytic and neuronal metabolisms to fulfill the anabolic and energy needs linked to brain activation.

Mitochondrial energetics is impaired in vivo in aged skeletal muscle

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging‐related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with 31P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness (‘elasticity’) of mitochondrial oxidative phosphorylation to contraction‐induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging‐related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon.

Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes

Acetyl-CoA produced in mitochondria from carbohydrate or amino acid catabolism needs to reach the cytosol to initiate de novo synthesis of fatty acids. All eukaryotes analyzed so far use a citrate/malate shuttle to transfer acetyl group equivalents from the mitochondrial matrix to the cytosol. Here we investigate how this acetyl group transfer occurs in the procyclic life cycle stage of Trypanosoma brucei, a protozoan parasite responsible of human sleeping sickness and economically important livestock diseases. Deletion of the potential citrate lyase gene, a critical cytosolic enzyme of the citrate/malate shuttle, has no effect on de novo biosynthesis of fatty acids from 14C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyl-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyl-CoA synthetase gene encoding a cytosolic enzyme (AceCS), which is essential for cell viability. Repression of AceCS by inducible RNAi results in a 20-fold reduction of 14C-incorporation from radiolabeled glucose or acetate into de novo synthesized fatty acids. Thus, we demonstrate that the essential cytosolic enzyme AceCS of T. brucei is responsible for activation of acetate into acetyl-CoA to feed de novo biosynthesis of lipids. To date, Trypanosoma is the only known eukaryotic organism that uses acetate instead of citrate to transfer acetyl groups over the mitochondrial membrane for cytosolic lipid synthesis.

Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability.

Background The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei. Methodology/Principal Findings A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway. Conclusions/Significance Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.