A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

International audienceBACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. RESULTS: SOD1(G93A) mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice). Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A) mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. CONCLUSIONS: These findings suggest that the protection against SOD1(G93A) offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons

Caractérisation de la fonction in vivo de la dynéine cytoplasmique par l'utilisation de souris mutantes

La dynéine cytoplasmique est un moteur moléculaire dont la principale fonction est de transporter des mitochondries, des vésicules ou des protéines, le long des microtubules de la périphérie vers le centre de la cellule. Dans les neurones, la dynéine est responsable du transport axonal rétrograde, un processus essentiel pour la survie des neurones qui permet la communication entre les différents compartiments cellulaires. Des altérations dans le transport axonal, en particulier dans le transport axonal rétrograde, ont été retrouvées dans la plupart des maladies neurodégénératives, et pourraient être liées à un mauvais fonctionnement de la dynéine. Ce travail de thèse a permis de mettre en évidence qu une altération du transport axonal rétrograde ne conduit pas à la perte des motoneurones (Dupuis, 2009), principale caractéristique de la SLA. Cependant nous avons montré que le striatum, région impliquée dans la motricité fine des mouvements est affectée par un dysfonctionnement de la dynéine (Braunstein, 2011). Nous nous sommes intéressés aux conséquences de la perte de fonction de la dynéine au niveau périphérique, notamment dans le métabolisme lipidique (Eschbach, 2011) et dans le maintien de la fonction mitochondriale (manuscrit en cours). Enfin, nous avons étudié les mécanismes responsables de l amélioration de la survie de modèles de SLA par la mutation dynéine (Fergani, 2011). Ce travail de thèse a permis de mettre en évidence qu une altération du transport axonal rétrograde n est pas suffisante pour conduire à la perte neuronale mais qu elle s accompagne néanmoins d une atrophie du striatum.Cytoplasmic dynein is a molecular motor that drives cargoes, including whole organelles such as mitochondria, from the periphery of the cell to the perinuclear region by using microtubules as railroad tracks. In Neurons, Dynein is involved in retrograde axonal transport, which is a process required for neurons survival. Alterations in axonal transport, in both anterograde and retrograde axonal transport, were found in neurodegenerative diseases. This thesis work aims at showing that an axonal transport alteration is not sufficient to induce motor neuron disease (Dupuis, 2009), but sufficient to provoke a striatal dysfunction (Braunstein, 2010).Besides this neuronal aspect, dynein seems to be involved in lipids metabolism since dynein mutant mice present also a metabolic phenotype. Indeed, they showed an increase of fat mass in correlation with age due to a defective lipolysis associated with an increase oxidative stress (Eschbach, 2011). In parallel, we observed that dynein is required for the mitochondrial functions (manuscript ongoing). Finally, we determined the mechanisms of protective effect of dynein mutation in ALS mice (Fergani, 2011). In conclusion, this thesis work aims at showing that an axonal transport alteration is not sufficient to induce motor neuron disease, but sufficient to provoke a striatal dysfunction. Concerning the white adipose tissue, dynein seems to be involved in the lipolysis process. In addition, our results suggest that dynein is required for the mitochondrial function. Our current hypothesis is that this mitochondrial dysfunction could provoke the observed phenotype in both striatum and adipose tissue.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Wärmeentwicklung bei der Keimung : Experimente zur Bestimmung der Keimungswärme

Bekanntlich findet infolge von Stoffwechselaktivität bei der Samenkeimung eine Wärmeentwicklung statt. Bei entsprechender Wärmeisolierung von einer größeren Anzahl keimender Samen, ist die Wärmeentwicklung als Temperaturerhöhung zu messen. Mit entsprechenden Experimenten wird aufgezeigt, dass die detektierten Werte bei Versuchsansätzen wie z. B. bei TROLL, 1973; KRÜGER, 1978; SCHWARZMAIER, 1986 und auch bei KUHN & PROBST, 1983 nicht allein von der Stoffwechselaktivität der keimenden Samen stammen, sondern ein beachtlicher Anteil mikrobiellem Stoffwechsel zuzuschreiben ist. Außerdem wird aufgezeigt, dass nach Desinfektion des Saatgutes (Erbsen, Mais und Mungobohnen) Temperaturen von über 60 °C während der Keimung nicht erreicht werden können

A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. Results SOD1(G93A) mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice). Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A) mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. Conclusions These findings suggest that the protection against SOD1(G93A) offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons.</p

Metabolites of Cannabigerol Generated by Human Cytochrome P450s Are Bioactive

The phytocannabinoid cannabigerol (CBG) is the central biosynthetic precursor to many cannabinoids, including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Though the use of CBG has recently witnessed a widespread surge because of its beneficial health effects and lack of psychoactivity, its metabolism by human cytochrome P450s is largely unknown. Herein, we describe comprehensive in vitro and in vivo cytochrome P450 (CYP)-mediated metabolic studies of CBG, ranging from liquid chromatography tandem mass spectrometry-based primary metabolic site determination, synthetic validation, and kinetic behavior using targeted mass spectrometry. These investigations revealed that cyclo-CBG, a recently isolated phytocannabinoid, is the major metabolite that is rapidly formed by selected human cytochrome P450s (CYP2J2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9). Additionally, in vivo studies with mice administered with CBG supported these studies, where cyclo-CBG is the major metabolite as well. Spectroscopic binding studies along with docking and modeling of the CBG molecule near the heme in the active site of P450s confirmed these observations, pointing at the preferred site selectivity of CBG metabolism at the prenyl chain over other positions. Importantly, we found out that CBG and its oxidized CBG metabolites reduced inflammation in BV2 microglial cells stimulated with LPS. Overall, combining enzymological studies, mass spectrometry, and chemical synthesis, we showcase that CBG is rapidly metabolized by human P450s to form oxidized metabolites that are bioactive

Metabolites of Cannabigerol (CBG) Generated by Human Cytochrome P450s are Bioactive

The phytocannabinoid cannabigerol (CBG) is the central biosynthetic precursor to many cannabinoids, including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Though the use of CBG has recently witnessed a widespread surge because of its beneficial health effects and lack of psychoactivity, its metabolism by human cytochrome P450s is largely unknown. Herein, we describe comprehensive in vitro and in vivo cytochrome P450 (CYP)-mediated metabolic studies of CBG, ranging from LC-MS/MS-based primary metabolic site determination, synthetic validation, and kinetic behavior using targeted mass spectrometry. These investigations revealed that cyclo-CBG, a recently isolated phytocannabinoid, is the major metabolite that is rapidly formed by selected human cytochrome P450s (CYP2J2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9). Additionally, in vivo studies with mice administered with CBG, supported these studies, where cyclo-CBG is the major metabolite as well. Spectroscopic binding studies along with docking and modeling of CBG molecule near the heme in the active site of P450s confirmed these observations, pointing at the preferred site-selectivity of CBG metabolism at the prenyl chain over other positions. Importantly, we found out that CBG and its oxidized CBG metabolites reduced inflammation in BV2 microglial cells stimulated with LPS. Overall, combining enzymological studies, mass spectrometry, and chemical synthesis, we showcase that CBG is rapidly metabolized by human P450s to form oxidized metabolites that are bioactive. The study reveals the structure-activity relationship of CBG metabolites and analogs to their anti-inflammatory activity

Strongly enhanced IL-10 production using stable galectin-1 homodimers

Galectin-1 is the homodimeric form of a protein, which is present in a dynamic equilibrium with the beta-galactoside monomeric form and has potent anti-inflammatory and immunomodulating effects. These favorable effects are probably related to the induction of apoptosis in activated T cells and the induction of IL-10, which have been demonstrated to be characteristic for the dimeric form of the protein. Based on these findings it can be speculated that the in vivo effects of galectin-1 can be improved by the generation of stable galectin-1 homodimers (dGal). To test this hypothesis we produced leucine-zipper based stable galectin-1 homodimers and tested its apoptosis inducing effects on MOLT-4 cells and its immunomodulatory effects in vitro on PBMC of five independent donors. Phosphatidylserine exposure and a drop in mitochondrial membrane potential was strongly enhanced on MOLT-4 cells upon treatment with dGal as compared to wtGal. The minimal effective concentration was 20-fold reduced as compared to the minimal effective wtGal concentration. dGal showed enhanced induction of IL-10 on total PBMC as compared to treatment with wild-type protein (wtGal). The minimal effective dGal concentration was 100-fold lower than that of wtGal. Of the purified cell populations monocytes are the strongest IL-10 producers, whereas T cells induce IL-10 at a lower level and no induction is observed in B cells. Besides induction of IL-10, dGal caused an increase in IL-1 beta production in all donors and a reduction of IL-2 production in 3 out of 5 donors, whereas no consistent changes were observed for other inflammatory cytokines. In summary, we demonstrated that dGal shows enhanced effects at strongly reduced concentrations. Application of dGal may therefore serve as an improved treatment of chronic inflammatory diseases. (c) 2006 Elsevier Ltd. All rights reserved

Muscle mitochondrial uncoupling dismantles neuromuscular junction and triggers distal degeneration of motor neurons.

BACKGROUND:Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS. METHODOLOGY/PRINCIPAL FINDINGS:We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model. CONCLUSIONS/SIGNIFICANCE:These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases

The Golgi-localized, gamma ear-containing, ARF-binding (GGA) protein family alters alpha synuclein (alpha-syn) oligomerization and secretion

Several age-related neurodegenerative disorders are associated with protein misfolding and aggregation of toxic peptides. α-synuclein (α-syn) aggregation and the resulting cytotoxicity is a hallmark of Parkinson's disease (PD) as well as dementia with Lewy bodies. Rising evidence points to oligomeric and pre-fibrillar forms as the pathogenic species, and oligomer secretion seems to be crucial for the spreading and progression of PD pathology. Recent studies implicate that dysfunctions in endolysosomal/autophagosomal pathways increase α-syn secretion. Mutation in the retromer-complex protein VPS35, which is involved in endosome to Golgi transport, was suggested to cause familial PD. GGA proteins regulate vesicular traffic between Golgi and endosomes and might work as antagonists for retromer complex mediated transport. To investigate the role of the GGAs in the α-syn oligomerization and/or secretion process we utilized protein-fragment complementation assays (PCA). We here demonstrate that GGAs alter α-syn oligomer secretion and α-syn oligomer-mediated toxicity. Specifically, we determined that GGA3 modifies extracellular α-syn species in an exosome-independent manner. Our data suggest that GGA3 drives α-syn oligomerization in endosomal compartments and thus facilitates α-syn oligomer secretion. Preventing the early events in α-syn oligomer release may be a novel approach to halt disease spreading in PD and other synucleinopathies.status: publishe