Efficacy of the combination of milbemycin oxime and afoxolaner for the control of myiasis by Musca domestica in desert tortoises (Gopherus berlandieri).

The efficacy of the combination of milbemycin oxime and afoxolaner was evaluated in desert tortoises infested with fly larvae. Oral administration of the combination of milbemycin oxime and afoxolaner eliminated the infestation without generating any evident adverse effects on the tortoises.

L'efficacité de la combinaison de milbémycine oxime et d'afoxolaner a été évaluée chez des tortues du désert infestées par des larves de mouches. L'administration orale de la combinaison de milbémycine oxime et d'afoxolaner traite l'infestation sans générer d'effets indésirables évidents sur les tortues.

A eficácia da associação de milbemicina oxima e afoxolaner foi avaliada em tartarugas do deserto infestadas por larvas de moscas. A administração oral da combinação de afoxolaner e milbemicina oxima eliminou a infestação sem gerar nenhum efeito adverso evidente às tartarugas.

Se evaluó la eficacia de la combinación de milbemicina oxima y afoxolaner en tortugas del desierto infestadas con larvas de mosca. La administración oral de la combinación de milbemicina oxima y afoxolaner eliminó la infestación sin generar efectos adversos evidentes en las tortugas.

Effectiveness of lotilaner against ticks of the genus Amblyomma spp. in three naturally infested cane toads (Rhinella horribilis).

The efficacy of lotilaner was evaluated in three tick-infested cane toads. A single oral administration of lotilaner eliminated all ticks from all three toads from Day (D)1. Environmental samples collected from toad enclosures were negative for ticks until D30.

L'efficacité du lotilaner a été évaluée chez trois crapauds buffle infestés de tiques. Une seule administration orale de lotilaner a éliminé toutes les tiques des trois crapauds à partir du jour (J)1. Les échantillons environnementaux prélevés dans les enclos des crapauds étaient négatifs pour les tiques jusqu'à J30.

Se evaluó la eficacia de lotilaner en tres sapos de caña infestados por garrapatas. Una sola administración oral de lotilaner eliminó todas las garrapatas de los tres sapos desde el día (D) 1. Las muestras ambientales recolectadas de los recintos de sapos fueron negativas para garrapatas hasta el D30.

A eficácia do lotialaner foi avaliada em três sapos-boi infestados por carrapatos. Uma única administração de lotialaner eliminou todos os carrapatos de todos os sapos no Dia (D) 1. Amostras ambientais coletadas dos terrários dos sapos foram todas negativas para carrapatos até o D30.

AGC1 Deficiency: Pathology and Molecular and Cellular Mechanisms of the Disease.

AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named "early infantile epileptic encephalopathy 39" (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.

ßOHB Protective Pathways in Aralar-KO Neurons and Brain: An Alternative to Ketogenic Diet.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether ß-hydroxybutyrate (ßOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that ßOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring ßOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of ßOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of ßOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by ßOHB administration, a 4-d ßOHB treatment to aralar-KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of ßOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation.SIGNIFICANCE STATEMENTAralar deficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressing aralar, its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Here, we find that ßOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. ßOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded by aralar deficiency, presumably through citrate-malate shuttle. In aralar-knock-out (KO) mice, ßOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The ßOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.

Dermatophytosis caused by Trichophyton benhamiae in a dog.

Trichophyton benhamiae was diagnosed in a 9-year-old female dog by histopathological evaluation, fungal culture and confirmation by sequencing of the internal transcribed spacer region of ribosomal DNA. Successful therapy was achieved with itraconazole, bathing with miconazole and chlorhexidine shampoo, and topical application of sodium hypochlorite as a rinse.

Trichophyton benhamiae a été diagnostiqué chez une chienne de 9 ans par examen histopathologique, culture fongique et confirmation par séquençage de la région ITS (internal trasbcriber spacer) de l'ADN ribosomique. Une guérison thérapeutique a été obtenue par de l'itraconazole, des shampooings de miconazole et chlorhexidine et application topique d'hypochlorite de sodium en rinçage.

Trichophyton benhamiae foi diagnosticado em uma cadela de 9 anos através de avaliação histopatológica, cultura de fungos e confirmação por sequenciamento da região espaçadora transcrita interna do DNA ribossomal. Sucesso terapêutico foi obtido com o uso de itraconazol, banho com shampoo à base de miconazol e clorexidina e aplicação tópica de hipoclorito de sódio.

Se diagnosticó infección por Trichophyton benhamiae en una perra de 9 años mediante evaluación histopatológica, cultivo de hongos y confirmación mediante secuenciación de la región espaciadora transcrita interna del DNA ribosómico. Se logró un tratamiento exitoso con itraconazol, baños con champú de miconazol y clorhexidina y aplicación tópica de hipoclorito de sodio como enjuague.

Two 20-Residue-Long Peptides Derived from Plasmodium vivax Merozoite Surface Protein 10 EGF-Like Domains Are Involved in Binding to Human Reticulocytes.

Plasmodium parasites' invasion of their target cells is a complex, multi-step process involving many protein-protein interactions. Little is known about how complex the interaction with target cells is in Plasmodium vivax and few surface molecules related to reticulocytes' adhesion have been described to date. Natural selection, functional and structural analysis were carried out on the previously described vaccine candidate P. vivax merozoite surface protein 10 (PvMSP10) for evaluating its role during initial contact with target cells. It has been shown here that the recombinant carboxyl terminal region (rPvMSP10-C) bound to adult human reticulocytes but not to normocytes, as validated by two different protein-cell interaction assays. Particularly interesting was the fact that two 20-residue-long regions (388DKEECRCRANYMPDDSVDYF407 and 415KDCSKENGNCDVNAECSIDK434) were able to inhibit rPvMSP10-C binding to reticulocytes and rosette formation using enriched target cells. These peptides were derived from PvMSP10 epidermal growth factor (EGF)-like domains (precisely, from a well-defined electrostatic zone) and consisted of regions having the potential of being B- or T-cell epitopes. These findings provide evidence, for the first time, about the fragments governing PvMSP10 binding to its target cells, thus highlighting the importance of studying them for inclusion in a P. vivax antimalarial vaccine.

Smoking prevention with narrative messages. An experimental study on the joint effect of audience-character similarity and narrative voice. / Prevención del tabaquismo con mensajes narrativos. Estudio experimental sobre el efecto conjunto de la similitud con el protagonista y la voz narrativa.

This study focuses on smoking prevention using narrative messages. In particular, the role of two narrative attributes that can indirectly influence the intention to quit smoking, self-efficacy expectations and the perceived effectiveness of the preventive response were analysed. An experimental study was carried out (N = 680, 50% women and age range 18-55 years) with a 2 (narrative voice: first- vs. third-person message) x 2 (audience-protagonist similarity: low vs. high) between-subjects factorial design. Results showed that the optimal reception condition (first-person narrative with a highly similar protagonist to the audience) induced the highest levels of identification with the protagonist (a former smoker who described the process of quitting smoking and subsequent the improvements he has experienced). Mediational analyses showed that the optimal reception condition exerted significant indirect effects on the dependent variables, due to the increase in identification and reactance reduction. In addition, the optimal reception condition also exerted a significant indirect effect on the perceived effectiveness of the preventive response that was explained by stronger identification and weaker counterarguing. The present study opens an innovative line of research on the construction of narrative messages for smoking prevention. The relevance of the characteristics of these messages is highlighted in order to activate mediating processes that facilitate persuasion.

El presente trabajo se centra en la prevención del tabaquismo utilizando mensajes narrativos. En particular, se analiza el papel de dos características de los mensajes narrativos que pueden influir, de manera indirecta, en la intención de dejar de fumar, las expectativas de auto-eficacia y la percepción de la eficacia de la respuesta preventiva. Para ello, se llevó a cabo una investigación experimental (N = 680, 50% mujeres y rango de edad 18-55 años) con un diseño factorial 2 (voz narrativa: mensaje en primera vs. tercera persona) x 2 (similitud con el protagonista: baja vs. alta). Los resultados mostraron que la condición óptima de recepción (narración en primera persona protagonizada por un personaje similar a la audiencia) indujo niveles más elevados de identificación con el protagonista (un exfumador describía el proceso de abandono del tabaco y las mejoras que ha experimentado desde entonces). Los análisis mediacionales mostraron que la condición óptima de recepción ejercía efectos indirectos significativos sobre las variables dependientes que se debían al aumento de la identificación y la reducción de la reactancia. Además, la condición óptima de recepción también ejerció un efecto indirecto significativo sobre la eficacia percibida de la respuesta preventiva que se explicaba por el incremento de la identificación y la reducción de la contra-argumentación. El presente trabajo abre una línea de estudio sobre la construcción de mensajes narrativos para la prevención del tabaquismo. Se pone de manifiesto la relevancia de las características que dichos mensajes deben tener para que se activen procesos mediadores que faciliten la persuasión.

De Novo Mutations in SLC25A24 Cause a Disorder Characterized by Early Aging, Bone Dysplasia, Characteristic Face, and Early Demise.

A series of simplex cases have been reported under various diagnoses sharing early aging, especially evident in congenitally decreased subcutaneous fat tissue and sparse hair, bone dysplasia of the skull and fingers, a distinctive facial gestalt, and prenatal and postnatal growth retardation. For historical reasons, we suggest naming the entity Fontaine syndrome. Exome sequencing of four unrelated affected individuals showed that all carried the de novo missense variant c.649C>T (p.Arg217Cys) or c.650G>A (p.Arg217His) in SLC25A24, a solute carrier 25 family member coding for calcium-binding mitochondrial carrier protein (SCaMC-1, also known as SLC25A24). SLC25A24 allows an electro-neutral and reversible exchange of ATP-Mg and phosphate between the cytosol and mitochondria, which is required for maintaining optimal adenine nucleotide levels in the mitochondrial matrix. Molecular dynamic simulation studies predict that p.Arg217Cys and p.Arg217His narrow the substrate cavity of the protein and disrupt transporter dynamics. SLC25A24-mutant fibroblasts and cells expressing p.Arg217Cys or p.Arg217His variants showed altered mitochondrial morphology, a decreased proliferation rate, increased mitochondrial membrane potential, and decreased ATP-linked mitochondrial oxygen consumption. The results suggest that the SLC25A24 mutations lead to impaired mitochondrial ATP synthesis and cause hyperpolarization and increased proton leak in association with an impaired energy metabolism. Our findings identify SLC25A24 mutations affecting codon 217 as the underlying genetic cause of human progeroid Fontaine syndrome.

Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

BACKGROUND/AIMS: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. METHODS: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. RESULTS: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172P-Cdk4/Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR-720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells. CONCLUSION: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cells.

Successful treatment of Ophionyssus natricis with afoxolaner in two Burmese pythons (Python molurus bivittatus).

The efficacy of afoxolaner was evaluated in two captive Burmese python snakes, which were naturally infested with Ophionyssus natricis mites. The administration of a single oral dose of afoxolaner eliminated live O. natricis mites from both snakes by Day 3. Environmental samples collected from the snakes' terrariums were negative for dead mites by Day 30.

L'efficacité de l'afoxolaner a été évaluée chez deux serpents python birmans captifs, naturellement infestés par des acariens Ophionyssus natricis. L'administration d'une dose orale unique d'afoxolaner a éliminé les acariens O. natricis vivants des deux serpents à jour 3. Les échantillons environnementaux prélevés dans les terrariums des serpents étaient négatifs pour les acariens morts à jour 30.

Se evaluó la eficacia de afoxolaner en dos serpientes pitón birmanas cautivas, que estaban naturalmente infestadas con ácaros Ophionyssus natricis. La administración de una sola dosis oral de afoxolaner eliminó los ácaros O. natricis vivos de ambas serpientes en el día 3. Las muestras ambientales recolectadas de los terrarios de las serpientes dieron negativo para ácaros muertos en el día 30.

A eficácia do afoxolaner foi avaliada em duas cobras python birmanesas de cativeiro, que foram naturalmente infestadas com ácaros Ophionyssus natricis. A administração de uma única dose oral de afoxolaner eliminou ácaros O. natricis vivos de ambas as cobras no dia 3. As amostras ambientais coletadas dos terrários das cobras foram negativas para ácaros mortos no dia 30.

The Response to Stimulation in Neurons and Astrocytes.

The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this high oxidative index, contrasts with the metabolic traits of the major cell types in the brain studied in culture, neurons and astrocytes, including the selective use of the malate-aspartate shuttle (MAS) in neurons and the glycerol-phosphate shuttle in astrocytes. Metabolic interactions among these cell types may partly explain the high oxidative index of the brain. In vivo, neuronal activation results in a decrease in the oxygen glucose index, which has been attributed to a stimulation of glycolysis and lactate production in astrocytes in response to glutamate uptake (astrocyte-neuron lactate shuttle, ANLS). Recent findings indicate that this is accompanied with a stimulation of pyruvate formation and astrocyte respiration, indicating that lactate formation is not the only astrocytic response to neuronal activation. ANLS proposes that neurons utilize lactate produced by neighboring astrocytes. Indeed, neurons can use lactate to support an increase in respiration with different workloads, and this depends on the Ca2+ activation of MAS. However, whether this activation operates in the brain, particularly at high stimulation conditions, remains to be established.

Effect of oral afoxolaner on naturally occurring infestations of peacocks by the louse Goniodes pavonis.

BACKGROUND: Ectoparasitism of ornamental birds, including captive species kept in zoos, represents a serious health problem. Up to 13 different species of lice have been reported to affect peacocks worldwide and heavy infestation may cause anaemia. Because of this, alternatives to the prevailing treatments have been sought including use of isoxazolines. This class of drugs has been used successfully in poultry without adverse effects on health or production. OBJECTIVE: To evaluate the effect of afoxolaner on the peacock louse (Goniodes pavonis). ANIMALS: Twenty-three peacocks (Pavo cristatus) with naturally occurring infestation with G. pavonis. METHODS AND MATERIALS: The peacocks were divided in two groups; one was treated once orally with 2.5 mg/kg afoxolaner and the other group received no treatment. Samples were collected using the acetate tape technique, for identification of lice by microscopy. Concomitantly, blood samples were taken to evaluate the haematocrit before and after the intervention. RESULTS: Treatment with afoxolaner significantly decreased the number of peacocks positive for lice (P = 0.02) compared to the control group, in which the number of positive birds did not decrease. The haematocrit improved in the afoxolaner-treated group from a baseline of 46.4%-54.7% at 35 days post-treatment, whereas it decreased in untreated birds (44.6%-40.7%). No adverse effects attributed to afoxolaner treatment were observed. CONCLUSIONS AND CLINICAL IMPORTANCE: Oral administration of afoxolaner is an effective treatment for G. pavonis infestation of peacocks.

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of Ser51 P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185 P-JNK1/2/JNK1/2, Thr172 P-AMPKα, Ser413 P-Foxo3a, Thr308 P-AKt/AKt and Thr32 P-Foxo3a/Foxo3a ratios, and reduction of Ser2481 P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of Thr308 P-AKt/AKt and Ser473 P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim EL , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM EL upregulation.

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients.

PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines.

Calcium regulation of mitochondrial carriers.

Mitochondrial function is regulated by calcium. In addition to the long known effects of matrix Ca(2+), regulation of metabolite transport by extramitochondrial Ca(2+) represents an alternative Ca(2+)-dependent mechanism to regulate mitochondrial function. The Ca(2+) regulated mitochondrial transporters (CaMCs) are well suited for that role, as they contain long N-terminal extensions harboring EF-hand Ca(2+) binding domains facing the intermembrane space. They fall in two groups, the aspartate/glutamate exchangers, AGCs, major components of the NADH malate aspartate shuttle (MAS) and urea cycle, and the ATP-Mg(2+)/Pi exchangers or short CaMCs (APCs or SCaMCs). The AGCs are activated by relatively low Ca(2+) levels only slightly higher than resting Ca(2+), whereas all SCaMCs studied so far require strong Ca(2+) signals, above micromolar, for activation. In addition, AGCs are not strictly Ca(2+) dependent, being active even in Ca(2+)-free conditions. Thus, AGCs are well suited to respond to small Ca(2+) signals and that do not reach mitochondria. In contrast, ATP-Mg(2+)/Pi carriers are inactive in Ca(2+) free conditions and activation requires Ca(2+) signals that will also activate the calcium uniporter (MCU). By changing the net content of adenine nucleotides of the matrix upon activation, SCaMCs regulate the activity of the permeability transition pore, and the Ca(2+) retention capacity of mitochondria (CRC), two functions synergizing with those of the MCU. The different Ca(2+) activation properties of the two CaMCs are discussed in relation to their newly obtained structures. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar +/- mice. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1-13C)-Glucose.

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1-13C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30-35 %) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina.

Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell's ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.