Andrei O. Chertov

Rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of DC-SIGN

Here, we describe the isolation and characterization of the rhesus macaque homolog for human DC-SIGN, a dendritic cell-specific C-type lectin. mac-DC-SIGN is 92% identical to hu-DC-SIGN. mac-DC-SIGN preserves the virus transmission function of hu-DC-SIGN, capturing and efficiently transducing simian and human immunodeficiency virus to target CD4+ T cells. Surprisingly, however, mac-DC-SIGN plays no discernable role in the ability of rhesus macaque dendritic cells to capture and transmit primate lentiviruses. Expression and neutralization analyses suggest that this process is DC-SIGN independent in macaque, although the participation of other lectin molecules cannot be ruled out. The ability of primate lentiviruses to effectively use human and rhesus dendritic cells in virus transmission without the cells becoming directly infected suggests that these viruses have taken advantage of a conserved dendritic cell mechanism in which DC-SIGN family molecules are significant contributors but not the only participants.

Flow of energy in the outer retina in darkness and in light

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor’s synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Roles of glucose in photoreceptor survival.

Background: The aim of this study is to understand the energy requirements of photoreceptor neurons. Results: Glucose withdrawal causes photoreceptor death. Mitochondrial fuels and autophagy can enhance survival. Conclusion: Mitochondrial activity and substrates for anabolic activity are required for photoreceptor survival. Significance: Understanding the energy requirements of photoreceptors will contribute to understanding the basis of retinal disease. Vertebrate photoreceptor neurons have a high demand for metabolic energy, and their viability is very sensitive to genetic and environmental perturbations. We investigated the relationship between energy metabolism and cell death by evaluating the metabolic effects of glucose deprivation on mouse photoreceptors. Oxygen consumption, lactate production, ATP, NADH/NAD+, TCA cycle intermediates, morphological changes, autophagy, and viability were evaluated. We compared retinas incubated with glucose to retinas deprived of glucose or retinas treated with a mixture of mitochondrion-specific fuels. Rapid and slow phases of cell death were identified. The rapid phase is linked to reduced mitochondrial activity, and the slower phase reflects a need for substrates for cell maintenance and repair.

Inhibition of mitochondrial pyruvate transport by Zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina

Background: Pyruvate transport into mitochondria is a key step in energy metabolism. Zaprinast is a well known phosphodiesterase inhibitor. Results: Zaprinast has a strong influence on pyruvate transport into mitochondria. Conclusion: Inhibition of the mitochondrial pyruvate carrier by Zaprinast causes accumulation of aspartate at the expense of glutamate. Significance: Maintenance of normal amino acid levels in the retina relies on pyruvate transport into mitochondria. Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing 13C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.

Cytosolic reducing power preserves glutamate in retina

Significance This report shows that the reducing power in the environment influences oxidation of glutamate in a neuronal tissue. Glutamate is a neurotransmitter, and it is especially important as a metabolite because it is required for synthesis of glutathione, other amino acids, and proteins. Glutamate also is a key intermediate in glutamine-dependent anaplerosis, now considered to be a principal source of citric acid cycle intermediates in cancer cells. Our analyses also show that the reducing power in the environmental can influence glutamate oxidation in cancer cells. Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using 13C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria. Isolation of glutamate from the oxidative pathway relies on cytosolic NADH/NAD+, which is influenced by extracellular glucose, lactate, and pyruvate.

Rho Signaling Participates in Membrane Fluidity Homeostasis

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeasts sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.

Energy Metabolism in the Vertebrate Retina

The fundamental need for efficient and well-controlled energy metabolism in living organisms is ubiquitous. However, vertebrate retinas have many fascinating adaptations that give them the ability to regulate energy production and anabolic activity to meet a unique set of metabolic demands.