We explored the role of hypocretins in human narcolepsy through histopathology of six narcolepsy brains and mutation screening of Hcrt, Hcrtr1 and Hcrtr2 in 74 patients of various human leukocyte antigen and family history status. One Hcrt mutation, impairing peptide trafficking and processing, was found in a single case with early onset narcolepsy. In situ hybridization of the perifornical area and peptide radioimmunoassays indicated global loss of hypocretins, without gliosis or signs of inflammation in all human cases examined. Although hypocretin loci do not contribute significantly to genetic predisposition, most cases of human narcolepsy are associated with a deficient hypocretin system.

Mounting evidence from in vitro experiments indicates that lactate is an efficient energy substrate for neurons and that it may significantly contribute to maintain synaptic transmission, particularly during periods of intense activity. Since lactate does not cross the blood-brain barrier easily, blood-borne lactate cannot be a significant source. In vitro studies by several laboratories indicate that astrocytes release large amounts of lactate. In 1994, we proposed a mechanism whereby lactate could be produced by astrocytes in an activity-dependent, glutamate-mediated manner. Over the last 2 years we have obtained further evidence supporting the notion that a transfer of lactate from astrocytes to neurons might indeed take place. In this article, we first review data showing the presence of mRNA encoding for two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Second, by using monoclonal antibodies selectively directed against the two distinct lactate dehydrogenase isoforms, LDH₁ and LDH₅, a specific cellular distribution between neurons and astrocytes is revealed which suggests that a population of astrocytes is a lactate ‘source’ while neurons may be a lactate ‘sink’. Third, we provide biochemical evidence that lactate is interchangeable with glucose to support oxidative metabolism in cortical neurons. This set of data is consistent with the existence of an activity-dependent astrocyte-neuron lactate shuttle for the supply of energy substrates to neurons.

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

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Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Downregulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes. INTRODUCTIONReactive oxygen species (ROS) are generated either in a nonregulated manner as side products of several enzymatic systems (e.g., cyclooxygenases, nitric oxide [NO] synthases, mitochondrial cytochromes) or in a regulated way as main products of superoxide producing enzymes, the NADPH oxidases. In the mouse, the family of NADPH oxidases includes NOX1, NOX2 (gp91 phox ), NOX3, and NOX4.Excessive cellular generation of ROS, such as superoxide anions (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ), is potentially destructive and is used by phagocytes to kill invading microorganisms. Under normal conditions, scavenging mechanisms (e.g., superoxide dismutase, catalase, glutathioneglutathione peroxidase system) remove excessive amounts of ROS. Under stress conditions, however, the production of ROS may exceed the reducing capacity of the cell and damage cellular functions. Small amounts of ROS, on the other hand, can function as intracellular second messengers and activate signaling cascades involved in growth and differentiation of many cell types (for review see Rhee, 1999;Laloi et al., 2004). For example, the MAP kinase-signaling pathway is sensitive to ROS (Torres and Forman, 2003). Moreover, distinct signaling pathways have differential sensitivity to oxidative stress, leading to dose-dependent effect on, for example, cardiomyocytes on which ROS can induce hypertrophy or apoptosis (Kwon et al., 2003). Transcription factors such as NF-B, p53, and AP-1 are redox-sensitive and can be directly modified by ROS, providing a link with the control of gene expression (Morel and Barouki, 1999).Cardiac differentiation can be studied by differentiating mouse embryonic stem cells (ESC) into embryoid bodies (EB), where the appearance of spontaneously beating cardiomyocytes is observed after 7-8 d of culture. This system thus provides a unique experimental model to study the role of ROS and ROS-generating enzymes in the regulation of cardiomyocyte growth and differentiation in vitro. Previous reports have shown a link between ROS and...