Altered lactate metabolism in Huntington's disease is dependent on GLUT3 expression.

AIMS: Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive abnormalities in cognitive function, mental state, and motor control. HD is characterized by a failure in brain energy metabolism. It has been proposed that monocarboxylates, such as lactate, support brain activity. During neuronal synaptic activity, ascorbic acid released from glial cells stimulates lactate and inhibits glucose transport. The aim of this study was to evaluate the expression and function of monocarboxylate transporters (MCTs) in two HD models. METHODS: Using immunofluorescence, qPCR, and Western blot analyses, we explored mRNA and protein levels of MCTs in the striatum of R6/2 animals and HdhQ7/111 cells. We also evaluated MCT function in HdhQ7/111 cells using radioactive tracers and the fluorescent lactate sensor Laconic. RESULTS: We found no significant differences in the mRNA or protein levels of neuronal MCTs. Functional analyses revealed that neuronal MCT2 had a high catalytic efficiency in HD cells. Ascorbic acid did not stimulate lactate uptake in HD cells. Ascorbic acid was also unable to inhibit glucose transport in HD cells because they exhibit decreased expression of the neuronal glucose transporter GLUT3. CONCLUSION: We demonstrate that stimulation of lactate uptake by ascorbic acid is a consequence of inhibiting glucose transport. Supporting this, lactate transport stimulation by ascorbic acid in HD cells was completely restored by overexpressing GLUT3. Therefore, alterations in GLUT3 expression could be responsible for inefficient use of lactate in HD neurons, contributing to the metabolic failure observed in HD.

A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays.

Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.

Beyond the redox imbalance: Oxidative stress contributes to an impaired GLUT3 modulation in Huntington's disease.

Failure in energy metabolism and oxidative damage are associated with Huntington's disease (HD). Ascorbic acid released during synaptic activity inhibits use of neuronal glucose, favouring lactate uptake to sustain brain activity. Here, we observe a decreased expression of GLUT3 in STHdhQ111 cells (HD cells) and R6/2 mice (HD mice). Localisation of GLUT3 is decreased at the plasma membrane in HD cells affecting the modulation of glucose uptake by ascorbic acid. An ascorbic acid analogue without antioxidant activity is able to inhibit glucose uptake in HD cells. The impaired modulation of glucose uptake by ascorbic acid is directly related to ROS levels indicating that oxidative stress sequesters the ability of ascorbic acid to modulate glucose utilisation. Therefore, in HD, a decrease in GLUT3 localisation at the plasma membrane would contribute to an altered neuronal glucose uptake during resting periods while redox imbalance should contribute to metabolic failure during synaptic activity.

A failure in energy metabolism and antioxidant uptake precede symptoms of Huntington's disease in mice.

Huntington's disease has been associated with a failure in energy metabolism and oxidative damage. Ascorbic acid is a powerful antioxidant highly concentrated in the brain where it acts as a messenger, modulating neuronal metabolism. Using an electrophysiological approach in R6/2 HD slices, we observe an abnormal ascorbic acid flux from astrocytes to neurons, which is responsible for alterations in neuronal metabolic substrate preferences. Here using striatal neurons derived from knock-in mice expressing mutant huntingtin (STHdhQ cells), we study ascorbic acid transport. When extracellular ascorbic acid concentration increases, as occurs during synaptic activity, ascorbic acid transporter 2 (SVCT2) translocates to the plasma membrane, ensuring optimal ascorbic acid uptake for neurons. In contrast, SVCT2 from cells that mimic HD symptoms (dubbed HD cells) fails to reach the plasma membrane under the same conditions. We reason that an early impairment of ascorbic acid uptake in HD neurons could lead to early metabolic failure promoting neuronal death.

Ascorbic acid-dependent GLUT3 inhibition is a critical step for switching neuronal metabolism.

Intracellular ascorbic acid is able to modulate neuronal glucose utilization between resting and activity periods. We have previously demonstrated that intracellular ascorbic acid inhibits deoxyglucose transport in primary cultures of cortical and hippocampal neurons and in HEK293 cells. The same effect was not seen in astrocytes. Since this observation was valid only for cells expressing glucose transporter 3 (GLUT3), we evaluated the importance of this transporter on the inhibitory effect of ascorbic acid on glucose transport. Intracellular ascorbic acid was able to inhibit (3)H-deoxyglucose transport only in astrocytes expressing GLUT3-EGFP. In C6 glioma cells and primary cultures of cortical neurons, which natively express GLUT3, the same inhibitory effect on (3)H-deoxyglucose transport and fluorescent hexose 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was observed. Finally, knocking down the native expression of GLUT3 in primary cultured neurons and C6 cells using shRNA was sufficient to abolish the ascorbic acid-dependent inhibitory effect on uptake of glucose analogs. Uptake assays using real-time confocal microscopy demonstrated that ascorbic acid effect abrogation on 2-NBDG uptake in cultured neurons. Therefore, ascorbic acid would seem to function as a metabolic switch inhibiting glucose transport in neurons under glutamatergic synaptic activity through direct or indirect inhibition of GLUT3.

A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid.

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K(+)]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD(+)/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.