Stable isotope tracing reveals disturbed cellular energy and glutamate metabolism in hippocampal slices of aged male mice.

Neurons and astrocytes work in close metabolic collaboration, linking neurotransmission to brain energy and neurotransmitter metabolism. Dysregulated energy metabolism is a hallmark of the aging brain and may underlie the progressive age-dependent cognitive decline. However, astrocyte and neurotransmitter metabolism remains understudied in aging brain research. In particular, how aging affects metabolism of glutamate, being the primary excitatory neurotransmitter, is still poorly understood. Here we investigated critical aspects of cellular energy metabolism in the aging male mouse hippocampus using stable isotope tracing in vitro. Metabolism of [U-13C]glucose demonstrated an elevated glycolytic capacity of aged hippocampal slices, whereas oxidative [U-13C]glucose metabolism in the TCA cycle was significantly reduced with aging. In addition, metabolism of [1,2-13C]acetate, reflecting astrocyte energy metabolism, was likewise reduced in the hippocampal slices of old mice. In contrast, uptake and subsequent metabolism of [U-13C]glutamate was elevated, suggesting increased capacity for cellular glutamate handling with aging. Finally, metabolism of [15N]glutamate was maintained in the aged slices, demonstrating sustained glutamate nitrogen metabolism. Collectively, this study reveals fundamental alterations in cellular energy and neurotransmitter metabolism in the aging brain, which may contribute to age-related hippocampal deficits.

Progressive Mitochondrial Dysfunction of Striatal Synapses in R6/2 Mouse Model of Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disorder characterized by synaptic dysfunction and loss of white matter volume especially in the striatum of the basal ganglia and to a lesser extent in the cerebral cortex. Studies investigating heterogeneity between synaptic and non-synaptic mitochondria have revealed a pronounced vulnerability of synaptic mitochondria, which may lead to synaptic dysfunction and loss. OBJECTIVE: As mitochondrial dysfunction is a hallmark of HD pathogenesis, we investigated synaptic mitochondrial function from striatum and cortex of the transgenic R6/2 mouse model of HD. METHODS: We assessed mitochondrial volume, ROS production, and antioxidant levels as well as mitochondrial respiration at different pathological stages. RESULTS: Our results reveal that striatal synaptic mitochondria are more severely affected by HD pathology than those of the cortex. Striatal synaptosomes of R6/2 mice displayed a reduction in mitochondrial mass coinciding with increased ROS production and antioxidants levels indicating prolonged oxidative stress. Furthermore, synaptosomal oxygen consumption rates were significantly increased during depolarizing conditions, which was accompanied by a marked increase in mitochondrial proton leak of the striatal synaptosomes, indicating synaptic mitochondrial stress. CONCLUSION: Overall, our study provides new insight into the gradual changes of synaptic mitochondrial function in HD and suggests compensatory mitochondrial actions to maintain energy production in the HD brain, thereby supporting that mitochondrial dysfunction do indeed play a central role in early disease progression of HD.

Functional Differences between Synaptic Mitochondria from the Striatum and the Cerebral Cortex.

Mitochondrial dysfunction has been shown to play a major role in neurodegenerative disorders such as Huntington's disease, Alzheimer's disease and Parkinson's disease. In these and other neurodegenerative disorders, disruption of synaptic connectivity and impaired neuronal signaling are among the early signs. When looking for potential causes of neurodegeneration, specific attention is drawn to the function of synaptic mitochondria, as the energy supply from mitochondria is crucial for normal synaptic function. Mitochondrial heterogeneity between synaptic and non-synaptic mitochondria has been described, but very little is known about possible differences between synaptic mitochondria from different brain regions. The striatum and the cerebral cortex are often affected in neurodegenerative disorders. In this study we therefore used isolated nerve terminals (synaptosomes) from female mice, striatum and cerebral cortex, to investigate differences in synaptic mitochondrial function between these two brain regions. We analyzed mitochondrial mass, citrate synthase activity, general metabolic activity and mitochondrial respiration in resting as well as veratridine-activated synaptosomes using glucose and/or pyruvate as substrate. We found higher mitochondrial oxygen consumption rate in both resting and activated cortical synaptosomes compared to striatal synaptosomes, especially when using pyruvate as a substrate. The higher oxygen consumption rate was not caused by differences in mitochondrial content, but instead corresponded with a higher proton leak in the cortical synaptic mitochondria compared to the striatal synaptic mitochondria. Our results show that the synaptic mitochondria of the striatum and cortex differently regulate respiration both in response to activation and variations in substrate conditions.

Glutamate Transporters in the Blood-Brain Barrier.

The amino acid L-glutamate serves a number of roles in the central nervous system, being an excitatory neurotransmitter, metabolite, and building block in protein synthesis. During pathophysiological events, where L-glutamate homeostasis cannot be maintained, the increased brain interstitial fluid concentration of L-glutamate causes excitotoxicity. A tight control of the brain interstitial fluid L-glutamate levels is therefore imperative, in order to maintain optimal neurotransmission and to avoid such excitotoxicity. The blood-brain barrier, i.e., the endothelial lining of the brain capillaries, regulates the exchange of nutrients, gases, and metabolic waste products between plasma and brain interstitial fluid. It has been suggested that brain capillary endothelial cells could play an important role in L-glutamate homeostasis by mediating brain-to-blood L-glutamate efflux. Both in vitro and in vivo studies have demonstrated blood-to-brain transport of L-glutamate, at least during pathological events. A number of studies have shown that brain endothelial cells express excitatory amino acid transporters, which may account for abluminal concentrative uptake of L-glutamate into the capillary endothelial cells. The mechanisms underlying transendothelial L-glutamate transport are however still not well understood. The present chapter summarizes the current knowledge on blood-brain barrier L-glutamate transporters and the suggested pathways for the brain-to-blood L-glutamate efflux.

Impaired Hippocampal Glutamate and Glutamine Metabolism in the db/db Mouse Model of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a risk factor for the development of Alzheimer's disease, and changes in brain energy metabolism have been suggested as a causative mechanism. The aim of this study was to investigate the cerebral metabolism of the important amino acids glutamate and glutamine in the db/db mouse model of T2DM. Glutamate and glutamine are both substrates for mitochondrial oxidation, and oxygen consumption was assessed in isolated brain mitochondria by Seahorse XFe96 analysis. In addition, acutely isolated cerebral cortical and hippocampal slices were incubated with [U-13C]glutamate and [U-13C]glutamine, and tissue extracts were analyzed by gas chromatography-mass spectrometry. The oxygen consumption rate using glutamate and glutamine as substrates was not different in isolated cerebral mitochondria of db/db mice compared to controls. Hippocampal slices of db/db mice exhibited significantly reduced 13C labeling in glutamate, glutamine, GABA, citrate, and aspartate from metabolism of [U-13C]glutamate. Additionally, reduced 13C labeling were observed in GABA, citrate, and aspartate from [U-13C]glutamine metabolism in hippocampal slices of db/db mice when compared to controls. None of these changes were observed in cerebral cortical slices. The results suggest specific hippocampal impairments in glutamate and glutamine metabolism, without affecting mitochondrial oxidation of these substrates, in the db/db mouse.

Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis.

Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous 13 C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of 13 C-labeled acetate as an astrocyte specific metabolic substrate. Recent studies have, however, challenged the arguments used to anchor this astrocyte specificity of acetate and glutamate. The aim of the current study was to evaluate the specificity of acetate and glutamate as astrocyte substrates in brain slices. Acutely isolated hippocampal and cerebral cortical slices from female NMRI mice were incubated in media containing [1,2-13 C]acetate or [U-13 C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority of glutamine 13 C-labeling from [1,2-13 C]acetate as intended. However, 13 C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-13 C]acetate in the presence of MSO exceeded the level probable from exclusive labeling of the astrocytic pool, which likewise suggests neuronal acetate metabolism. Approximately 50% of glutamate was uniformly labeled in slices incubated with [U-13 C]glutamate in the presence of MSO, suggesting that neurons exhibit substantial uptake of exogenously provided glutamate. © 2017 Wiley Periodicals, Inc.

In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.

The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm(2) , and (14) C-D-mannitol permeability values of 0.88 ± 0.13 × 10(-6) cm s(-1) . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K(m) of 69 ± 15 µM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 µM and J(max) of 28 ± 3.1 pmol min(-1) cm(-2) for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), -2 (Slc1a2), and -3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and -3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations.

Brain glycogen and its role in supporting glutamate and GABA homeostasis in a type 2 diabetes rat model.

The number of people suffering from diabetes is hastily increasing and the condition is associated with altered brain glucose homeostasis. Brain glycogen is located in astrocytes and being a carbohydrate reservoir it contributes to glucose homeostasis. Furthermore, glycogen has been indicated to be important for proper neurotransmission under normal conditions. Previous findings from our laboratory suggested that glucose metabolism was reduced in type 2 diabetes, and thus we wanted to investigate more specifically how brain glycogen metabolism contributes to maintain energy status in the type 2 diabetic state. Also, our objective was to elucidate the contribution of glycogen to support neurotransmitter glutamate and GABA homeostasis. A glycogen phosphorylase (GP) inhibitor was administered to Sprague-Dawley (SprD) and Zucker Diabetic Fatty (ZDF) rats in vivo and after one day of treatment [1-¹³C]glucose was used to monitor metabolism. Brain levels of ¹³C labeling in glucose, lactate, alanine, glutamate, GABA, glutamine and aspartate were determined. Our results show that inhibition of brain glycogen metabolism reduced the amounts of glutamate in both the control and type 2 diabetes models. The reduction in glutamate was associated with a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in the control but not the type 2 diabetes model. In the type 2 diabetes model GABA levels were increased suggesting that brain glycogen serves a role in maintaining a proper ratio between excitatory and inhibitory neurotransmitters in type 2 diabetes. Both the control and the type 2 diabetic states had a compensatory increase in glucose-derived ¹³C processed through the TCA cycle following inhibition of glycogen degradation. Finally, it was indicated that the type 2 diabetes model might have an augmented necessity for compensatory upregulation at the glycolytic level.

Branched-chain amino acids increase arterial blood ammonia in spite of enhanced intrinsic muscle ammonia metabolism in patients with cirrhosis and healthy subjects.

Branched-chain amino acids (BCAA) are used in attempts to reduce blood ammonia in patients with cirrhosis and intermittent hepatic encephalopathy based on the hypothesis that BCAA stimulate muscle ammonia detoxification. We studied the effects of an oral dose of BCAA on the skeletal muscle metabolism of ammonia and amino acids in 14 patients with cirrhosis and in 7 healthy subjects by combining [(13)N]ammonia positron emission tomography (PET) of the thigh muscle with measurements of blood flow and arteriovenous (A-V) concentrations of ammonia and amino acids. PET was used to measure the metabolism of blood-supplied ammonia and the A-V measurements were used to measure the total ammonia metabolism across the thigh muscle. After intake of BCAA, blood ammonia increased more than 30% in both groups of subjects (both P < 0.05). Muscle clearance of blood-supplied ammonia (PET) was unaffected (P = 0.75), but the metabolic removal rate (PET) increased significantly because of increased blood ammonia in both groups (all P < 0.05). The total ammonia clearance across the leg muscle (A-V) increased by more than 50% in both groups, and the flux (A-V) of ammonia increased by more than 45% (all P < 0.05). BCAA intake led to a massive glutamine release from the muscle (cirrhotic patients, P < 0.05; healthy subjects, P = 0.12). In conclusion, BCAA enhanced the intrinsic muscle metabolism of ammonia but not the metabolism of blood-supplied ammonia in both the patients with cirrhosis and in the healthy subjects.

Paracellular tightness and claudin-5 expression is increased in the BCEC/astrocyte blood-brain barrier model by increasing media buffer capacity during growth.

Most attempts to develop in vitro models of the blood-brain barrier (BBB) have resulted in models with low transendothelial electrical resistances (TEER), as compared to the native endothelium. The aim of the present study was to investigate the impact of culture pH and buffer concentration on paracellular tightness of an established in vitro model of the BBB consisting of bovine brain capillary endothelial cells (BCEC) co-cultured with rat astrocytes. BCEC and rat astrocytes were isolated and co-cultured using astrocyte-conditioned media with cAMP increasing agonists and dexamethasone. The co-culture had average TEER values from 261 ± 26 Ω cm² to 760 ± 46 Ω cm² dependent on BCEC isolation batches. Furthermore, mRNA of occludin, claudin-1, claudin-5, JAM-1, and ZO-1 were detected. Increased buffer concentration by addition of HEPES, MOPS, or TES to the media during differentiation increased the TEER up to 1,638 ± 256 Ω cm² independent of the type of buffer. This correlated with increased expression of claudin-5, while expression of the other tight junction proteins remained unchanged. Thus, we show for the first time that increased buffer capacity of the medium during differentiation significantly increases tightness of the BCEC/astrocyte in vitro BBB model. This regulation may be mediated by increased claudin-5 expression. The observations have practical implications for generating tighter BBB cell culture models, and may also have physiological implications, if similar sensitivity to pH-changes can be demonstrated in vivo.

Low cerebral oxygen consumption and blood flow in patients with cirrhosis and an acute episode of hepatic encephalopathy.

BACKGROUND & AIMS: It is unclear whether patients with hepatic encephalopathy (HE) have disturbed brain oxygen metabolism and blood flow. METHODS: We measured cerebral oxygen metabolism rate (CMRO(2)) by using (15)O-oxygen positron emission tomography (PET); and cerebral blood flow (CBF) by using (15)O-water PET in 6 patients with liver cirrhosis and an acute episode of overt HE, 6 cirrhotic patients without HE, and 7 healthy subjects. RESULTS: Neither whole-brain CMRO(2) nor CBF differed significantly between cirrhotic patients without HE and healthy subjects, but were both significantly reduced in cirrhotic patients with HE (P < .01). CMRO(2) was 0.96 +/- 0.07 mumol oxygen/mL brain tissue/min (mean +/- SEM) in cirrhotic patients with HE, 1.34 +/- 0.08 in cirrhotic patients without HE, and 1.35 +/- 0.05 in healthy subjects; and CBF was 0.29 +/- 0.01 mL blood/mL brain tissue/min in patients with HE, 0.47 +/- 0.02 in patients without HE, and 0.49 +/- 0.03 in healthy subjects. CMRO(2) and CBF were correlated, and both variables correlated negatively with arterial ammonia concentration. Analysis of regional values, using individual magnetic resonance co-registrations, showed that the reductions in CMRO(2) and CBF in patients with HE were essentially generalized throughout the brain. CONCLUSIONS: The observations imply that reduced cerebral oxygen consumption and blood flow in cirrhotic patients with an acute episode of overt HE are associated with HE and not cirrhosis as such, and that the primary event in the pathogenesis of HE could be inhibition of cerebral energy metabolism by increased blood ammonia.