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.

Low cerebral energy metabolism in hepatic encephalopathy reflects low neuronal energy demand. Role of ammonia-induced increased GABAergic tone.

Hepatic encephalopathy (HE) is a frequent and devastating but generally reversible neuropsychiatric complication secondary to chronic and acute liver failure. During HE, brain energy metabolism is markedly reduced and it remains unclear whether this is due to external or internal energy supply limitations, or secondary to depressed neuronal cellular functions - and if so, which mechanisms that are in play. The extent of deteriorated cerebral function correlates to blood ammonia levels but the metabolic link to ammonia is not clear. Early studies suggested that high levels of ammonia inhibited key tricarboxylic acid (TCA) cycle enzymes thus limiting mitochondrial energy production and oxygen consumption; however, later studies by us and others showed that this is not the case in vivo. Here, based on a series of translational studies from our group, we advocate the view that the low cerebral energy metabolism of HE is likely to be caused by neuronal metabolic depression due to an elevated GABAergic tone rather than by restricted energy availability. The increased GABAergic tone seems to be secondary to synthesis of large amounts of glutamine in astrocytes for detoxification of ammonia with the glutamine acting as a precursor for elevated neuronal synthesis of vesicular GABA.

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.

Decreased Glucose Metabolism and Glutamine Synthesis in the Retina of a Transgenic Mouse Model of Alzheimer's Disease.

Visual changes are some of the earliest symptoms that patients with Alzheimer's disease (AD) experience. Pathophysiological processes such as amyloid-ß plaque formation, vascular changes, neuroinflammation, and loss of retinal ganglion cells (RGCs) have been detected in the retina of AD patients and animal models. However, little is known about the molecular processes that underlie retinal neurodegeneration in AD. The cellular architecture and constant sensory activity of the retina impose high metabolic demands. We thus hypothesized that energy metabolism might be compromised in the AD retina similarly to what has been observed in the AD brain. To address this question, we explored cellular alterations and retinal metabolic activity in the 5 × FAD mouse model of AD. We used 8-month-old female 5 × FAD mice, in which the AD-related pathology has been shown to be apparent. We observed that RGC density is selectively affected in the retina of 5 × FAD mice. To map retinal metabolic activity, we incubated isolated retinal tissue with [U-13C] glucose and analyzed tissue extracts by gas chromatography-mass spectrometry. We found that the retinas of 5 × FAD mice exhibit glucose hypometabolism. Moreover, we detected decreased glutamine synthesis in 5 × FAD retinas but no changes in the expression of markers of Müller glia, the main glial cell type responsible for glutamate uptake and glutamine synthesis in the retina. These findings suggest that AD presents with metabolic alterations not only in the brain but also in the retina that may be detrimental to RGC activity and survival, potentially leading to the visual impairments that AD patients suffer.

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus.

GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20-25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals.

Hypermetabolism and impaired endothelium-dependent vasodilation in mesenteric arteries of type 2 diabetes mellitus db/db mice.

Besides being a metabolic disease, diabetes is considered a vascular disease as many of the complications relate to vascular pathologies. The aim of this study was to investigate how vascular tone and reactivity and vascular cell metabolism were affected in type 2 diabetes mellitus and whether ß-hydroxybutyrate could have a positive effect as alternative energy substrate. Isolated mesenteric arteries of db/db and control mice were incubated in media containing [U-13C]glucose or [U-13C]ß-hydroxybutyrate, and tissue extracts were analysed by mass spectrometry. Functional characterization was performed by wire myography to assess vasodilation and vasocontraction. Hypermetabolism of glucose and ß-hydroxybutyrate was observed for mesenteric arteries of db/db mice; however, hypermetabolism was significant only with ß-hydroxybutyrate as energy substrate. The functional characterization showed impaired endothelial-dependent vasodilation in mesenteric arteries of the db/db mice, whereas the contractility was unaffected. This study provides evidence that the endothelial cells are impaired, whereas the vascular smooth muscle cells are more robust and seemed less affected in the db/db mouse. Furthermore, the results indicate that hypermetabolism of energy substrates may be due to adaptive changes in the mesenteric arteries.

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.

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.