Exploring Valine Metabolism in Astrocytic and Liver Cells: Lesson from Clinical Observation in TBI Patients for Nutritional Intervention.

The utilization of alternative energy substrates to glucose could be beneficial in traumatic brain injury (TBI). Recent clinical data obtained in TBI patients reported valine, ß-hydroxyisobutyrate (ibHB) and 2-ketoisovaleric acid (2-KIV) as three of the main predictors of TBI outcome. In particular, higher levels of ibHB, 2-KIV, and valine in cerebral microdialysis (CMD) were associated with better clinical outcome. In this study, we investigate the correlations between circulating and CMD levels of these metabolites. We hypothesized that the liver can metabolize valine and provide a significant amount of intermediate metabolites, which can be further metabolized in the brain. We aimed to assess the metabolism of valine in human-induced pluripotent stem cell (iPSC)-derived astrocytes and HepG2 cells using 13C-labeled substrate to investigate potential avenues for increasing the levels of downstream metabolites of valine via valine supplementation. We observed that 94 ± 12% and 84 ± 16% of ibHB, and 94 ± 12% and 87 ± 15% of 2-KIV, in the medium of HepG2 cells and in iPSC-derived astrocytes, respectively, came directly from valine. Overall, these findings suggest that both ibHB and 2-KIV are produced from valine to a large extent in both cell types, which could be of interest in the design of optimal nutritional interventions aiming at stimulating valine metabolism.

Therapeutic potential of glutathione-enhancers in stress-related psychopathologies.

The mammalian brain has high energy demands, which may become higher in response to environmental challenges such as psychogenic stress exposure. Therefore, efficient neutralization of reactive oxygen species that are produced as a by-product of ATP synthesis is crucial for preventing oxidative damage and ensuring normal energy supply and brain function. Glutathione (GSH) is arguably the most important endogenous antioxidant in the brain. In recent years, aberrant GSH levels have been implicated in different psychiatric disorders, including stress-related psychopathologies. In this review, we examine the available data supporting a role for GSH levels and antioxidant function in the brain in relation to anxiety and stress-related psychopathologies. Additionally, we identify several promising compounds that could raise GSH levels in the brain by either increasing the availability of its precursors or the expression of GSH-regulating enzymes through activation of Nuclear factor erythroid-2-related factor 2 (Nrf2). Given the high tolerability and safety profile of these compounds, they may represent attractive new opportunities to complement existing therapeutic manipulations against stress-related psychopathologies.

Differential Metabolism of Medium-Chain Fatty Acids in Differentiated Human-Induced Pluripotent Stem Cell-Derived Astrocytes.

Medium-chain triglyceride (MCT) ketogenic diets increase ketone bodies, which are believed to act as alternative energy substrates in the injured brain. Octanoic (C8:0) and decanoic (C10:0) acids, which produce ketone bodies through ß-oxidation, are used as part of MCT ketogenic diets. Although the ketogenic role of MCT is well-established, it remains unclear how the network metabolism underlying ß-oxidation of these medium-chain fatty acids (MCFA) differ. We aim to elucidate basal ß-oxidation of these commonly used MCFA at the cellular level. Human-induced pluripotent stem cell-derived (iPSC) astrocytes were incubated with [U-13C]-C8:0 or [U-13C]-C10:0, and the fractional enrichments (FE) of the derivatives were used for metabolic flux analysis. Data indicate higher extracellular concentrations and faster secretion rates of ß-hydroxybutyrate (ßHB) and acetoacetate (AcAc) with C8:0 than C10:0, and an important contribution from unlabeled substrates. Flux analysis indicates opposite direction of metabolic flux between the MCFA intermediates C6:0 and C8:0, with an important contribution of unlabeled sources to the elongation in the C10:0 condition, suggesting different ß-oxidation pathways. Finally, larger intracellular glutathione concentrations and secretions of 3-OH-C10:0 and C6:0 were measured in C10:0-treated astrocytes. These findings reveal MCFA-specific ketogenic properties. Our results provide insights into designing different MCT-based ketogenic diets to target specific health benefits.

Discovery and validation of temporal patterns involved in human brain ketometabolism in cerebral microdialysis fluids of traumatic brain injury patients.

BACKGROUND: Traumatic brain injury (TBI) is recognized as a metabolic disease, characterized by acute cerebral glucose hypo-metabolism. Adaptive metabolic responses to TBI involve the utilization of alternative energy substrates, such as ketone bodies. Cerebral microdialysis (CMD) has evolved as an accurate technique allowing continuous sampling of brain extracellular fluid and assessment of regional cerebral metabolism. We present the successful application of a combined hypothesis- and data-driven metabolomics approach using repeated CMD sampling obtained routinely at patient bedside. Investigating two patient cohorts (n = 26 and n = 12), we identified clinically relevant metabolic patterns at the acute post-TBI critical care phase. METHODS: Clinical and CMD metabolomics data were integrated and analysed using in silico and data modelling approaches. We used both unsupervised and supervised multivariate analysis techniques to investigate structures within the time series and associations with patient outcome. FINDINGS: The multivariate metabolite time series exhibited two characteristic brain metabolic states that were attributed to changes in key metabolites: valine, 4-methyl-2-oxovaleric acid (4-MOV), isobeta-hydroxybutyrate (iso-bHB), tyrosyine, and 2-ketoisovaleric acid (2-KIV). These identified cerebral metabolic states differed significantly with respect to standard clinical values. We validated our findings in a second cohort using a classification model trained on the cerebral metabolic states. We demonstrated that short-term (therapeutic intensity level (TIL)) and mid-term patient outcome (6-month Glasgow Outcome Score (GOS)) can be predicted from the time series characteristics. INTERPRETATION: We identified two specific cerebral metabolic patterns that are closely linked to ketometabolism and were associated with both TIL and GOS. Our findings support the view that advanced metabolomics approaches combined with CMD may be applied in real-time to predict short-term treatment intensity and long-term patient outcome.

Alterations of Brain Energy Metabolism in Type 2 Diabetic Goto-Kakizaki Rats Measured In Vivo by 13C Magnetic Resonance Spectroscopy.

Type 2 diabetes (T2D) is associated with deterioration of brain structure and function. Here, we tested the hypothesis that T2D induces a reorganization of the brain metabolic networks that support brain function. For that, alterations of neuronal and glial energy metabolism were investigated in a T2D model, the Goto-Kakizaki (GK) rat. 13C magnetic resonance spectroscopy in vivo at 14.1 T was used to detect 13C labeling incorporation into carbons of glutamate, glutamine, and aspartate in the brain of GK (n = 7) and Wistar (n = 13) rats during intravenous [1,6-13C]glucose administration. Labeling of brain glucose and amino acids over time was analyzed with a two-compartment mathematical model of brain energy metabolism to determine the rates of metabolic pathways in neurons and glia. Compared to controls, GK rats displayed lower rates of brain glutamine synthesis (- 32%, P < 0.001) and glutamate-glutamine cycle (- 40%, P < 0.001), and mitochondrial tricarboxylic acid (TCA) cycle rate in neurons (- 7%, P = 0.036). In contrast, the TCA cycle rate of astrocytes was larger in GK rats than controls (+ 21%, P = 0.042). We conclude that T2D alters brain energy metabolism and impairs the glutamate-glutamine cycle between neurons and astrocytes, in line with diabetes-induced neurodegeneration and astrogliosis underlying brain dysfunction.

Astrocytic and neuronal oxidative metabolism are coupled to the rate of glutamate-glutamine cycle in the tree shrew visual cortex.

Astrocytes play an important role in glutamatergic neurotransmission, namely by clearing synaptic glutamate and converting it into glutamine that is transferred back to neurons. The rate of this glutamate-glutamine cycle (VNT ) has been proposed to couple to that of glucose utilization and of neuronal tricarboxylic acid (TCA) cycle. In this study, we tested the hypothesis that glutamatergic neurotransmission is also coupled to the TCA cycle rate in astrocytes. For that we investigated energy metabolism by means of magnetic resonance spectroscopy (MRS) in the primary visual cortex of tree shrews (Tupaia belangeri) under light isoflurane anesthesia at rest and during continuous visual stimulation. After identifying the activated cortical volume by blood oxygenation level-dependent functional magnetic resonance imaging, 1 H MRS was performed to measure stimulation-induced variations in metabolite concentrations. Relative to baseline, stimulation of cortical activity for 20 min caused a reduction of glucose concentration by -0.34 ± 0.09 µmol/g (p < 0.001), as well as a -9% ± 1% decrease of the ratio of phosphocreatine-to-creatine (p < 0.05). Then 13 C MRS during [1,6-13 C]glucose infusion was employed to measure fluxes of energy metabolism. Stimulation of glutamatergic activity, as indicated by a 20% increase of VNT , resulted in increased TCA cycle rates in neurons by 12% ( VTCAn, p < 0.001) and in astrocytes by 24% ( VTCAg, p = 0.007). We further observed linear relationships between VNT and both VTCAn and VTCAg. Altogether, these results suggest that in the tree shrew primary visual cortex glutamatergic neurotransmission is linked to overall glucose oxidation and to mitochondrial metabolism in both neurons and astrocytes.

How Energy Metabolism Supports Cerebral Function: Insights from 13C Magnetic Resonance Studies In vivo.

Cerebral function is associated with exceptionally high metabolic activity, and requires continuous supply of oxygen and nutrients from the blood stream. Since the mid-twentieth century the idea that brain energy metabolism is coupled to neuronal activity has emerged, and a number of studies supported this hypothesis. Moreover, brain energy metabolism was demonstrated to be compartmentalized in neurons and astrocytes, and astrocytic glycolysis was proposed to serve the energetic demands of glutamatergic activity. Shedding light on the role of astrocytes in brain metabolism, the earlier picture of astrocytes being restricted to a scaffold-associated function in the brain is now out of date. With the development and optimization of non-invasive techniques, such as nuclear magnetic resonance spectroscopy (MRS), several groups have worked on assessing cerebral metabolism in vivo. In this context, 1H MRS has allowed the measurements of energy metabolism-related compounds, whose concentrations can vary under different brain activation states. 1H-[13C] MRS, i.e., indirect detection of signals from 13C-coupled 1H, together with infusion of 13C-enriched glucose has provided insights into the coupling between neurotransmission and glucose oxidation. Although these techniques tackle the coupling between neuronal activity and metabolism, they lack chemical specificity and fail in providing information on neuronal and glial metabolic pathways underlying those processes. Currently, the improvement of detection modalities (i.e., direct detection of 13C isotopomers), the progress in building adequate mathematical models along with the increase in magnetic field strength now available render possible detailed compartmentalized metabolic flux characterization. In particular, direct 13C MRS offers more detailed dataset acquisitions and provides information on metabolic interactions between neurons and astrocytes, and their role in supporting neurotransmission. Here, we review state-of-the-art MR methods to study brain function and metabolism in vivo, and their contribution to the current understanding of how astrocytic energy metabolism supports glutamatergic activity and cerebral function. In this context, recent data suggests that astrocytic metabolism has been underestimated. Namely, the rate of oxidative metabolism in astrocytes is about half of that in neurons, and it can increase as much as the rate of neuronal metabolism in response to sensory stimulation.

Energy metabolism in the rat cortex under thiopental anaesthesia measured In Vivo by 13 C MRS.

Barbiturates, commonly used as general anaesthetics, depress neuronal activity and thus cerebral metabolism. Moreover, they are likely to disrupt the metabolic support of astrocytes to neurons, as well as the uptake of nutrients from circulation. By employing 13 C magnetic resonance spectroscopy (MRS) in vivo at high magnetic field, we characterized neuronal and astrocytic pathways of energy metabolism in the rat cortex under thiopental anaesthesia. The neuronal tricarboxylic acid (TCA) cycle rate was 0.46 ± 0.02 µmol/g/min, and the rate of the glutamate-glutamine cycle was 0.09 ± 0.02 µmol/g/min. In astrocytes, the TCA cycle rate was 0.16 ± 0.02 µmol/g/min, accounting for a quarter of whole brain glucose oxidation, pyruvate carboxylase rate was 0.02 ± 0.01 µmol/g/min, and glutamine synthetase was 0.12 ± 0.01 µmol/g/min. Relative to previous experiments under light α-chloralose anaesthesia, thiopental reduced oxidative metabolism in neurons and even more so in astrocytes. Interestingly, total oxidative metabolism in the cortex under thiopental anaesthesia surpassed the rate of pyruvate production by glycolysis, indicating substantial utilisation of substrates other than glucose, likely plasma lactate. © 2017 Wiley Periodicals, Inc.

Lactate and glutamate dynamics during prolonged stimulation of the rat barrel cortex suggest adaptation of cerebral glucose and oxygen metabolism.

A better understanding of BOLD responses stems from a better characterization of the brain's ability to metabolize glucose and oxygen. Non-invasive techniques such as functional magnetic resonance spectroscopy (fMRS) have thus been developed allowing for the reproducible assessment of metabolic changes during barrel cortex (S1BF) activations in rats. The present study aimed at further exploring the role of neurotransmitters on local and temporal changes in vascular and metabolic function in S1BF. fMRS and fMRI data were acquired sequentially in α-chloralose anesthetized rats during 32-min rest and trigeminal nerve stimulation periods. During stimulation, concentrations of lactate (Lac) and glutamate (Glu) increased in S1BF by 0.23±0.05 and 0.34±0.05µmol/g respectively in S1BF. Dynamic analysis of metabolite concentrations allowed estimating changes in cerebral metabolic rates of glucose (ΔCMRGlc) and oxygen (ΔCMRO2). Findings confirmed a prevalence of oxidative metabolism during prolonged S1BF activation. Habituation led to a significant BOLD magnitude decline as a function of time while both total ΔCMRGlc and ΔCMRO2 remained constant revealing adaptation of glucose and oxygen metabolisms to support ongoing trigeminal nerve stimulation.

Investigating the Role of Glutamate and GABA in the Modulation of Transthalamic Activity: A Combined fMRI-fMRS Study.

The Excitatory-Inhibitory balance (EIB) between glutamatergic and GABAergic neurons is known to regulate the function of thalamocortical neurocircuits. The thalamus is known as an important relay for glutamatergic and GABAergic signals ascending/descending to/from the somatosensory cortex in rodents. However, new investigations attribute a larger role to thalamic nuclei as modulators of information processing within the cortex. In this study, functional Magnetic Resonance Spectroscopy (fMRS) was used to measure glutamate (Glu) and GABA associations with BOLD responses during activation of the thalamus to barrel cortex (S1BF) pathway at 9.4T. In line with previous studies in humans, resting GABA and Glu correlated negatively and positively respectively with BOLD responses in S1BF. Moreover, a significant negative correlation (R = -0.68, p = 0.0024) between BOLD responses in the thalamus and the barrel cortex was found. Rats with low Glu levels and high resting GABA levels in S1BF demonstrated lower BOLD responses in S1BF and high amplitude BOLD responses in the thalamus themselves linked to the release of high GABA levels during stimulation. In addition, early analysis of resting state functional connectivity suggested EIB controlled thalamocortical neuronal synchrony. We propose that the presented approach may be useful for further characterization of diseases affecting thalamocortical neurotransmission.

Compartmentalised energy metabolism supporting glutamatergic neurotransmission in response to increased activity in the rat cerebral cortex: A 13C MRS study in vivo at 14.1 T.

Many tissues exhibit metabolic compartmentation. In the brain, while there is no doubt on the importance of functional compartmentation between neurons and glial cells, there is still debate on the specific regulation of pathways of energy metabolism at different activity levels. Using (13)C magnetic resonance spectroscopy (MRS) in vivo, we determined fluxes of energy metabolism in the rat cortex under α-chloralose anaesthesia at rest and during electrical stimulation of the paws. Compared to resting metabolism, the stimulated rat cortex exhibited increased glutamate-glutamine cycle (+67 nmol/g/min, +95%, P < 0.001) and tricarboxylic (TCA) cycle rate in both neurons (+62 nmol/g/min, +12%, P < 0.001) and astrocytes (+68 nmol/g/min, +22%, P = 0.072). A minor, non-significant modification of the flux through pyruvate carboxylase was observed during stimulation (+5 nmol/g/min, +8%). Altogether, this increase in metabolism amounted to a 15% (67 nmol/g/min, P < 0.001) increase in CMRglc(ox), i.e. the oxidative fraction of the cerebral metabolic rate of glucose. In conclusion, stimulation of the glutamate-glutamine cycle under α-chloralose anaesthesia is associated to similar enhancement of neuronal and glial oxidative metabolism.

Assessment of brain metabolite correlates of adeno-associated virus-mediated over-expression of human alpha-synuclein in cortical neurons by in vivo (1) H-MR spectroscopy at 9.4 T.

In this study, we used proton-localized spectroscopy ((1) H-MRS) for the acquisition of the neurochemical profile longitudinally in a novel rat model of human wild-type alpha-synuclein (α-syn) over-expression. Our goal was to find out if the increased α-syn load in this model could be linked to changes in metabolites in the frontal cortex. Animals injected with AAV vectors encoding for human α-syn formed the experimental group, whereas green fluorescent protein expressing animals were used as the vector-treated control group and a third group of uninjected animals were used as naïve controls. Data were acquired at 2, 4, and 8 month time points. Nineteen metabolites were quantified in the MR spectra using LCModel software. On the basis of 92 spectra, we evaluated any potential gender effect and found that lactate (Lac) levels were lower in males compared to females, while the opposite was observed for ascorbate (Asc). Next, we assessed the effect of age and found increased levels of GABA, Tau, and GPC+PCho. Finally, we analyzed the effect of treatment and found that Lac levels (p = 0.005) were specifically lower in the α-syn group compared to the green fluorescent protein and control groups. In addition, Asc levels (p = 0.05) were increased in the vector-injected groups, whereas glucose levels remained unchanged. This study indicates that the metabolic switch between glucose-lactate could be detectable in vivo and might be modulated by Asc. No concomitant changes were found in markers of neuronal integrity (e.g., N-acetylaspartate) consistent with the fact that α-syn over-expression in cortical neurons did not result in neurodegeneration in this model. We acquired the neurochemical profile longitudinally in a rat model of human wild-type alpha-synuclein (α-syn) over-expression in cortical neurons. We found that Lactate levels were reduced in the α-syn group compared to the control groups and Ascorbate levels were increased in the vector-injected groups. No changes were found in markers of neuronal integrity consistent with the fact that α-syn over-expression did not result in frank neurodegeneration.

Imaging of prolonged BOLD response in the somatosensory cortex of the rat.

Blood oxygenation level-dependent (BOLD) functional MRI is a widely employed methodology in experimental and clinical neuroscience, although its nature is not fully understood. To gain insights into BOLD mechanisms and take advantage of the new functional methods, it is of interest to investigate prolonged paradigms of activation suitable for long experimental protocols and to observe any long-term modifications induced by these functional challenges. While different types of sustained stimulation paradigm have been explored in human studies, the BOLD response is typically limited to a few minutes in animal models, due to fatigue, anesthesia effects and physiological instability. In the present study, the rat forepaw was electrically stimulated for 2 h, which resulted in a prolonged and localized cortical BOLD response over that period. The stimulation paradigm, including an inter-stimulus interval (ISI) of 10 s, that is 25% of the total time, was applied at constant or variable frequency over 2 h. The steady-state level of the BOLD response was reached after 15-20 min of stimulation and was maintained until the end of the stimulation. On average, no substantial loss in activated volume was observed at the end of the stimulation, but less variability in the fraction of remaining activated volume and higher steady-state BOLD amplitude were observed when stimulation frequency was varied between 2 and 3 Hz every 5 min. We conclude that the combination of ISI and variable stimulus frequency reproducibly results in robust, prolonged and localized BOLD activation.

Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9) associated with early-onset parkinsonism.

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism.

PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P(5)-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity.

Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1.

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.