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-20th 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metabolism, the earlier picture of astrocytes being restricted to a scaffold-associated function in the brain is now out of date.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 provide 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 somatosensory stimulation

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 P5-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 integrit

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 β-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 β-oxidation of these medium-chain fatty acids (MCFA) differ. We aim to elucidate basal β-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 β-hydroxybutyrate (βHB) 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 β-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

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

Study of cortical energy metabolism during sensory stimulation-induced brain activity by ¹³C magnetic resonance spectroscopy in vivo

Cerebral function is associated with high metabolic activity that is supported by continuous supply of oxygen and glucose from the blood. Coupling between brain energy metabolism and neuronal activity has been studied extensively during the past decades. Notably, glucose metabolism was demonstrated to result from cellular cooperation between neurons and astrocytes. Although both cell types regulate synaptic transmission via the glutamate-glutamine cycle, the actual contribution of glial and neuronal oxidative metabolism is still unclear. Several research groups have tried to quantify cerebral energy metabolism in the living brain using methods, such as positron emission tomography, autoradiography and proton magnetic resonance spectroscopy (1H MRS). Yet, these methods lack chemical specificity and do not provide quantitative information specific to either cell types. The development of tracers detectable by MRS, such as 13C isotope, along with high magnetic field and dedicated hardware systems has improved sensitivity and has motivated the design of advanced metabolic modeling, in which cell types can be compartmentalized and which associated metabolic rates can be assessed independently of any prior estimations. The work of this thesis aimed at focally increasing neuronal activity and estimating the resulting changes in glial and neuronal oxidative metabolism using direct 13C MRS along with [1,6-13C]glucose infusion in vivo at 14.1 T. 4h-somatosensory stimulation paradigm was first developed in rats under alpha-chloralose. The protocol consisted on intermittently electrically stimulating both fore- and hindpaws, which resulted in a sustained and localized blood oxygenation level-dependent signal as assessed by functional magnetic resonance imaging. The relatively large activated area in the cortex allowed monitoring local 13C isotope incorporation over 4h into specific positions of glutamate, glutamine and aspartate. The analysis of the turnover curves with a two-compartment model of brain energy metabolism revealed an increase of both glial (VTCAg,+68 nmol/g/min,+22%) and neuronal (VTCAn,+62 nmol/g/min,+12%) oxidative metabolism associated with 95% increase (VNT,+67 nmol/g/min) in glutamate-glutamine cycle rate, which represents glutamatergic neurotransmission rate. The total increase in glucose oxidative metabolism was of 15% (CMRglc(ox), +67 nmol/g/min). In randomly delivering lines in 4 orientations and 2 directions, 4h-continuous stimulation of tree shrew primary visual cortex under light isoflurane anesthesia resulted in a decrease in both brain glucose concentration (-17%;-0.34 µmol/g) and phosphocreatine/creatine ratio (-9%;-0.07) after 16 minutes of stimulation onset, which recovered 21 minutes after stimulation offset. At the individual level, a nearly one-to-one relationships between VNT and CMRglc(ox) was observed. VTCAn and VTCAg were also coupled to VNT. At the group level, 20% increase in VNT (+0.038±0.042 µmol/g/min) resulted in 24% (+0.063±0.057 µmol/g/min) and 12% (+0.061±0.032 µmol/g/min) increase in VTCAg and VTCAn, respectively, resulting in 14% increase in CMRglc(ox) (+0.058±0.032 µmol/g/min). To conclude, this work demonstrates that a significant fraction of glucose is also oxidized in astrocytes and that both neuronal and astrocytic metabolism can be stimulated by and coupled to the glutamate-glutamine cycle rate

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, β-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

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-minute 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

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