Preface: Special issue: 14th International Conference on Brain Energy Metabolism: Energy substrates and microbiome govern brain bioenergetics and cognitive function with aging.

This Preface introduces the Special Issue entitled, "Energy Substrates and Microbiome Govern Brain Bioenergetics and Cognitive Function with Aging", which is comprised of manuscripts contributed by invited speakers and program/organizing committee members who participated in the 14th International Conference on Brain Energy Metabolism (ICBEM) held on October 24-27, 2022 in Santa Fe, New Mexico, USA. The conference covered the latest developments in research related to neuronal energetics, emerging roles for glycogen in higher brain functions, the impact of dietary intervention on aging, memory, and Alzheimer's disease, roles of the microbiome in gut-brain signaling, astrocyte-neuron interactions related to cognition and memory, novel roles for mitochondria and their metabolites, and metabolic neuroimaging in aging and neurodegeneration. The special issue contains 25 manuscripts on these topics plus three tributes to outstanding scientists who have made important contributions to brain energy metabolism and participated in numerous ICBEM conferences. In addition, two of the manuscripts describe important directions and the rationale for future research in many thematic areas covered by the conference.

Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

A tribute to John Edmond: His leadership role in founding the International Conference on Brain Energy Metabolism (ICBEM) meetings and key contributions to substrate metabolism in brain cells.

This is a tribute to John Edmond, professor emeritus of biological chemistry in the David Geffen School of Medicine at UCLA, a renowned neurochemist who had a leadership role in founding the ICBEM meeting series in 1993. John was known for his very warm and engaging personality and his innovative approaches to studying the developing brain and auditory system. He was a brilliant scientist and a fun and delightful person. Without John Edmond's enthusiasm and contributions, we would not have the biennial ICBEM meetings which as noted by Dienel et al. "have had a high impact on conceptual and experimental advances" … "in the energetics and metabolism underlying neural functions"… and "on promoting collaborative interactions among neuroscientists." Sadly, John Edmond passed away on February 18, 2022, following a cerebral hemorrhage. He will be greatly missed by his colleagues and friends.

A tribute to Leif Hertz: The historical context of his pioneering studies of the roles of astrocytes in brain energy metabolism, neurotransmission, cognitive functions, and pharmacology identifies important, unresolved topics for future studies.

Leif Hertz, M.D., D.Sc. (honoris causa) (1930-2018), was one of the original and noteworthy participants in the International Conference on Brain Energy Metabolism (ICBEM) series since its inception in 1993. The biennial ICBEM conferences are organized by neuroscientists interested in energetics and metabolism underlying neural functions; they have had a high impact on conceptual and experimental advances in these fields and on promoting collaborative interactions among neuroscientists. Leif made major contributions to ICBEM discussions and understanding of metabolic and signaling characteristics of astrocytes and their roles in brain function. His studies ranged from uptake of K+ from extracellular fluid and its stimulation of astrocytic respiration, identification, and regulation of enzymes specifically or preferentially expressed in astrocytes in the glutamate-glutamine cycle of excitatory neurotransmission, a requirement for astrocytic glycogenolysis for fueling K+ uptake, involvement of glycogen in memory consolidation in the chick, and pharmacology of astrocytes. This tribute to Leif Hertz highlights his major discoveries, the high impact of his work on astrocyte-neuron interactions, and his unparalleled influence on understanding the cellular basis of brain energy metabolism. His work over six decades has helped integrate the roles of astrocytes into neurotransmission where oxidative and glycogenolytic metabolism during neurotransmitter glutamate turnover are key aspects of astrocytic energetics. Leif recognized that brain astrocytic metabolism is greatly underestimated unless the volume fraction of astrocytes is taken into account. Adjustment for pathway rates expressed per gram tissue for volume fraction indicates that astrocytes have much higher oxidative rates than neurons and astrocytic glycogen concentrations and glycogenolytic rates during sensory stimulation in vivo are similar to those in resting and exercising muscle, respectively. These novel insights are typical of Leif's astute contributions to the energy metabolism field, and his publications have identified unresolved topics that provide the neuroscience community with challenges and opportunities for future research.

A tribute to Sebastián Cerdán and his key contributions to brain metabolism.

This is a tribute to Sebastián Cerdán, a brilliant and innovative NMR spectroscopist whose studies contributed greatly to the fundamental information to the understanding of brain metabolism, particularly in regard to multinuclear magnetic resonance spectroscopy (MRS) techniques. Sebastián Cerdán sadly passed away in May 2022. He was a wonderful mentor and colleague who will be greatly missed.

Choline Improves Neonatal Hypoxia-Ischemia Induced Changes in Male but Not Female Rats.

Choline is an essential nutrient with many roles in brain development and function. Supplementation of choline in early development can have long-lasting benefits. Our experiments aimed to determine the efficacy of choline supplementation in a postnatal day (PND) 10 rat model of neonatal hypoxia ischemia (HI) at term using both male and female rat pups. Choline (100 mg/kg) or saline administration was initiated the day after birth and given daily for 10 or 14 consecutive days. We determined choline's effects on neurite outgrowth of sex-specific cultured cerebellar granule cells after HI with and without choline. The magnitude of tissue loss in the cerebrum was determined at 72 h after HI and in adult rats. The efficacy of choline supplementation in improving motor ability and learning, tested using eyeblink conditioning, were assessed in young adult male and female rats. Overall, we find that choline improves neurite outgrowth, short-term histological measures and learning ability in males. Surprisingly, choline did not benefit females, and appears to exacerbate HI-induced changes.

Metabolism of [1,6-13 C]glucose in the cerebellum of 18-day-old rats: Comparison with cerebral metabolism.

There is little information on metabolism in developing cerebellum despite the known importance of this region in cognition and motor tasks. Ex vivo 1 H- and 13 C-NMR spectroscopy were used to determine metabolism during late postnatal development in cerebellum and cerebrum from 18-day-old rat pups after intraperitoneal (i.p.) injection of [1,6-13 C]glucose. The concentration of several metabolites in cerebellum was distinctly different than cerebrum; alanine, glutamine, creatine and myo-inositol were higher in cerebellum than cerebrum, the concentrations of lactate, GABA, aspartate and N-acetylaspartate (NAA) were lower in cerebellum than in cerebrum, and levels of glutamate, succinate, choline and taurine were similar in both brain regions. The incorporation of label from the metabolism of [1,6-13 C]glucose into most isotopomers of glutamate (GLU), glutamine (GLN), GABA and aspartate was lower in cerebellum than in cerebrum. Incorporation of label into the C2 position of lactate via the pyruvate recycling pathway was found in both brain regions. The ratio of newly synthesized GLN/GLU was significantly higher in cerebellum than in cerebrum indicating relatively active metabolism via glutamine synthetase in cerebellar astrocytes at postnatal day 18. This is the first study to determine metabolism in the cerebellum and cerebrum of male and female rat brain.

Fundamentals of CNS energy metabolism and alterations in lysosomal storage diseases.

The brain has a very high requirement for energy. Adult brain relies on glucose as an energy substrate, whereas developing brain can utilize alternative substrates as well as glucose for energy and for the biosynthesis of lipids and proteins required for brain development. Metabolism provides the energy required to support all cellular functions and brain development and building blocks for macromolecules. Lysosomes are organelles involved in breakdown of biological compounds including proteins and complex lipids in the body and brain. Recent studies suggest that lysosomal dysfunction can damage neurons and/or alter neurotransmitter homeostasis. Several studies also implicate mitochondrial dysfunction in the pathophysiology of brain damage in lysosomal storage diseases. This manuscript provides a brief review of energy metabolism and the key pathways involved in metabolism in brain. Roles of lysosomes related to metabolism and neurotransmission are discussed, and evidence for mitochondrial dysfunction in several lysosomal storage diseases is presented. This article is part of the Special Issue "Lysosomal Storage Disorders".

White Matter Alterations in Fmr1 Knockout Mice during Early Postnatal Brain Development.

Fragile X syndrome (FXS) is the most commonly inherited form of intellectual disability ascribed to the autism spectrum disorder. Studies with FXS patients have reported altered white matter volume compared to controls. The Fmr1 knockout (KO) mouse, a model for FXS, showed evidence of delayed myelination during postnatal brain development. In this study, we examined several white matter regions in the male Fmr1 KO mouse brain compared to male wild-type (WT) mice at postnatal days (PND) 18, 21, 30, and 60, which coincide with critical stages of myelination and postnatal brain development. White matter volume, T2 relaxation time, and magnetization transfer ratio (MTR) were measured using magnetic resonance imaging and myelin content was determined with histological staining of myelin. Differences in the developmental accumulation of white matter and myelin between Fmr1 KO and WT mice were observed in the corpus callosum, external and internal capsules, cerebral peduncle, and fimbria. Alterations were more predominant in the external and internal capsules and fimbria of Fmr1 KO mice, where the MTR was lower at PND 18, then elevated at PND 30, and again lower at PND 60 compared to the corresponding regions in WT mice. The pattern of changes in MTR were similar to those observed in myelin staining and could be related to the altered protein synthesis that is a hallmark of FXS. While no significant changes in white matter volumes and T2 relaxation time between the Fmr1 KO and WT mice were observed, the altered pattern of myelin staining and MTR, particularly in the external capsule, reflecting the abnormalities associated with myelin content is suggestive of a developmental delay in the white matter of Fmr1 KO mouse brain. These early differences in white matter during critical developmental stages may contribute to altered brain networks in the Fmr1 KO mice.

Effect of Acetyl-L-carnitine Used for Protection of Neonatal Hypoxic-Ischemic Brain Injury on Acute Kidney Changes in Male and Female Rats.

Neonatal hypoxia-ischemia (HI) is a common cause of brain injury in infants. Acute kidney injury frequently occurs after birth asphyxia and is associated with adverse outcome. Treatment with acetyl-L-carnitine (ALCAR) after HI protects brain and improves outcome. Rat pups underwent carotid ligation and 75 min hypoxia on postnatal day 7 to determine effects of HI on kidney which is understudied in this model. HI + ALCAR pups were treated at 0, 4 and 24 h after HI. The organic cation/carnitine transporter 2 (OCTN2), transports ALCAR and functions to reabsorb carnitine and acylcarnitines from urine. At 24 h after injury OCTN2 levels were significantly decreased in kidney from HI pups, 0.80 ± 0.04 (mean ± SEM, p < 0.01), compared to sham controls 1.03 ± 0.04, and HI + ALCAR pups 1.11 ± 0.06. The effect of HI on the level of pyruvate dehydrogenase (PDH) was determined since kidney has high energy requirements. At 24 h after HI, kidney PDH/ß-actin ratios were significantly lower in HI pups, 0.98 ± 0.05 (mean ± SEM, p < 0.05), compared to sham controls 1.16 ± 0.06, and HI + ALCAR pups 1.24 ± 0.03, p < 0.01. Treatment of pups with ALCAR after HI prevented the decrease in renal OCTN2 and PDH levels at 24 h after injury. Protection of PDH and OCTN2 after HI would improve energy metabolism in kidney, maintain tissue carnitine levels and overall carnitine homeostasis which is essential for neonatal health.

L-Carnitine and Acetyl-L-carnitine Roles and Neuroprotection in Developing Brain.

L-Carnitine functions to transport long chain fatty acyl-CoAs into the mitochondria for degradation by ß-oxidation. Treatment with L-carnitine can ameliorate metabolic imbalances in many inborn errors of metabolism. In recent years there has been considerable interest in the therapeutic potential of L-carnitine and its acetylated derivative acetyl-L-carnitine (ALCAR) for neuroprotection in a number of disorders including hypoxia-ischemia, traumatic brain injury, Alzheimer's disease and in conditions leading to central or peripheral nervous system injury. There is compelling evidence from preclinical studies that L-carnitine and ALCAR can improve energy status, decrease oxidative stress and prevent subsequent cell death in models of adult, neonatal and pediatric brain injury. ALCAR can provide an acetyl moiety that can be oxidized for energy, used as a precursor for acetylcholine, or incorporated into glutamate, glutamine and GABA, or into lipids for myelination and cell growth. Administration of ALCAR after brain injury in rat pups improved long-term functional outcomes, including memory. Additional studies are needed to better explore the potential of L-carnitine and ALCAR for protection of developing brain as there is an urgent need for therapies that can improve outcome after neonatal and pediatric brain injury.

Sex-dependent mitochondrial respiratory impairment and oxidative stress in a rat model of neonatal hypoxic-ischemic encephalopathy.

Increased male susceptibility to long-term cognitive deficits is well described in clinical and experimental studies of neonatal hypoxic-ischemic encephalopathy. While cell death signaling pathways are known to be sexually dimorphic, a sex-dependent pathophysiological mechanism preceding the majority of secondary cell death has yet to be described. Mitochondrial dysfunction contributes to cell death following cerebral hypoxic-ischemia (HI). Several lines of evidence suggest that there are sex differences in the mitochondrial metabolism of adult mammals. Therefore, this study tested the hypothesis that brain mitochondrial respiratory impairment and associated oxidative stress is more severe in males than females following HI. Maximal brain mitochondrial respiration during oxidative phosphorylation was two-fold more impaired in males following HI. The endogenous antioxidant glutathione was 30% higher in the brain of sham females compared to males. Females also exhibited increased glutathione peroxidase (GPx) activity following HI injury. Conversely, males displayed a reduction in mitochondrial GPx4 protein levels and mitochondrial GPx activity. Moreover, a 3-4-fold increase in oxidative protein carbonylation was observed in the cortex, perirhinal cortex, and hippocampus of injured males, but not females. These data provide the first evidence for sex-dependent mitochondrial respiratory dysfunction and oxidative damage, which may contribute to the relative male susceptibility to adverse long-term outcomes following HI. Lower basal GSH levels, lower post-hypoxic mitochondrial glutathione peroxidase (mtGPx) activity, and mitochondrial glutathione peroxidase 4 (mtGPx4) protein levels may contribute to the susceptibility of the male brain to oxidative damage and mitochondrial dysfunction following neonatal hypoxic-ischemia (HI). Treatment of male pups with acetyl-L-carnitine (ALCAR) protects against the loss of mtGPx activity, mtGPx4 protein, and increases in protein carbonylation after HI. These findings provide novel insight into the pathophysiology of sexually dimorphic outcomes following HI.

Neuroprotective Effects of Acetyl-L-Carnitine on Neonatal Hypoxia Ischemia-Induced Brain Injury in Rats.

Perinatal hypoxia ischemia (HI) is a significant cause of brain injury in surviving infants. Although hypothermia improves outcomes in some infants, additional therapies are needed since about 40% of infants still have a poor outcome. Acetyl-L-carnitine (ALCAR), an acetylated derivative of L-carnitine, protected against early changes in brain metabolites and mitochondrial function after HI on postnatal day (PND) 7 in a rat pup model of near-term HI injury. However, its efficacy in long-term structural and functional outcomes remains unexplored. We determined the efficacy of ALCAR therapy administered to rat pups after HI at PND 7, using both longitudinal in vivo magnetic resonance imaging and behavioral tests, in male and female rats. HI led to sex-specific behavioral impairment, with males exhibiting more global functional deficits than females. Interestingly, HI reduced the volume of the contralateral hemisphere in males only, suggesting that the brain injury is more diffuse in males than in females. Treatment with ALCAR improved both morphological and functional outcomes in both male and female rats. These results suggest that ALCAR may be a potential therapy for clinical use since the treatment attenuated the moderate injury produced under the experimental conditions used and improved the functional outcome in preclinical studies.

Glutamate oxidation in astrocytes: Roles of glutamate dehydrogenase and aminotransferases.

The cellular distribution of transporters and enzymes related to glutamate metabolism led to the concept of the glutamate-glutamine cycle. Glutamate is released as a neurotransmitter and taken up primarily by astrocytes ensheathing the synapses. The glutamate carbon skeleton is transferred back to the presynaptic neurons as the nonexcitatory amino acid glutamine. The cycle was initially thought to function with a 1:1 ratio between glutamate released and glutamine taken up by neurons. However, studies of glutamate metabolism in astrocytes have shown that a considerable proportion of glutamate undergoes oxidative degradation; thus, quantitative formation of glutamine from the glutamate taken up is not possible. Oxidation of glutamate is initiated by transamination catalyzed by an aminotransferase, or oxidative deamination catalyzed by glutamate dehydrogenase (GDH). We discuss methods available to elucidate the enzymes that mediate this conversion. Methods include pharmacological tools such as the transaminase inhibitor aminooxyacetic acid, studies using GDH knockout mice, and siRNA-mediated knockdown of GDH in astrocytes. Studies in brain slices incubated with [15 N]glutamate demonstrated activity of GDH in astrocytes in situ. These results, in conjunction with reports in the literature, support the conclusion that GDH is active in astrocytes both in culture and in vivo and that this enzyme plays a significant role in glutamate oxidation. Oxidative metabolism of glutamate, primarily mediated by GDH, but also by transamination by aspartate aminotransferase, provides considerably more energy than is required to maintain the activity of the high-affinity glutamate transporters needed for efficient removal of glutamate from the synaptic cleft. © 2016 Wiley Periodicals, Inc.

A new role for α-ketoglutarate dehydrogenase complex: regulating metabolism through post-translational modification of other enzymes.

This Editorial highlights a study by Gibson et al. published in this issue of JNeurochem, in which the authors reveal a novel role for the α-ketoglutarate dehydrogenase complex (KGDHC) in post-translational modification of proteins. KGDHC may catalyze post-translational modification of itself as well as several other proteins by succinylation of lysine residues. The authors' report of an enzyme responsible for succinylation of key mitochondrial enzymes represents a major step toward our understanding of the complex functional metabolome. TCA, tricarboxylic acid; KG, α-ketoglutarate; KGDHC, α-ketoglutarate dehydrogenase complex; FUM, fumarase; MDH, malate dehydrogenase; ME, malic enzyme; GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SIRT3, silent information regulator 3; SIRT5, silent information regulator 5.

In vivo longitudinal proton magnetic resonance spectroscopy on neonatal hypoxic-ischemic rat brain injury: Neuroprotective effects of acetyl-L-carnitine.

PURPOSE: This study evaluated the longitudinal metabolic alterations after neonatal hypoxia-ischemia (HI) in rats and tested the neuroprotective effect of acetyl-L-carnitine (ALCAR) using in vivo proton short-TE Point-RESolved Spectroscopy method. METHODS: Rice-Vannucci model was used on 7-day-old Sprague-Dawley rats. Data were acquired from contralateral and ipsilateral cortex and hippocampus, respectively at 4 time points (24-h, 72-h, 7-days, 28-days) post-HI. The effect of subcutaneous administration of ALCAR (100 mg/kg) immediately after HI, at 4-h, 24-h, and 48-h post-HI was determined. RESULTS: Significant reductions in glutathione (P < 0.005), myo-inositol (P < 0.002), taurine (P < 0.001), and total creatine (P < 0.005) were observed at 24-h postinjury compared with the control group in the ipsilateral hippocampus of the HI rat pups. ALCAR-treated-HI rats had lower levels of lactate and maintained total creatine at 24-h and had smaller lesion size compared with the HI only rats. CONCLUSION: Severe oxidative, osmotic stress, impaired phosphorylation, and a preference for anaerobic glycolysis were found in the ipsilateral hippocampus in the HI pups at 24-h postinjury. ALCAR appeared to have a neuroprotective effect if administered early after HI by serving as an energy substrate and promote oxidative cerebral energy producing and minimize anaerobic glycolysis.

Metabolic Alterations in Developing Brain After Injury: Knowns and Unknowns.

Brain development is a highly orchestrated complex process. The developing brain utilizes many substrates including glucose, ketone bodies, lactate, fatty acids and amino acids for energy, cell division and the biosynthesis of nucleotides, proteins and lipids. Metabolism is crucial to provide energy for all cellular processes required for brain development and function including ATP formation, synaptogenesis, synthesis, release and uptake of neurotransmitters, maintaining ionic gradients and redox status, and myelination. The rapidly growing population of infants and children with neurodevelopmental and cognitive impairments and life-long disability resulting from developmental brain injury is a significant public health concern. Brain injury in infants and children can have devastating effects because the injury is superimposed on the high metabolic demands of the developing brain. Acute injury in the pediatric brain can derail, halt or lead to dysregulation of the complex and highly regulated normal developmental processes. This paper provides a brief review of metabolism in developing brain and alterations found clinically and in animal models of developmental brain injury. The metabolic changes observed in three major categories of injury that can result in life-long cognitive and neurological disabilities, including neonatal hypoxia-ischemia, pediatric traumatic brain injury, and brain injury secondary to prematurity are reviewed.

Longitudinal in vivo developmental changes of metabolites in the hippocampus of Fmr1 knockout mice.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is studied in the Fmr1 knockout (KO) mouse, which models both the anatomical and behavioral changes observed in FXS patients. In vitro studies have shown many alterations in synaptic plasticity and increased density of immature dendritic spines in the hippocampus, a region involved in learning and memory. In this study, magnetic resonance imaging (MRI) and (1) H magnetic resonance spectroscopy (MRS) were used to determine in vivo longitudinal changes in volume and metabolites in the hippocampus during the critical period of early myelination and synaptogenesis at post-natal days (PND) 18, 21, and 30 in Fmr1 KO mice compared with wild-type (WT) controls. MRI demonstrated an increase in volume of the hippocampus in the Fmr1 KO mouse compared with controls. MRS revealed significant developmental changes in the ratios of hippocampal metabolites N-acetylaspartate (NAA), myo-inositol (Ins), and taurine to total creatine (tCr) in Fmr1 KO mice compared with WT controls. Ins was decreased at PND 30, and taurine was increased at all ages studied in Fmr1 KO mice compared with controls. An imbalance of brain metabolites in the hippocampus of Fmr1 KO mice during the critical developmental period of synaptogenesis and early myelination could have long-lasting effects that adversely affect brain development and contribute to ongoing alterations in brain function.