Mitochondrial Metabolism in Astrocytes Regulates Brain Bioenergetics, Neurotransmission and Redox Balance

1The central nervous system (CNS) is a complex, integrated structure composed of diverse cell types. Neurons are highly specialized cells in charge of transmitting, processing, and storing information and thus have been considered the functional units of the CNS. However, research within the last few decades has demonstrated the critical roles non-neuronal cells play in CNS function. The non-neuronal cells of the CNS include macroglia (astrocytes, ependymal cells and oligodendrocytes) and microglia, which support essential functions such as the maintenance of neurotransmitter communication, metabolism, trophic support, formation of myelin sheath, wound healing, and immune surveillance.

2Astrocytes originate from the neuroectoderm. Recent estimates suggest they account for between 19 and 40% of the glial population (with an estimated glial to neuron ratio of ∼1:1), and a strong regional variation within the CNS. Astrocytes are a heterogenous population of cells with a diverse array of morphological, functional and molecular properties. While the cellular identity of astrocytes is determined by their gene expression profile post-differentiation (intrinsic patterning), the surrounding environment and neuronal circuitry further shapes, modifies, and maintains the form and functions of differentiated astrocytes (extrinsic factors). Structurally, astrocytes contain multiple radial processes that create diverse interfaces with other glia, neurons, and capillary endothelial cells. Astrocytes’ fine processes and endfeet envelope neuronal cell bodies, synapses, and blood vessels. In addition, an intracellular interface with other astrocytes or oligodendrocytes is generated via gap junctions (connexin channels), creating an extensive interconnected network throughout the brain. Gap junctions exchange ions and small molecules between cells (<1 kDa in size) including; inositol triphosphate (IP3), K+, Ca2+, ATP, glucose, glutamate, glutathione (GSH), and cyclic AMP; highlighting its signaling and buffering role within the CNS (Figure 3).

3Astrocytes are involved in a number of processes associated with brain function and disease progression. Astrocytes are major secretory cells, releasing factors or transmitters (gliotransmitters) including neurotransmitters (and their precursors), modulators, peptides, hormones, trophic (growth) factors, and metabolites. Astrocyte secreted factors have been shown to be involved in synapse formation, function and plasticity, neuronal growth, differentiation and survival, as well as in the regulation of the vascular tone and blood flow in the brain. Astrocytes have also been shown to mediate synaptic pruning by phagocytosis.

4Astrocytes become “reactive” as a response to pathological conditions or to perturbations in cellular homeostasis. Astrocyte reactivity is a loose term that primarily refers to an enlargement in the cell body and processes (hypertrophy), and an increase in the levels of the glial fibrillary acidic protein (GFAP), that relates to a pathological stimulus in the CNS. Unlike microglia, reactive astrocytes are not believed to be proliferative outside of conditions of glial scar formation, which is a specific, irreversible form of astrocyte reactivity. Reactive astrocytes contribute to the production and release of cytokines and the neuroinflammatory process during injury and neurodegeneration. Reactive astrocytes should not be confused with activated astrocytes, as the latter pertains to astrocyte responses to neurotransmission.

5Astrocytes can also be considered the master regulators of brain metabolism. In this review paper, we discuss the importance of astrocytes’ metabolism in the regulation of neurotransmitter recycling and synthesis, central carbon metabolism and bioenergetics, as well as redox homeostasis and antioxidant/xenobiotic defense. Our goal is to highlight the metabolic niche astrocytes fill within overall brain metabolism.

6While the mass of the human brain accounts for approximately 2% of total body weight, its oxidative metabolism accounts for ∼20% of the body’s total. Most of this oxidative energy (∼75–80%) is utilized at the synapse to maintain and restore ionic gradients, and for the uptake and recycling of neurotransmitters (;;;; Figure 3.4). By comparison, the metabolic processes in astrocytes have been estimated to account for between 5 and 15% of the total ATP expenditure in the brain. Glucose is the primary energy substrate in the adult brain, however, alternate energy sources can fuel brain function. Lactate transport across the blood-brain barrier (BBB) (Figure 3.1) provides ∼8–10% of the brain’s energy requirements under basal conditions, and it has been estimated to supply ∼20–25% of energy during energy demanding activities. As plasma lactate concentrations rise, lactate uptake in the CNS increases coinciding with a decrease in glucose uptake. When required, an important pool of lactate is always available as the extracellular levels of lactate (0.5–1.5 mM) are similar to those of glucose.

7Metabolism in astrocytes and neurons is interconnected, and it has been clearly demonstrated that neurons depend upon astrocytes for a variety of metabolic processes. The in vivo energy demands of astrocytes can be fulfilled in the absence of oxidative phosphorylation (OXPHOS), with an observed increase in lactate production in mutants of Cox10 (part of the cytochrome c oxidase complex and required for complex IV function), indicating that astrocytes are glycolytic (Figures 1.1, 3.2; ). Glucose has been reported as being the preferential energy substrate of astrocytes over lactate, though glucose uptake does not match the theoretical bioenergetic output. Inside the cell, glucose is phosphorylated generating glucose-6-phosphate (P), which is subsequently converted to fructose-6P. Glucose-6P can also be used for glycogen synthesis or in the pentose phosphate pathway (PPP) (Figure 1.1), while fructose-6P can be either metabolized to pyruvate or initiate the hexosamine pathway with uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) as its final product and mediator of protein GlcNAcylation. Downstream of glycolysis, glyceraldehyde-3P (G3P) can be metabolized for de novo synthesis of serine (Figure 1.1). Serine is also a precursor for the synthesis of glycine (folate cycle) and cysteine (through the trans-sulfuration pathway, TSP) (Figures 1.1, 4.4, 4.5), both of which contribute to the production of GSH (Figure 3.2), which is essential for redox buffering. Serine and glycine are major sources for the transfer of one-carbon units through folate intermediates for their use in purine and thymidylate synthesis, nicotinamide adenine dinucleotide phosphate (NADPH) production and methylation reactions (Figure 4.4; ).

8Pyruvate entry into the tricarboxylic acid (TCA or Krebs) cycle (Figure 2.1) is limited by a reduced activity of pyruvate dehydrogenase (PDH) in astrocytes due to its phosphorylation via pyruvate dehydrogenase (PDK), creating a surplus of pyruvate due to the high levels of glucose consumption (Figure 1.2). In contrast to astrocytes, glycolysis in neurons is restricted by the continuous degradation of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3 (PFKFB3), which catalyzes the synthesis of fructose-2,6-P an allosteric activator of phosphofructokinase 1 (PFK1). Accordingly, glucose utilization in neurons is directed to PPP in order to regenerate NADPH for the reductive recycling of GSH upon oxidative stress (Figure 3.3;;;; ). The astrocyte-neuron lactate shuttle (ANLS) hypothesis states that a large portion of glucose metabolism in astrocytes is directed to lactate production and this is subsequently shuttled to neurons as fuel for OXPHOS (Figures 3.2, 3.3;;;; ). This shuttle seems to involve sequestration within the endoplasmic reticulum for its delivery from perivascular endfeet to perisynaptic processes. Under hypoxic conditions astrocytes have the ability to upregulate glycolytic enzymes including the monocarboxylate transporter MCT4 involved in the transfer of lactate to neurons via the activation of the hypoxia-inducible factor-1α (HIF-1α). Nitric oxide can also upregulate glucose transporter-1 (GLUT1), hexokinase-2 and MCT4 via HIF-1α. However, other studies have reported that selective loss of HIF-1α in astrocytes protects neurons from hypoxic injury. Recent studies also demonstrate that during energy-demanding conditions or aging neurons have the capacity to upregulate glucose metabolism, and these findings have been used to argue against the ANLS hypothesis (Figure 3.3;;;; ).

9Astrocytes also store energy in the form of glycogen, which can be broken down to lactate. Yet due to the limited amount of glycogen stored in astrocytes (∼3–12 μmol per g of tissue, 1–6% of total glucose; ), its contribution to the maintenance of either neuronal or astrocyte energy-dependent processes under stress conditions is still unclear. There is strong evidence supporting a robust oxidative metabolic capacity in astrocytes (1/3 of total brain glucose and 1/2 of total brain lactate oxidative metabolism). Microarray and proteomic analyses have shown that most enzymes in the TCA cycle are expressed at higher levels in astrocytes than in neurons, and immuno-electron microscopy revealed that mitochondria is present at higher density in fine astrocytic processes encompassing synapses (Figure 3.6; ). Nevertheless, astrocytes deficient in mitochondrial respiration survive as glycolytic cells, both in vitro and in vivo. What then is the physiological importance of mitochondrial metabolism in astrocytes? We next discuss the particularities of mitochondrial central carbon metabolism in these glial cells and its importance for neurotransmission and redox balance.

10Oxidative metabolism in mitochondria is primarily fueled by pyruvate, fatty acids (FAs)/ketone bodies, and glutamine/glutamate. Pyruvate generated from glycolysis is actively transported into the mitochondrial matrix via the mitochondrial pyruvate carrier (MPC1) to be subsequently decarboxylated and combined with coenzyme A (CoA) (Figure 2.1). Oxidative decarboxylation of pyruvate to acetyl-CoA (Ac-CoA) is mediated by PDH and is coupled to the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. Subsequently, citrate synthase catalyzes the condensation of oxaloacetate (OAA) with Ac-CoA to generate citrate. Pyruvate decarboxylation is the primary carbon source for the TCA cycle and is sufficient to sustain oxidative carbon flux during energy consumption, as the last step in the cycle regenerates OAA to be combined further with another Ac-CoA molecule generated by PDH (a closed loop) (Figure 2.1). However, TCA cycle intermediates also provide precursors for the biosynthesis of several classes of molecules. TCA intermediates leave the cycle via cataplerotic reactions linked to biosynthetic processes, while anapletoric reactions supplement carbons back into to the TCA cycle. A balance between anaplerosis and cataplerosis controls the entry and exit of carbons into the TCA cycle, and is essential for biosynthetic processes (Figure 2.1; ).

11Pyruvate carboxylase (PC) is a major anaplerotic enzyme in the mitochondria that generates OAA from pyruvate in an ATP-dependent manner (Figure 2.1), replenishing carbons lost by the export of OAA, citrate, and alpha-ketoglutarate (αKG) from the TCA cycle, for gluconeogenesis, FA, and amino acid synthesis, respectively (Figures 1.2, 1.6, 2.1, 2.6). It has been estimated that 3–10% of glucose utilization in the brain is via PC, and in astrocytes, 33–50% of pyruvate entering mitochondria is utilized for anaplerotic reactions instead of producing energy. Furthermore, the metabolism of mitochondrial pyruvate through PC is reported to be 4.3-fold higher (39.3 nmol/h/106 cell) than through PDH (9.1 nmol/h/106 cell).

12In astrocytes’ gluconeogenesis, OAA is converted to phosphoenolpyruvate (PEP) in the mitochondria by phosphoenolpyruvate carboxykinase (PEPCK2), as the cytosolic activity of the cytosolic isoform (PEPCK1) has not been detected (Figure 2.1; ). While PEP has been shown to exit mitochondria, the precise mechanism has not been determined, with the citrate/isocitrate carrier (CIC) and adenine nucleotide transporter (ANT) being proposed as likely intermediators. In the brain, gluconeogenesis is astrocyte-specific as they express fructose 1,6-biphosphatase (FBP), which generates fructose-6P from fructose-1,6P (Figure 1.1). Astrocytes also express glucose 6-phosphatase (G6PC) (Figure 1.1), but because they normally store glycogen, it has been suggested that gluconeogenesis in astrocytes is primarily involved in glycogenesis, and that G6PC may be silent under physiological conditions becoming activated at times of stress. Gluconeogenesis in astrocytes has been demonstrated with aspartate, glutamate, alanine, and lactate as precursors and is becoming more recognized as an important alternative source of glucose for neurons during stress conditions.

13In the brain, it has been long recognized that PC is primarily expressed in astrocytes and to a lesser extent in microglia, oligodendrocytes and ependymal cells. However, as mentioned above, PDH activity in astrocytes is reduced by its phosphorylation and thus an additional carbon source is proposed to exist to supplement Ac-CoA to the TCA cycle (Figure 2.1; ). FA oxidation (FAO) has been proposed to be the preferable pathway for Ac-CoA synthesis in astrocytes (Figures 1.4, 2.2), but its physiological implications are still unclear.

14Ketone bodies are water-soluble molecules derived from FA metabolism in the liver, supplying energy to peripheral tissues including the brain. Ketone bodies have been shown to be produced by astrocytes as well. During starvation up to ∼70% of the brain’s energy can be supplied by ketone bodies. Ketone bodies are produced in the mitochondria from Ac-CoA in three sequential steps mediated by: (1) Ac-CoA acetyltransferase 1 (ACAT1) also known as acetoacetyl (AcAc)-CoA thiolase or thiolase II (Ac-CoA → AcAc-CoA, a reversible reaction); (2) β-hydroxy-β-methyl glutaryl (HMG)-CoA synthase (HMCS1, AcAc-CoA → HMG-CoA); and (3) HMG-CoA lyase [HMGCL, HMG-CoA → acetoacetate (AcAc)]. However, in astrocytes, AcAc is primarily formed by the action of 3-oxo-acid-CoA transferase (OXCT1 or SCOT) or AcAc-CoA hydrolase (ACAA2) on AcAc-CoA and not by the action of HMGCL and HMCS1. AcAc is then decarboxylated to acetone or metabolized to β-hydroxybutyrate (βHB) via βHB dehydrogenase (BDH) (Figure 2.3). Ketone bodies can cross the BBB and plasma membranes via MCT1, 2 and 4 (Figures 1.5, 3.1, 3.4;; ). At high concentrations, ketone bodies have been proposed to support basal and activity-dependent needs of neurons and glutamatergic signaling, while decreasing glucose utilization. Within the cell, ketone body transport across the mitochondrial membrane has not been fully characterized. While the mitochondrial pyruvate carrier (MPC1) has been implicated in ketone body transport, inhibition of the MPC1 does not abolish ketone body accumulation in the mitochondria, indicating that an undetermined transporter system or diffusion are involved (Figure 2.6;;;; ). In mitochondria, ketolysis starts when BDH converts βHB back to acetoacetate, which is activated to AcAc-CoA by OXCT1. AcAc-CoA is then degraded by ACAT1 generating Ac-CoA that can be incorporated into the TCA cycle (Figure 2.3). Neurons, astrocytes and oligodendroglia have been shown to be fully capable of using ketone bodies as metabolic fuel in vitro.

15Ketone bodies are not toxic to the brain unless associated with ketoacidosis, a complication linked to diabetes mellitus (hyperglycemia) or alcoholism where the circulation levels of ketone bodies increase to > 10 mM. The increase in ketone bodies arises primarily from metabolism outside the brain and to date it is unclear if the toxicity is mediated by ketone body accumulation (ketosis) or acidosis. A number of investigations have reported the beneficial effects of ketosis by regulating astrocytic metabolism, glutamate-glutamine cycle, glutamine synthetase (GS) activity, the function of vesicular glutamate transporters, excitatory amino acid transporters (EAAT), sodium-potassium adenosine triphosphatase (Na+/K+ ATPase), potassium channels (Kir4.1 and KATP), aquaporin-4, connexins, as well as on astrogliosis. It has also been clearly shown that production of ketone bodies is used as a signaling mechanism to regulate food intake. While astrocytes exposed to ketone bodies observe significant morphological changes, these changes do not seem to correlate with gliosis or inflammation.

16In addition to ketone bodies, FAs are also energy substrates for the brain, and unlike ketone bodies, they can diffuse across the BBB or utilize transport systems for their influx. The main source of FAs crossing into the brain are non-esterified long chain FAs (LCFAs, 13–21 carbons) complexed with albumin and circulating lipoproteins. Transport of non-esterified FAs across the BBB is mediated by passive diffusion (FA flip-flop) or by FA transporters including FA transport proteins (FATP1 and 4, with intrinsic acyl-CoA synthetase activity) and the FA translocase (FAT/CD36) (Figure 3.1;;;;; ). The exact transport mechanisms that mediate the uptake of FAs into neurons or astrocytes are still unclear. A recent report suggests that FATP1 and 4 mediate FA uptake in glial cells. Importantly, while neurons are capable of synthesizing significant amounts of lipids during bioenergetic excess, they are reported to be dependent on an apolipoprotein E (ApoE) shuttle system to transfer them to astrocytes to avoid neurodegenerative phenotypes (Figure 3.6;, ).

17In the brain, FAO occurs primarily in astrocytes. Two recent studies comparing mitochondrial transcriptomes and proteomes between different neural cell types corroborated the increased concentration of enzymes involved in FA metabolism and transport in astrocytes compared to neurons. Specifically, enzymes associated with LCFA transport into mitochondria (carnitine palmitoyltransferases or CPTs) and enzymes that support the requirement of FAO for anaplerotic reactions and the replenishment of TCA cycle intermediates (PDK4/2 and PC) where found enriched in astrocytes. FAO was also demonstrated to coexist with glycolysis and FAO enzymes seemed to be more abundant in adult vs. fetal astrocytes.

18To utilize FAs metabolically (FAO), FAs must be esterified to CoA in an ATP-dependent process by acyl-CoA synthetases (ACS), a process that is often linked to, or performed shortly after, FA transport by FATPs (Figures 1.3, 4.2;; ). In the brain, signaling through the peroxisome proliferation activated receptor beta (PPARβ, predominantly expressed in neurons but also glia) increases the expression of ACS2, in turn increasing the oxidation of exogenously supplied FAs in neurons. Astrocytes have an increased expression of both PPARα and ACS1 compared to neurons, which has been linked to the upregulation of genes involved in FAO and ketogenic processes.

19Carnitine palmitoyltransferase 1a (CPT1a) transfers the acyl groups from esterified FAs (acyl-CoA) to L-carnitine (Figure 4.1), producing acyl-carnitine esters which are transported across the outer and inner mitochondrial membranes via the voltage-dependent anion channel (VDAC) and the carnitine/acyl-carnitine translocase (CACT) transport, respectively. Then, CPT2 converts acyl-carnitine back to acyl-CoA in the mitochondrial matrix (Figure 4.1;;; ). While both astrocytes and oligodendrocytes uptake saturated and unsaturated FAs, CTP1a seems to be found primarily in astrocytes and neural progenitor cells, while absent in neurons, microglia, and oligodendrocytes. As such, carnitine deficiency causes a metabolic encephalopathy that is characterized by astrocytic swelling and mitochondrial expansion, corroborating that FAO is an essential metabolic component of astrocytic function. CPT1a activity is negatively regulated by malonyl-CoA, a molecule synthesized by Ac-CoA carboxylase (ACC) from cytosolic Ac-CoA. Malonyl-CoA is also a principal component of FA synthesis, making this molecule the regulator of FAO and lipogenesis (Figure 4.3; ). ACC is inhibited by phosphorylation via the energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK), which links energy deficiency with mitochondrial FAO (Figure 4.3). Recurrent low glucose exposure has been shown to activate AMPK and increase FAO dependency in astrocytes. In another study, astrocyte activation with the ciliary neurotrophic factor (CNTF) was also shown to activate AMPK and increases FAO and ketolysis. In contrast, upon hypoxic conditions or high concentrations of FAs, ketogenesis is induced in astrocytes and this is regulated as well by AMPK signaling.

20In the mitochondria, acyl-CoAs are principally broken down into Ac-CoA by FAO in a series of sequential reactions that include: (1) an initial flavin adenine dinucleotide (FAD)-dependent dehydrogenation of acyl-CoAs by acyl-CoA dehydrogenase (ACD or ACAD) that generates FADH2; (2) a subsequent hydration step mediated by enoyl-CoA hydratase (ECH) forming 3-hydroxy(OH)acyl-CoA; (3) generation of 3-ketoacyl-CoA via 3-OHacyl-CoA dehydrogenase (HAD) in an NAD+-dependent manner regenerating NADH; and (4) cleavage and release of Ac-CoA via thiolase I leaving an acyl-CoA two carbon atoms shorter that re-enters the pathway. The last three steps are carried by a heteroctamer protein complex called the mitochondrial trifunctional protein composed of alpha [HADHA (ECH/HAD)] and beta [HADHB (thiolase)] subunits (Figure 2.2;;; ). In the brain, 95% of HADHA co-localizes with the astrocyte biomarker GFAP and ACD is also predominantly find in astrocytes.

21For FAs/acyl-CoAs with odd numbered carbons, FAO will yield propionyl (Prop)-CoA rather than Ac-CoA from the final carbons of the acyl-CoA. Prop-CoA can either be converted to succinyl (Succ)-CoA in an ATP-dependent manner by a series of reactions providing an anaplerotic input to the TCA cycle, or act as a co-substrate in the ketolytic conversion of AcAc-CoA to acetoacetate by OXCT1 (Figure 2.3). In the event that the β-carbon of a FA is methylated, α-oxidation occurs to remove one carbon unit as formic acid [formic acid is converted to carbon dioxide (CO2)], shifting the methylated carbon from the β to α position, allowing β-oxidation to resume. The steps in α-oxidation include: (1) hydroxylation of the α-carbon by phytanoyl-CoA hydroxylase (PhyH/Pahx) utilizing the conversion of αKG to succinate as a driving force; (2) decarboxylation by 2-hydroxyphytanoyl-CoA lyase (2-HPCL) releasing formyl-CoA and a fatty aldehyde; (3) oxidation of the fatty aldehyde by aldehyde dehydrogenase generating NADH and an acyl-CoA one carbon shorter than the original product.

22A large quantity of ATP can be generated from FAO (one molecule of palmitic acid provides approximately 115 ATPs vs. 32 ATPs generated from glucose oxidation). Despite its high-energy potential, FAs have long been considered a poor energy substrate in the brain. The brain as a whole was thought to be limited in FAO capacity as thiolase I, ACD and ECH activities are 0.7, 50, and 19% that of the heart mitochondria, though it must be noted that glial and neuronal forms of the enzymes were not assayed independently (Figure 2.2). While there are three known carnitine palmitoyltransferase 1 (CPT1) isoforms, CPT1a has a low expression in the whole brain and is not expressed in neurons, CTP1b is not expressed in the brain, and CPT1c localizes exclusively to the endoplasmic reticulum in neurons and has not been shown to participate in FAO but has been implicated in ROS management. Lower CPT1a expression and activity restricts mitochondrial oxidation of FAs in neurons, except for those with 12 or fewer carbons, which can passively diffuse across the mitochondrial inner membrane (Figures 1.3, 2.2, 4.2). Yet the brain has been demonstrated to metabolize carbons from FAs in vivo, accounting for ∼20% of the total Ac-CoA pool when labeled octanoate is supplied to the bloodstream, which occurs predominantly in astrocytes, based on comparisons of astrocytes, oligodendrocytes and neurons in primary cultures.

23Astrocytes have a high buffering capacity against ROS compared to neurons, and FAO is proposed to induce higher amounts of ROS formation. One cycle of FAO generates one molecule of FADH2 and NADH, increasing the total FADH2/NADH ratio per Ac-CoA created compared to glycolysis (Figure 2.2). When ACD transfers electrons from acyl-CoA to FAD, it is coupled to the subsequent transfer of electrons from the resultant FADH2 to the electron transferring flavoprotein (ETF). The follow-up transfer of electrons from ETF to ubiquinone (coenzyme Q) by ETF dehydrogenase creates a backpressure of electrons flowing through complex I (donated from NADH) and complex II (donated from succinate) to ubiquinone that enhances the probability of leakage and formation of ROS (Figure 2.5). Thus, it has been proposed that restricting FAO protects neurons against oxidative damage beyond the stages of development. In the brain, inhibition of FAO by methyl palmoxirate has been shown to reduce the concentrations of non-enzymatically oxidized metabolites derived from poly-unsaturated FAs (PUFA), indicating that ROS are being generated from FAO.

24However, the mechanism of ROS generation from FA substrates is not universal, and depends upon the length and structure of the FA. Very long chain FAs (VLCFAs, 22 or more carbons) that are not esterified have been shown to integrate into artificial phospholipid bilayers, desorpting at slower rates than shorter FAs and disrupting membrane fluidity. This integration and disruption by VLCFAs has been shown to occur in the inner mitochondrial membrane, decreasing the membrane potential by protonophoric activity, and reducing ROS generated from reverse electron transport (RET). The protonophoric property, and its resulting decrease in ROS from hyperpolarization, has been demonstrated in medium chain FAs (MCFAs 6–12 carbons), LCFAs and branched chain FAs. This protonophoric capacity of FAs decreases as the length of the FA decreases, while branched FAs such as phytanic acid show an increased protonophoric activity compared to their straight chain FA counterparts. Additionally, application of carnitine esters as substrates demonstrated that RET was not occurring in the mitochondria, furthering that RET is not a prominent form of ROS production during FAO.

25While protonophoric properties of FAs can mitigate the effects of hyperpolarization/RET, FAs can promote ROS generated through forward electron transport and by interfering with electron transport. Isolated mitochondria increased ROS production proportionally to FA exposure in the presence of the recoupling agent carboxyatractyloside and during uncoupling with CCCP, indicating that ROS was being generated independently of the protonophoric effects. Interestingly, branched phytanic acid and poly unsaturated arachidonic acid demonstrated stronger ROS effects than unbranched saturated FAs in the aforementioned studies. Mitochondria exposed to low concentrations of LCFAs were also found to generate ROS at complex III and in a membrane potential-independent manner at the ETF-ETF-oxidoreductase. As described in, despite the evidence of electron transfer interference by FAs, the mechanism is not well understood.

26FAs are stored within cells as energy-rich triacylglycerides localized in lipid droplets, removing excess free FAs from the cytoplasm, which are toxic and disrupt mitochondrial membrane integrity. In addition, LDs deliver FAs into mitochondria for their consumption as an alternative energy source during periods of nutrient depletion. Neurons do not typically make LDs, while astrocytes do, and it has been shown that toxic FAs produced in hyperactive neurons are transferred to astrocytic LDs where they are consumed by via mitochondrial FAO. Importantly, under nutrient deprivation conditions, the survival of astrocytes depends on LD-fueled FAO.

27Peroxisomes are located throughout the brain but are primarily found in astrocytes and oligodendrocytes. VLCFAs cannot be transported into the mitochondria via the carnitine shuttle and thus, are broken down first by peroxisomes. As in mitochondria, FAs must be esterified to CoA before undergoing FAO in peroxisomes (Figures 1.4, 4). Because peroxisomes lack an electron transport chain (ETC), electrons from FADH2 are transferred directly to O2 creating hydrogen peroxide (H2O2) that is scavenged by catalase (CAT). Within the peroxisome, FAs are shortened to Ac-CoA, Prop-CoA, and a wide range of LCFAs, MCFAs, and short chain FAs (SCFAs, 6 carbons or less). A variety of export systems exist within the peroxisomes, each with different affinities for different lengths of FAs, including carnitine-transferases [carnitine acetyltransferases (CRATs), carnitine octanoyltransferases (CROTs)], and thiolases [acyl-CoA thioesterases (ACOTs)] (Figure 1.4; ). Additionally, peroxisomes have been shown to possess ketogenic enzymes, implicating peroxisomes as another potential source of ketone bodies and cholesterol.

28Dysfunction of peroxisomal processes has been linked to demyelination, oxidative stress, inflammation, cell death, and abnormalities in neuronal migration and differentiation. Defects in the ATP binding cassette subfamily D member 1 (ABCD1) transporter that translocates very LCFAs into peroxisomes (Figure 1.4), leads to the accumulation of these fats in cells and tissues. VLCFAs and branched chain FAs can interfere directly with the ETC by opening the permeability transition pore, disrupting Ca2+ balance and depolarizing the mitochondria. LCFAs activate PPAR transcription factors that stimulate both FAO and ketogenesis as well as mitochondrial and peroxisome biogenesis. Recent reports have also demonstrated that peroxisome biogenesis requires the generation of pre-peroxisomes from mitochondrial derived vesicles.

29Previous studies have demonstrated that astrocytes have the machinery to metabolize MCFAs, LCFAs and VLCFAs as evidenced not only by the higher expression of FAO-related enzymes but also by the higher content of peroxisomes. For example, the MCFA octanoate (C8) that composes 13% of the normal free fatty acid pool and can cross the BBB is avidly metabolized by astrocytes. In brain slices, astrocytes have been shown to contain palmitate (C16) and oleate (C18) LCFAs.

30Alterations in FA metabolism in astrocytes have been reported in different disorders. A recent report demonstrated that in a Huntington’s disease mouse model, astrocytes within the striatum consume FAs as an alternative fuel, which results in increased oxidative damage. Astrocytes carrying the ApoE4 allele, the strongest genetic risk factor for the development of Alzheimer’s disease, observe profound alterations in FA metabolism and LD formation. Stimulation of FAO protects against ischemic injury as well. Pharmacological modulation of FAO has been suggested as a therapeutic approach against glioblastoma cells. Many chemical compounds are considered to be inhibitors or activators of FAO enzymes. However, the current knowledge on their specificities, potencies and metabolic impacts is limited and controversial. For example, at high concentrations, etomoxir, the inhibitor of CPT1a, has off-target effects linked to the inhibition of the ETC. Other FAO modulators include the CPT1a inhibitor oxfenicine and PPARs agonists/antagonists. Accordingly, the impairment in FAO observed in astrocytes derived from induced pluripotent stem cells isolated from Alzheimer’s disease patients is corrected by PPARβ/δ agonists. The specificity of the metabolic modifiers ranolazine (Ranexa) and trimetazidine, initially described as partial FAO inhibitors that target the HAD (or 3-KAT) activity of the trifunctional protein HADHA, has been recently questioned.

31Astrocyte processes extending to synaptic clefts play an important role in regulating synaptic transmission, particularly glutamatergic signaling. After its vesicle-mediated release, the excitatory neurotransmitter glutamate is taken from the synaptic cleft by astrocytes in a Na+ dependent manner via EAAT1 (glutamate aspartate transporter, GLAST) or EAAT2 (glutamate transporter 1, GLT-1) (Figure 3.4). In the cytosol, glutamate is then converted into non-excitatory glutamine via GS, and transferred back to neurons via the Na+-coupled neutral amino acid transporters (SNATs) to be converted back into glutamate in neurons via phosphate-activated glutaminase (PAG) (Figure 3.4;;;; ). In astrocytes, despite the presence of a weak mitochondrial targeting sequence, GS is localized in the cytosol because the mitochondrial membrane potential is not negative enough to drive GS into the mitochondria (Figures 1.6, 3.2; ). Neurons lack PC activity, restricting their ability to replenish TCA intermediates lost during neurotransmitter release (anaplerotic reaction). Thus, the glutamate-glutamine cycle between neurons and astrocytes, not only terminates/modulates glutamatergic signaling, preventing excitotoxicity triggered by excessive or prolonged exposure to glutamate, but it also replenishes the neurotransmitter pool in neurons (Figure 3.4; ).

32Not all the glutamate taken from the synaptic cleft by astrocytes becomes glutamine. There is evidence that a significant portion is incorporated into the TCA cycle via oxidative metabolism (Figure 2.4). Glutamate transport into the mitochondria is mediated via the aspartate/glutamate carrier antiporter (AGC or Aralar 1) or via the glutamate carrier (GC1 or GC2), a symporter for glutamate and H+. Controversy still exists about the contribution of AGC to the uptake of glutamate in astrocytic mitochondria, as it has been shown to be primarily expressed in neurons. However, while the AGC activity in astrocytes corresponds only to 7% of the total brain AGC activity, it is calculated to be twice the minimum required for glutamate production and degradation (Figure 2.6; ). AGC is also central to the malate-aspartate shuttle (Figure 1.6), a system that allows the transfer of NADH electrons generated in the cytosol, from processes such as glycolysis, to the mitochondria. On the other hand, knockdown of GC1 suggests that this carrier is the main gate for net glutamate entry into mitochondria for oxidative metabolism in astrocytes (Figure 2.6; ).

33In the mitochondria, glutamate is metabolized to αKG by transamination via aspartate aminotransferase (GOT2), and to a lesser extent by alanine (ALAT) and branched chain aminotransferases (BCAT) (Figure 2.4). In addition, αKG can also be synthesized from glutamate via glutamate dehydrogenase (GDH), which is also a source of NADPH. While glutamate transamination via GOT2 generates a truncated TCA cycle due to the requirement of OAA as a co-substrate, GDH-mediated dehydrogenation can serve as an anaplerotic reaction to replenish TCA intermediates (Figures 2.1, 2.4;;; ). Astrocytes deficient in GDH increase their reliance on GOT2 and their glycolytic input to PC, which is paralleled by an accumulation of intracellular glutamate, underlying the importance of GDH in anaplerosis. It should be noted that humans, unlike most other animals including mice and rats, have an additional GDH2 isoform that is expressed solely in astrocytes. Expression of GDH2 in rat cortical astrocytes increased glutamate uptake and metabolism, reducing their dependence on glucose and increasing their capacity to utilize branched chain amino acids (BCAA). Importantly, glutamate to glutamine metabolism is not determined by the extracellular glutamate concentration. However, the entry of glutamate into the TCA cycle seems to be triggered by high extracellular glutamate concentrations (high μM).

34The homeostasis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is similarly linked to the glutamate-glutamine cycle. GABA is synthesized in neurons from the decarboxylation of glutamate by glutamate decarboxylase (GAD). After its vesicular release, GABA is uptaken by astrocytes through the GABA transporter (GAT3) (Figure 3.4). GABA is then metabolized by GABA transaminase (GABA-T) with αKG as cofactor to produce succinate semialdehyde (SSA) and glutamate (Figure 2.4). SSA is metabolized to succinate via SSA-dehydrogenase (SSADH), which can then be incorporated back into the TCA cycle. This anaplerotic addition of succinate compensates the cataplerotic loss of carbons from the generation of glutamate by the GABA-T (Figure 2.1; ).

35In addition to metabolism, evidence indicates that astrocyte mitochondrial bioenergetics are important for the regulation of neurotransmitter balance. Both glutamate and GABA uptake systems in astrocytes are driven by Na+ gradients that are established by the electrogenic action of the Na+/K+ ATPase (Figure 3.4). PC and GS activities also require ATP as well, and are critical enzymes in replenishing neuronal glutamine/glutamate (FIgures 2.1, 3.4). Importantly, it has been demonstrated that mitochondria co-localize with EAAT1/2 and glycolytic enzymes on the plasma membrane of astrocytes, and that glutamate transporters physically interact with the Na+/K+ ATPase (Figure 3.6; ), suggesting that while mitochondrial-derived ATP is not required for astrocyte survival, functional mitochondrial metabolism is important for neurotransmitter uptake and homeostasis (Figures 3.2, 3.4, 3.6). In fact, astrocyte processes enveloping synaptic terminals contain abundant mitochondria, and inhibition of mitochondrial function by fluorocitrate in astrocytes increases glutamate excitotoxicity in co-cultures with neurons.

36Astrocytes contain higher levels of endogenous antioxidants and antioxidant systems being more resistant to oxidative stress than neurons, which is explained by the activation of the antioxidant response via the nuclear factor erythroid-2-related factor 2 (Nrf2) transcription factor. Astrocytes also have higher levels of NADPH and G6PD (glucose 6-phosphate dehydrogenase) glucose 6-phosphate dehydrogenase (G6PD). In the mitochondria, superoxide anion (O2•–) is generally considered to be generated in the matrix by electron leakage from complex I, and in both the matrix and the intermembrane space (IMS) by electron leakage from complex III. O2•– generated in the matrix and the IMS has been proposed to be released to the cytosol via the mitochondrial permeability transition pore (mPTP) and the VDAC, respectively. However, at least 10 different additional sites for O2•–/H2O2 production in the mitochondria have been described. The relative and absolute contribution of specific sites in the mitochondria to the production of ROS seems to depend on the availability of the substrate being oxidized. Of importance, one ROS can lead to the generation of distinct more reactive species. As such, O2•– can be dismutated in an enzymatic (superoxide dismutases [SODs]) or non-enzymatic manner to H2O2. While H2O2 is thought to diffuse across membranes, it has also been reported that it can be transported by aquaporins. Surprisingly, astrocytes have less efficient mitochondrial respiration and increased ROS formation when compared to neurons, in part due to a lower incorporation of complex I into supercomplexes in astrocytes caused by lower expression of the NADH-ubiquinone oxidoreductase core subunit S1 (NDUFS1).

37The physiological role of increased mitochondrial ROS production in astrocytes had not been explored before, but an elegant recent investigation using mitochondria-targeted antioxidants demonstrated that high ROS production in astrocytes regulates GSH metabolism via Nrf2, and conversely, represses glucose metabolism via the PPP to decrease NADPH-dependent ROS production by NADPH-dependent oxidases (Nox1 and 2), which together modulate the redox status and survival of neurons. Interestingly, glutamate (N-methyl-d-aspartate) receptors (NMDA-R) couple neurotransmission to Nrf2 activation, where NMDA-R signaling in astrocytes promotes Nrf2 phosphorylation by cyclin-dependent kinase 5 (Cdk5) and its translocation to the nucleus, a novel mechanisms of intercellular feedback communication from neurons to astrocytes.

38While both neurons and astrocytes can synthesize GSH, astrocytes have been reported to protect neurons via regulation of GSH metabolism. This protective effect is thought to be dependent on the supply of cysteine from astrocytes as a precursor for de novo GSH synthesis, and in part due to neurons being unable to uptake significant amounts of extracellular cystine. GSH has been proposed to be released from astrocytes via the ATP-binding cassette transporters subfamily C member 1 transporter (ABCC1, or multidrug-resistance-associated protein 1 [MRP1]) (Figures 1.6, 3.2;; ). Indeed, inhibition of MRP1 has been reported to reduce the extracellular accumulation of GSH in cultured astrocytes. We have failed to observe a reciprocal accumulation of GSH in astrocytess when MRP1 activity is reduced (unpublished data), but this effect might be ascribed to feedback inhibition of GSH synthesis by GSH.

39Once released, GSH is degraded by the γ-glutamyl transpeptidase (γGT) to produce l-cysteine-l-glycine (CysGly). CysGly is cleaved further by the neuronal aminopeptidase N (ApN) into the amino acids glycine and cysteine that are taken up by neurons for de novo GSH synthesis via the Na+-Cl–-dependent glycine transporter 1 (GlyT1) and EAAT3, respectively (Figures 3.2, 3.3;,;; ). The export of GSH should imply a loss of carbons that must be replenished in astrocytes to maintain cellular redox homeostasis. Cysteine acquisition in astrocytes is mediated by the Na+-dependent XAG-/EAAT3 and -independent Xc-/xCT uptake systems, but their exact contribution is still under debate. De novo synthesis via the TSP pathway is also an important source for cysteine in astrocytes, and was shown to be upregulated and utilized during oxidative stress (Figures 3.2, 4.5; ). In most cells, cysteine is considered the limiting substrate for GSH synthesis. However, glutamate might also be considered a limiting in astrocytes as it is metabolized to glutamine (Figure 1.6). Because glutamate synthesis depends on mitochondrial anaplerotic metabolism, mitochondrial function might as well be essential for GSH homeostasis (Figure 2.1, 2.4). Interestingly, GSH depletion upregulates mitochondrial activity and expression of complex I in astrocytes, but the exact mechanisms that regulate this phenomenon are still unclear.

40Central carbon metabolism in the mitochondria is also important for the maintenance of redox balance as it fuels the regeneration of NADPH. In addition to the anaplerotic activity of GDH mentioned above (Figure 2.4), within the mitochondria, the malic enzyme (ME) and isocitrate dehydrogenase isoform 2 (IDH2) regenerate NADPH coupled to the formation of OAA and αKG, respectively (Figures 2.1, 2.6). While higher levels of mitochondrial IDH activity have been reported in astrocytes, conflicting results exists in regards to the activity levels of mitochondrial ME. Metabolic flux analysis has also demonstrated that transfer of H from NADH via nicotinamide nucleotide transhydrogenase (NNT) contributes to the regeneration of the mitochondrial NADPH pool (Figure 2.5; ), which is highly expressed in the brain. A comparative analysis demonstrated that these three metabolic pathways actively contribute to NADPH replenishment in brain mitochondria, but other studies have reported a predominant role for the NNT pathway, particularly under metabolic stress.

41Folate metabolism supports one-carbon metabolism that serves to activate and transfer one-carbon units for biosynthetic processes. Mammals lack the capacity for folate synthesis whose requirements are largely met through dietary sources. Folate is not biologically active, but it is readily reduced in the intestinal mucosa or liver and converted into tetrahydrofolate (THF) molecules, such as 5-methyl-THF and 5-formy-THF. 5-methylTHF is the predominant form of folate in the blood and its transport in the brain across the BBB is mediated by the folate receptor alpha (FRα/FOLR1), the H+-coupled folate transporter (PCFT/SLC46A1), and the reduced folate carrier (RFC/SLC19A1), but whether the same mechanisms mediate folate transport in neural cells including astrocytes is unclear (Figure 3.1; ). Furthermore, because there is no dihydrofolate reductase in the brain to catalyze the formation of 5-methyl-THF, 5-methylTHF rather than folate is present in the CSF. Carrier-mediated energy-dependent transport of 5-methyl- and 5-formyl-THF has been described in astrocytes.

42While serine can be acquired from the diet, central carbon metabolism makes substantial contribution to serine availability and as a consequence to folate metabolism in both the cytosol and the mitochondria (Figure 4.4). THF and serine are transported into the mitochondria via SLC25A32 and sideroflexin-1 (SFXN1), respectively. Mitochondrial serine hydroxymethyltransferase 2 (SHMT2) transfers the β-carbon of serine to THF, generating 5,10-methylene-THF that can also be produced from glycine by the activity of glycine decarboxylase (GLDC), an abundant enzyme in astrocytes (Figure 4.4; ). GLDC deficiency induces neural tube defects and hydrocephaly. The bifunctional enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) or MTHFD2-like (MTHFD2L) then catalyzes the sequential production of 5,10-methenyl-THF and 10-formyl-THF. Finally, 10-formyl-THF is cleaved by MTHFD1L to generate formate, which can be exported to the cytosol (Figure 4.4). 10-formyl-THF can also be metabolized by 10-formyl-THF dehydrogenase (ALDH1L2). During this process both MTHFD2(L) and ALDH1L2 generate NADPH (; Figure 4.4). As such, knockdown of MTHFD2 decreases cellular NADPH/NADP+ and GSH/GSH disulfide (GSSG) ratios, and sensitizes cells to oxidative stress. The cytosolic counterpart of ALDH1L2, ALDH1L1 is expressed abundantly in astrocytes. ALDH1L2 mutations seem to induce astrocytic stress. Interestingly, complex I deficiency has been shown to decrease NADPH synthesis via ALDH1L2 due to the impaired generation of NAD+. Despite the bidirectionality of this pathway, most of the cytosolic one-carbon units derived from serine are catabolized in the mitochondria, as this seems to be favored by the more oxidative mitochondrial redox potential, when compared to the cytosol. However, when the mitochondrial folate pathway is lost the directionality of the cytosolic one-carbon flux is reversed to compensate the synthesis of NADPH.

43In the cytosol, the trifunctional enzyme MTHFD1 regenerates THF and the intermediate 5,10-methylene-THF links the folate pathway and thus, central carbon metabolism, with the remethylation of homocysteine within the TSP. As mentioned before the TSP is an important source for cysteine in astrocytes (Figures 3.2, 4.5; ). While remethylation of homocysteine to methionine can be accomplished by the activity of methionine synthase, in the liver and kidney mitochondria, betaine-homocysteine S-methyltransferase (BHMT) also produces methionine from homocysteine, linking the mitochondrial oxidation of choline to betaine with the TSP (Figure 4.5). Brain genetic variants of BHMT have been linked to schizophrenia. However, the presence of BHMT and its significance to the remethylation of homocysteine hasn’t been confirmed in astrocytes.

44The scientific process has been aptly described as blind people attempting to describe an elephant while only focusing on particular parts of the animal. Each person is contextually correct, but only when the data is combined can we achieve an accurate understanding of the whole. While there are far more parts of the proverbial elephant to explore to complete the picture, the recent evidence indicates that we must adjust our current understanding of brain metabolism, particularly as it pertains to astrocytes and neurons.

45Current research indicates that metabolism in the brain extends far beyond glucose. FAs, and their derived ketone bodies, are important sources of bioenergetic potential for the brain, particularly during development, as shown by the experiments in vivo and in vitro that follow its oxidation and integration into biomolecules. Glutamate is more than just a neurotransmitter kept in strict stoichiometry by the glutamate-glutamine cycle; it is a resource the brain uses for amino acid synthesis by transamination, for redox balance by generation of NADPH, and for anaplerosis to maintain the TCA cycle.

46The brain is far from a homogeneous body of cells, and the literature paints a clear picture of metabolic specialization and intracellular cooperation/dependence. In this review, we have centered our attention to astrocytic mitochondrial metabolism and its role in the regulation of brain bioenergetics, neurotransmission and redox balance via neuronal-astrocyte metabolic interactions. Astrocytes have been demonstrated to be functional without OXPHOS in vivo, to provide a GSH-dependent protective effect to neurons, to function as neutralizing and cycling agents of glutamatergic/GABAergic signaling, and to be synthesizers/exporters of OXPHOS bioenergetic molecules (lactate/ketone bodies). In contrast, neurons have a metabolic specialization toward OXPHOS, spurning FAO, restricting glucose oxidation while maintaining the PPP for redox homeostasis, and limiting their ability to conduct mitochondrial biosynthesis due to a lack of pyruvate carboxylase for anaplerosis. Together, neurons shoulder the bioenergetic burden of neurotransmission by controlled alterations in cell potential, while astrocytes conduct biosynthesis and high ROS oxidative processes to supplement neuronal functions.

47The key organelle in this differential specialization is the mitochondria. While the broad mitochondrial role in OXPHOS is well known in mammalian cells, specialization is evident within neurons and astrocytes. Astrocytes restrict the conversion of pyruvate to Ac-CoA by their phosphorylation of PDH, limiting their utilization of glucose for OXPHOS but instead directing these carbons to biosynthesis through the TCA cycle by PC. Additionally, the efficiency of the astrocyte ETC is far less than neurons, resulting in higher ROS generation in astrocyte compared to neurons. Astrocytic mitochondria have been demonstrated to utilize FAO for the production of ketone bodies, a process that has not been shown to be significant in neurons. Further, evidence shows that astrocytes localize mitochondria to endfeet processes in conjunction with glutamate transporters and enzymes related to glycolysis. Coupled with the evidence that OXPHOS is not required in astrocytes in vivo, this paints a unique picture for astrocyte mitochondria compared to the prototypical mammalian cell. It is a mitochondria specializing in synthesis at the expense of OXPHOS, while the neuronal mitochondria specializes in OXPHOS at the expense of synthesis.

48JR and CB contributed to the first draft and figures. AP, MP, and RF contributed to the edition of the final version of the manuscript and figures. All authors contributed to the article and approved the submitted version.

49The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.