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

In the brain, mitochondrial metabolism has been largely associated with energy production, and its dysfunction is linked to neuronal cell loss. However, the functional role of mitochondria in glial cells has been poorly studied. Recent reports have demonstrated unequivocally that astrocytes do not require mitochondria to meet their bioenergetics demands. Then, the question remaining is, what is the functional role of mitochondria in astrocytes? In this work, we review current evidence demonstrating that mitochondrial central carbon metabolism in astrocytes regulates overall brain bioenergetics, neurotransmitter homeostasis and redox balance. Emphasis is placed in detailing carbon source utilization (glucose and fatty acids), anaplerotic inputs and cataplerotic outputs, as well as carbon shuttles to neurons, which highlight the metabolic specialization of astrocytic mitochondria and its relevance to brain function.

Keywords: glycolysis, astrocytes, bioenergetics, redox, fatty acid oxidation, cataplerotic, anaplerotic, mitochondria

The 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 (von Bernhardi et al., 2016).

Astrocytes 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 (von Bartheld et al., 2016). Astrocytes are a heterogenous population of cells with a diverse array of morphological, functional and molecular properties (Garcia-Marin et al., 2007). While the cellular identity of astrocytes is determined by their gene expression profile post-differentiation (intrinsic patterning) (Song et al., 2002; Krencik et al., 2011), the surrounding environment and neuronal circuitry further shapes, modifies, and maintains the form and functions of differentiated astrocytes (extrinsic factors) (Koulakoff et al., 2008; Yang et al., 2009; Farmer et al., 2016; Ben Haim and Rowitch, 2017). 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 (Orthmann-Murphy et al., 2008). 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) (Niessen et al., 2000; Goldberg et al., 2002; Bedner et al., 2006; Lapato and Tiwari-Woodruff, 2018).

Astrocytes 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 (Chung et al., 2015; MacVicar and Newman, 2015; Verkhratsky et al., 2016).

Astrocytes 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 (Ben Haim et al., 2015).

Astrocytes 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.

While 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 (Rolfe and Brown, 1997; Attwell and Laughlin, 2001). 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 (Riveros et al., 1986; Wong-Riley, 1989; Attwell and Laughlin, 2001; Hyder et al., 2013; 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 (Attwell and Laughlin, 2001; Belanger et al., 2011). 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 (Boumezbeur et al., 2010), and it has been estimated to supply ∼20–25% of energy during energy demanding activities (van Hall et al., 2009). As plasma lactate concentrations rise, lactate uptake in the CNS increases coinciding with a decrease in glucose uptake (Smith et al., 2003; van Hall et al., 2009). 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 (Magistretti and Allaman, 2015).

Metabolism 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; Supplie et al., 2017). Glucose has been reported as being the preferential energy substrate of astrocytes over lactate, though glucose uptake does not match the theoretical bioenergetic output (Bouzier-Sore et al., 2006; Jakoby et al., 2014). 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 (Goncalves et al., 2018). 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; Yang and Vousden, 2016).

Pyruvate 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) (Bouzier-Sore et al., 2006; Halim et al., 2010; Jakoby et al., 2014), 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) (Almeida et al., 2004; Herrero-Mendez et al., 2009). 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; Delgado-Esteban et al., 2000; Vaughn and Deshmukh, 2008; Herrero-Mendez et al., 2009; Bolanos, 2016). 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; Pellerin and Magistretti, 1994; Magistretti and Allaman, 2015; Machler et al., 2016; Gonzalez-Gutierrez et al., 2019). This shuttle seems to involve sequestration within the endoplasmic reticulum for its delivery from perivascular endfeet to perisynaptic processes (Muller et al., 2018). 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) (Rosafio and Pellerin, 2014). Nitric oxide can also upregulate glucose transporter-1 (GLUT1), hexokinase-2 and MCT4 via HIF-1α (Brix et al., 2012).

However, other studies have reported that selective loss of HIF-1α in astrocytes protects neurons from hypoxic injury (Vangeison et al., 2008). 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; Patel et al., 2014; Lundgaard et al., 2015; Diaz-Garcia et al., 2017; Drulis-Fajdasz et al., 2018).

Astrocytes 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; Choi et al., 1999), its contribution to the maintenance of either neuronal or astrocyte energy-dependent processes under stress conditions is still unclear (Bak et al., 2018). 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) (Zielke et al., 2009). 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; Lovatt et al., 2007). Nevertheless, astrocytes deficient in mitochondrial respiration survive as glycolytic cells, both in vitro and in vivo (Supplie et al., 2017). 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.

Oxidative 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; Owen et al., 2002).

Pyruvate 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 (Weber and Barros, 2015). 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) (Sa et al., 2017).

In 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; Schmoll et al., 1995). While PEP has been shown to exit mitochondria (Garber and Ballard, 1969; Stark et al., 2009), the precise mechanism has not been determined, with the citrate/isocitrate carrier (CIC) and adenine nucleotide transporter (ANT) being proposed as likely intermediators (Stark and Kibbey, 2014). 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 (Bernard-Helary et al., 2002), and that G6PC may be silent under physiological conditions becoming activated at times of stress (Ghosh et al., 2005). 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 (Yip et al., 2016).

In 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 (Shank et al., 1985; Murin et al., 2009). 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; Halim et al., 2010). 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 (Halim et al., 2010; Adina-Zada et al., 2012; Panov et al., 2014).

Ketone 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 (Blazquez et al., 1998; Cullingford et al., 1998; Guzman and Blazquez, 2001; Thevenet et al., 2016). During starvation up to ∼70% of the brain’s energy can be supplied by ketone bodies (Owen et al., 1967). 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 (Auestad et al., 1991). 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; Halestrap and Price, 1999; Morris, 2005). At high concentrations, ketone bodies have been proposed to support basal and activity-dependent needs of neurons and glutamatergic signaling, while decreasing glucose utilization (Lopes-Cardozo et al., 1986; Kunnecke et al., 1993; Sibson et al., 1998; Magistretti et al., 1999; Chowdhury et al., 2014; Courchesne-Loyer et al., 2017). 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; Paradies and Papa, 1977; Halestrap, 1978; Booth and Clark, 1981; Achanta and Rae, 2017). 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 (Chechik et al., 1987).

Ketone 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 (Fedorovich et al., 2018). 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 (Valdebenito et al., 2016; Morris et al., 2020). It has also been clearly shown that production of ketone bodies is used as a signaling mechanism to regulate food intake (Le Foll and Levin, 2016). While astrocytes exposed to ketone bodies observe significant morphological changes, these changes do not seem to correlate with gliosis or inflammation (Gzielo et al., 2019).

In 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 (Hamilton and Brunaldi, 2007). 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; Gnaedinger et al., 1988; Purdon et al., 1997; Hamilton and Brunaldi, 2007; Ouellet et al., 2009; Mitchell et al., 2011). 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; Liu et al., 2015, 2017).

In the brain, FAO occurs primarily in astrocytes (Edmond et al., 1987; Auestad et al., 1991; Schonfeld and Reiser, 2013; Eraso-Pichot et al., 2018). 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 (Eraso-Pichot et al., 2018; Fecher et al., 2019). FAO was also demonstrated to coexist with glycolysis and FAO enzymes seemed to be more abundant in adult vs. fetal astrocytes (Eraso-Pichot et al., 2018).

To 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; Mashek et al., 2007; Anderson and Stahl, 2013). In the brain, signaling through the peroxisome proliferation activated receptor beta (PPARβ, predominantly expressed in neurons but also glia) increases the expression of ACS2 (Basu-Modak et al., 1999), in turn increasing the oxidation of exogenously supplied FAs in neurons (Marszalek et al., 2004). Astrocytes have an increased expression of both PPARα and ACS1 compared to neurons (Basu-Modak et al., 1999), which has been linked to the upregulation of genes involved in FAO and ketogenic processes (Cullingford et al., 2002).

Carnitine 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; Houten and Wanders, 2010; Panov et al., 2014; Romano et al., 2017). While both astrocytes and oligodendrocytes uptake saturated and unsaturated FAs (Hofmann et al., 2017), CTP1a seems to be found primarily in astrocytes and neural progenitor cells, while absent in neurons, microglia, and oligodendrocytes (Jernberg et al., 2017). 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 (Kimura and Amemiya, 1990; Calabrese et al., 2005; Jones et al., 2010). 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; Foster, 2012). 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 (Weightman Potter et al., 2019). In another study, astrocyte activation with the ciliary neurotrophic factor (CNTF) was also shown to activate AMPK and increases FAO and ketolysis (Escartin et al., 2007).

In contrast, upon hypoxic conditions or high concentrations of FAs, ketogenesis is induced in astrocytes and this is regulated as well by AMPK signaling (Blazquez et al., 1999; Takahashi et al., 2014).

In 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; Houten and Wanders, 2010; Panov et al., 2014; Romano et al., 2017). In the brain, 95% of HADHA co-localizes with the astrocyte biomarker GFAP (Sayre et al., 2017) and ACD is also predominantly find in astrocytes (Fecher et al., 2019).

For 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 (Jansen and Wanders, 2006).

A 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 (Yang et al., 1987), 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 (Jernberg et al., 2017), CTP1b is not expressed in the brain (Obici et al., 2003; Lavrentyev et al., 2004), and CPT1c localizes exclusively to the endoplasmic reticulum in neurons and has not been shown to participate in FAO (Wolfgang et al., 2006; Sierra et al., 2008) but has been implicated in ROS management (Lee and Wolfgang, 2012). Lower CPT1a expression and activity restricts mitochondrial oxidation of FAs in neurons (Bird et al., 1985), 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 (Ebert et al., 2003), which occurs predominantly in astrocytes, based on comparisons of astrocytes, oligodendrocytes and neurons in primary cultures (Edmond et al., 1987).

Astrocytes have a high buffering capacity against ROS compared to neurons (Sun et al., 2006), 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 (Schonfeld and Reiser, 2013, 2017). 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 (Chen et al., 2014).

However, 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 (Ho et al., 1995). 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) (Hein et al., 2008). The protonophoric property, and its resulting decrease in ROS from hyperpolarization, has been demonstrated in medium chain FAs (MCFAs 6–12 carbons) (Korshunov et al., 1998), LCFAs and branched chain FAs (Schonfeld and Wojtczak, 2007). This protonophoric capacity of FAs decreases as the length of the FA decreases (Schonfeld and Wojtczak, 2016), while branched FAs such as phytanic acid show an increased protonophoric activity compared to their straight chain FA counterparts (Komen et al., 2007; Schonfeld and Reiser, 2016). 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 (Schonfeld et al., 2010).

While 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 (Schonfeld and Wojtczak, 2007) and during uncoupling with CCCP (Cocco et al., 1999), 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 (Cocco et al., 1999; Schonfeld and Wojtczak, 2007). 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 (Seifert et al., 2010). As described in Schonfeld and Wojtczak (2008), despite the evidence of electron transfer interference by FAs, the mechanism is not well understood.