**3. Metabolic pathways during brain activation**

The resting brain is not really at rest. It demonstrates continuous cognitive and sensory activity. For example, when we listen music, auditive areas of the brain are selectively activated. Over 120 years ago, brain activation was shown to produce an increase en blood flow in specific brain areas. The increased blood flow has been shown to be accompanied by an increase in glucose utilization (Sokoloff et al., 1977). A mechanism that couples neuronal

brain. Astrocytes are able to oxidize fatty acids and ketone bodies, while neurons and

Ketone bodies are synthesized mainly in the liver. Ketone body synthesis is activated after exhaustion of cellular carbohydrate stores (glycogen). Fatty acids are broken down via oxidation to Acetyl-CoA, NADH and FADH2. Normally, Acetyl-CoA is completely oxidized via the TCA cycle. However, if the amount of Acetyl-CoA generated in fatty-acid oxidation increases disproportionately, the processing capacity of the TCA cycle will start to drop due to low levels of intermediates. Acetyl-CoA will then be used instead for biosynthesis of acetoacetate, -hydroxybutyrate and acetone (ketone bodies) through four enzymatic reactions (Figure 4). The utilization of ketones bodies is controlled by leptin

During starvation, during chronic feeding with high fat/low carbohydrate or in pathophysiological conditions as diabetes, glucose stores are depleted and ketone body synthesis is stimulated. Under these conditions, the concentration of ketone bodies in blood increases and MCT expression increases at the blood brain barrier. Within the brain, ketone bodies may be used because all brain cells express MCTs and because the enzymatic reactions of ketone body synthesis are reversible (except for spontaneous decarboxylation of acetoacetate to acetone). Thus brain cells obtain Acetyl-CoA from ketone bodies and Acetyl-CoA may be oxidized for those cells through the TCA cycle. The starving brain also extracts fatty acids from blood. Astrocytes can degrade fatty acids by -oxidation to provide neurons with ketone bodies (Edmond, 1992). Moreover, ketone body oxidation must be important during the first postnatal period. Knockout animals for the ketolytic enzyme succinyl-CoA: 3-oxoacid CoA-transferase exhibit normal prenatal development, but develop ketoacidosis, hypoglycemia, and reduced plasma lactate concentrations within the first 48 h of birth

A ketogenic diet has been proposed for treatment of epilepsy. This diet is a strict high fat, low protein, low carbohydrate diet, and is anticonvulsant in many drug-resistant epileptic children. The diet is also effective in mice. The mechanism by which this diet controls intractable seizures is unknown. The diet does not affect behavorial performance or synaptic plasticity. Ketone bodies not only function as an energy substrate, but also as intermediates for the synthesis of lipids and neurotransmitters. Because of this, a link has been proposed between ketone bodies, brain function and polyunsaturated fatty acids (Pifferi et al., 2008). Another proposed mechanism to explain the neuroprotective effect of the ketogenic diet is via mitochondrial improvement by scavenging of reactive species and regulation of gene expression to increase neuronal survival (Beskow et al., 2008). Ketone bodies are also proposed as protectors from neurotoxicity in other pathologies, such as Parkinson's disease

The resting brain is not really at rest. It demonstrates continuous cognitive and sensory activity. For example, when we listen music, auditive areas of the brain are selectively activated. Over 120 years ago, brain activation was shown to produce an increase en blood flow in specific brain areas. The increased blood flow has been shown to be accompanied by an increase in glucose utilization (Sokoloff et al., 1977). A mechanism that couples neuronal

through AMPK inhibition in the hypothalamic region (Narishima et al., 2011).

oligodendrocytes can only use ketone bodies.

(Cotter et al., 2011).

and Alzheimer's disease (Kashiwaya et al., 2000).

**3. Metabolic pathways during brain activation** 

metabolism with blood flow should thus exist. An increase in extracellular K+, adenosine, and lactate and related changes in pH are all a consequence of increased neuronal activity. All of them have been considered mediators of neurovascular coupling because they have vasoactive effects (Villringer & Dirnagl, 1995). Astroglial cells seem to be protagonists in the coupling of neuron metabolism and blood flow. Astrocytes, like neurons, are organized into networks, although glial cells are organized as a syncytium, linked to each other by gap junctions (Giaume et al., 2010). Neuronal cells exchange information through chemical and electrical synapses. Astrocyte gap junctions are regulated by extra- and intracelullar signals. Astrocytes release molecules that induce local constriction or dilation of smooth muscle cells surrounding endothelial cells forming arterioles (Attwell et al., 2010). Astrocytes also release molecules in response to neuronal activation. These molecules can modulate synaptic transmission and neuronal excitability (a concept referred to as the tripartite synapse, Perea & Araque, 2010). So, during brain activation, blood flow is increased due to several interactions between brain cells. We will discuss below the metabolic pathways that function during brain activation in neuronal and astroglial cells. We will then discuss the neuroglial interation and molecular mechanisms that modulate the interaction.

#### **3.1 Neuron metabolism during activity periods**

In a chemical synapse, neurotransmitters released from the presynaptic neuron bind to ionotropic receptors in the postsynaptic membrane increasing the open probability of these ion channels. The ion flow can produce membrane depolarization (excitatory action potentials) or hyperpolarization (inhibitory action potentials). Excitatory action potentials are produced by the sequential opening of voltage-sensitive Na+ channels, followed by the delayed opening of K+ channels. This permits movement of positive charges from and into the neuronal cytosol. According to the biophysical properties of neuronal membranes, a theoretical minimum positive charge transfer (carried by Na+) was predicted per propagating action potential of 121 nC/cm2. This was corroborated by Alle and colleagues (2009) who calculated that charging of membrane capacitance requires 153 nC/cm2 only 1.3 times more than the theoretical minimum. Action potentials are thus energy-efficient, minimizing their contribution to activity-dependent metabolism. But human brain has billions of neurons and each neuron maintains a large number of synaptic connections to other neurons. Even though action potentials are energy-efficient, this is consistent with the fact that neuronal activity accounts for 80% of brain energy consumption (Sibson et al., 1998). Most of the energy consumed in the brain is attributable to restoration of the membrane resting potential following depolarization. This is accomplished by the Na+/K+- ATPase (EC 3.6.1.3, Attwell & Laughlin, 2001). Other energy-consuming processes are neurotransmitter recycling and axonal and dendritic transport (Ames, 2000). Because glutamate synapses represent at least 80% of cortical synapses, glutamate-mediated neurotransmission consumes most of the energy expended in the brain.

Electrical stimulation opens Na+ channels in neuronal populations. An increase in intracellular Na+ concentration activates the Na+/K+-ATPase at its Na+-sensitive intracellular site. The activation of Na+/K+-ATPase is accompanied by a decrease in the ATP/ADP ratio and glycolysis activation (Figure 6, probably via allosteric activation of PFK1 by ADP). Glycolysis activation is accompanied by a Ca2+ increase and an increase in pyruvate and NADH/NAD+ concentrations. TCA and oxidative phosphorylation are also

Brain Energy Metabolism in Health and Disease 343

Fig. 5. Relationship between lipid metabolism and the TCA cycle. Under particular dietary conditions, such as lactation in newborns or fasting in adults, the ketone bodies acetoacetate and -hydroxybutyrate and circulating fatty acids can provide substrates to the TCA cycle after their conversion to Acetyl-CoA. Carbon atoms for lipid synthesis can be provided by glucose through citrate produced in the TCA cycle, a particularly relevant process for the

Glycogen stores may also be mobilized during astrocytic activation (Figure 7). During brain activation, neurotransmitters such as noradrenaline can stimulate PTG expression and thus, glycogen synthesis (Allaman et al., 2010). However, at the same time, Na+/K+-ATPase activation induces a decrease in the ATP/ADP ratio and glycogen catabolism activation. Serotonin and other neurotransmitters induce an increase in intracellular concentrations of Ca2+ (via IP3 production) and thus, activate glycogenolysis (Chen & Hertz, 1999). Activation of glycogen synthesis and degradation is termed the glycogen shunt (Walls et al., 2008). Glucose-6-phosphate from glycogen can be oxidized through glycolysis to produce lactate which is exported to neurons (Figure 7). It has, in fact, been demonstrated that glycogen is required for sustaining glutamatergic neurotransmission and for long-term memory

Besides glutathione, the brain also uses ascorbic acid to protect itself from oxidant species. Ascorbic acid is highly concentrated in brain (Kratzing et al. 1982). In aqueous solutions, ascorbic acid is a powerful reductant and is oxidized to dehydroascorbic acid. The regeneration of ascorbic acid from dehydroascorbic acid is not spontaneous. Reduction is mainly an enzymatically catalyzed reaction, which may be glutathione-dependent (Ishikawa et al. 1998). Astrocytes are thought to be involved in ascorbic acid recycling (Figure 10, Astuya et al. 2005). During synaptic activity, ascorbic acid is released from intracellular reservoirs (O'Neill et al. ,1984; Ghasemzadeh et al., 1991; Yusa, 2001). The molecular basis of ascorbic acid efflux is not yet well known. Neurons can take up ascorbic acid efficiently because they express SVCT2 (Castro et al., 2001). Ascorbic acid is oxidized within neurons

developing brain.

formation (Suzuki et al., 2011).

stimulated (Figure 6). Indeed, mitochondrial dehydrogenases are activated by an increase in intracellular Ca2+ (Figure 4). During glutamatergic activity, glutamate binds to ionotropic receptors and metabotropic channels in these cells. Both produce an increase in intracellular Ca2+. Glucose oxidation (determined by CO2 production) has been shown to be produced by an increased intracellular Na+ concentration in neuronal-enriched cultures. However, neuronal cells are unable to stimulate glycolysis via fructose 2,6-bisphosphate (see above, Herrero-Mendez et al., 2009). It has thus been proposed that, when energy needs are overcome by glycolytic activity stimulated by ADP, neuronal cells preferentially consume lactate. Astrocytes release lactate in response to synaptic activation (see below, Magistretti et al., 2000) and neuronal cells oxidize this lactate through the TCA and oxidative phosphorylation via a mechanism named the neuron lactate shuttle hypothesis (ANLSH, see below, Magistretti et al., 1999; Allaman et al., 2010). To facilitate lactate use, glycolysis must be inhibited. We will discuss below the details of neuron-glia coupling and the inhibition of glucose use.

The pentose phosphate pathway (PPP) supplies red blood cells with NADPH, which in turn maintains the reduced state of glutathione (GSH). GSH has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, GSH is maintained in the reduced form by the enzyme glutathione reductase. Regeneration of reduced *GSH* requires *NADPH.* Therefore, PPP is important for maintenance of adequate levels of GSH (Figure 6)*.* Neurons are thought to be particularly vulnerable to damage by reactive oxygen and nitrogen species. Nitrogen species stimulate the PPP rate (Bolaños et al., 2010). The brain is a specific source of oxidative species such as those coming from metabolism of excitatory amino acids and neurotransmitters. During glutamatergic activity, glutamate binds ionotropic receptors and metabotropic channels in these cells. Both produce an increase on intracellular Ca2+. The high and constant use of oxygen results in oxidative stress through the production of superoxide. Finally, other sources of free radicals are produced by Cytochrome P450 and monoamine oxidase activity. It is thus not surprising that neuronal cells oxidize glucose through the PPP (Figure 5 and Figure 6), especially, considering that neurons are unable to increase glycolysis activity via allosteric activation of PFK1 by fructose 2,6-bisphosphate (Figure 1, Herrero-Mendez et al., 2009).

#### **3.2 Metabolic activation in astrocytes**

Extracellular K+ activates the Na+/K+-ATPase via its extracellular K+-sensitive site in cultured astrocytes but not in neurons (Grisar et al. 1979; Hajek et al. 1996). Na+/K+-ATPase activation produces a decrease in the ATP/ADP ratio, and thus glycolysis activation (see above, Figure 7, Sokoloff et al., 1996). Extracellular K+ concentrations of between 5 and 12 mM increase glucose phosphorylation in cultured astrocytes (Hof et al., 1988). The Na+/K+- ATPase can also be activated by increased intracellular Na+ concentration (Denton et al., 1988). An increase in intracellular Na+ concentration in astrocytes can be stimulated by the presence of glutamate. Glutamate uptake is carried out by excitatory amino acid transporters. This transport by excitatory amino acid transporters is dependent on the electrochemical gradient of sodium ions. Glutamate uptake can induce metabolic activation in astrocytes and can also be used as energetic fuel. Glutamate can be transformed into αketoglutarate through a reaction catalyzed by the enzyme aspartate amino transferase (EC 2.6.1.1, Fonnum, 1967) and thus, glutamate carbons can be oxidized through the TCA.

stimulated (Figure 6). Indeed, mitochondrial dehydrogenases are activated by an increase in intracellular Ca2+ (Figure 4). During glutamatergic activity, glutamate binds to ionotropic receptors and metabotropic channels in these cells. Both produce an increase in intracellular Ca2+. Glucose oxidation (determined by CO2 production) has been shown to be produced by an increased intracellular Na+ concentration in neuronal-enriched cultures. However, neuronal cells are unable to stimulate glycolysis via fructose 2,6-bisphosphate (see above, Herrero-Mendez et al., 2009). It has thus been proposed that, when energy needs are overcome by glycolytic activity stimulated by ADP, neuronal cells preferentially consume lactate. Astrocytes release lactate in response to synaptic activation (see below, Magistretti et al., 2000) and neuronal cells oxidize this lactate through the TCA and oxidative phosphorylation via a mechanism named the neuron lactate shuttle hypothesis (ANLSH, see below, Magistretti et al., 1999; Allaman et al., 2010). To facilitate lactate use, glycolysis must be inhibited. We will discuss below the details of neuron-glia coupling and the inhibition of

The pentose phosphate pathway (PPP) supplies red blood cells with NADPH, which in turn maintains the reduced state of glutathione (GSH). GSH has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, GSH is maintained in the reduced form by the enzyme glutathione reductase. Regeneration of reduced *GSH* requires *NADPH.* Therefore, PPP is important for maintenance of adequate levels of GSH (Figure 6)*.* Neurons are thought to be particularly vulnerable to damage by reactive oxygen and nitrogen species. Nitrogen species stimulate the PPP rate (Bolaños et al., 2010). The brain is a specific source of oxidative species such as those coming from metabolism of excitatory amino acids and neurotransmitters. During glutamatergic activity, glutamate binds ionotropic receptors and metabotropic channels in these cells. Both produce an increase on intracellular Ca2+. The high and constant use of oxygen results in oxidative stress through the production of superoxide. Finally, other sources of free radicals are produced by Cytochrome P450 and monoamine oxidase activity. It is thus not surprising that neuronal cells oxidize glucose through the PPP (Figure 5 and Figure 6), especially, considering that neurons are unable to increase glycolysis activity via allosteric activation of

Extracellular K+ activates the Na+/K+-ATPase via its extracellular K+-sensitive site in cultured astrocytes but not in neurons (Grisar et al. 1979; Hajek et al. 1996). Na+/K+-ATPase activation produces a decrease in the ATP/ADP ratio, and thus glycolysis activation (see above, Figure 7, Sokoloff et al., 1996). Extracellular K+ concentrations of between 5 and 12 mM increase glucose phosphorylation in cultured astrocytes (Hof et al., 1988). The Na+/K+- ATPase can also be activated by increased intracellular Na+ concentration (Denton et al., 1988). An increase in intracellular Na+ concentration in astrocytes can be stimulated by the presence of glutamate. Glutamate uptake is carried out by excitatory amino acid transporters. This transport by excitatory amino acid transporters is dependent on the electrochemical gradient of sodium ions. Glutamate uptake can induce metabolic activation in astrocytes and can also be used as energetic fuel. Glutamate can be transformed into αketoglutarate through a reaction catalyzed by the enzyme aspartate amino transferase (EC 2.6.1.1, Fonnum, 1967) and thus, glutamate carbons can be oxidized through the TCA.

PFK1 by fructose 2,6-bisphosphate (Figure 1, Herrero-Mendez et al., 2009).

**3.2 Metabolic activation in astrocytes** 

glucose use.

Fig. 5. Relationship between lipid metabolism and the TCA cycle. Under particular dietary conditions, such as lactation in newborns or fasting in adults, the ketone bodies acetoacetate and -hydroxybutyrate and circulating fatty acids can provide substrates to the TCA cycle after their conversion to Acetyl-CoA. Carbon atoms for lipid synthesis can be provided by glucose through citrate produced in the TCA cycle, a particularly relevant process for the developing brain.

Glycogen stores may also be mobilized during astrocytic activation (Figure 7). During brain activation, neurotransmitters such as noradrenaline can stimulate PTG expression and thus, glycogen synthesis (Allaman et al., 2010). However, at the same time, Na+/K+-ATPase activation induces a decrease in the ATP/ADP ratio and glycogen catabolism activation. Serotonin and other neurotransmitters induce an increase in intracellular concentrations of Ca2+ (via IP3 production) and thus, activate glycogenolysis (Chen & Hertz, 1999). Activation of glycogen synthesis and degradation is termed the glycogen shunt (Walls et al., 2008). Glucose-6-phosphate from glycogen can be oxidized through glycolysis to produce lactate which is exported to neurons (Figure 7). It has, in fact, been demonstrated that glycogen is required for sustaining glutamatergic neurotransmission and for long-term memory formation (Suzuki et al., 2011).

Besides glutathione, the brain also uses ascorbic acid to protect itself from oxidant species. Ascorbic acid is highly concentrated in brain (Kratzing et al. 1982). In aqueous solutions, ascorbic acid is a powerful reductant and is oxidized to dehydroascorbic acid. The regeneration of ascorbic acid from dehydroascorbic acid is not spontaneous. Reduction is mainly an enzymatically catalyzed reaction, which may be glutathione-dependent (Ishikawa et al. 1998). Astrocytes are thought to be involved in ascorbic acid recycling (Figure 10, Astuya et al. 2005). During synaptic activity, ascorbic acid is released from intracellular reservoirs (O'Neill et al. ,1984; Ghasemzadeh et al., 1991; Yusa, 2001). The molecular basis of ascorbic acid efflux is not yet well known. Neurons can take up ascorbic acid efficiently because they express SVCT2 (Castro et al., 2001). Ascorbic acid is oxidized within neurons

Brain Energy Metabolism in Health and Disease 345

and the production of arachidonic acid and prostaglandins. These substances stimulate dilation or constriction of adjacent arterioles and permit that the correct nutrients and oxygen supply are present in order to sustain neurotransmission (Peppiat & Attwell, 2004).

As we discuss above, the key to neuron-glia metabolic coupling is the excitatory neurotransmitter glutamate and a rise in extracellular K+. Glutamatergic synaptic activity is necessary to maintain a low glutamate concentration in the extracellular space for efficient and successful synaptic transmission to occur and to prevent excitotoxicity. Glutamate uptake in astrocytes occurs via excitatory amino acid transporters. Within the cell, glutamate is then converted to glutamine by glutamine synthetase (EC 6.3.1.2). Glutamine is released into the extracellular space and is taken up by adjacent neurons that synthesize glutamate from glutamine in a reaction catalyzed by glutaminase (EC 3.5.1.2). This recycling of

glutamate is named the glutamate–glutamine cycle (Figure 10, Sibson et al., 1997).

Fig. 7. Metabolic activation of astrocytes. Synaptic activity produces an increase in

extracellular K+, which stimulates Na+K+-ATPase by binding of its extracellular K+-sensitive site. The excitatory neurotransmitter glutamate is taken up by astrocytes through excitatory amino acid transporters. This kind of transport produces an increase in intracellular Na+, which stimulates Na+K+-ATPase by binding of its intracellular Na+-sensitive site. Na+K+- ATPase activation produces a decrease in ATP/ADP ratio and, thus glycolysis and

glycogenolysis activation. In addition, glucose is oxidized by PPP to produce NADPH and to maintain the redox balance, reducing glutathione and ascorbic acid (Asc). Ascorbic acid released by astrocytes is taken up by neurons to protect themselves from oxidant species (ascorbic acid is oxidized in neurons). Oxidized ascorbic acid (dehydroascorbic acid, Asc+) is released from neurons and taken up by astrocytes through GLUT1. Finally, in astrocytes, glutamate is able to bind ionotropic receptors, which are predominantly calcium channels. This Ca2+ increase cooperates with TCA activation and produces arachidonic acid (AA) and prostaglandin (PG) which stimulate the constriction and dilation of capillaries, respectively.

**3.3 Neuron-glia metabolic coupling** 

Fig. 6. Neuron metabolism during synaptic activity. Electrical stimulation opens Na+ channels in neuronal cells. An increase in intracellular Na+ concentration is able to activate Na+K+--ATPase at its Na+-sensitive intracellular site (early phase). This activation produces a decrease in ATP/ADP ratio and thus, glycolysis activation by allosteric activation of PFK1 by ADP. Glycolysis activation is accompanied by Ca2+ increase and therefore, mitochondrial oxidative metabolism activation. In the late phase of synaptic activation, glycolysis activity is inhibited and lactate uptake is stimulated (see Figure 10 for details of inhibition of glucose utilization and stimulation of lactate transport in synaptically-active neurons). PPP is always present in neuronal cells because NADPH is required to maintain the redox balance in these cells. During synaptic activity, PPP becomes more important because synaptic activity produces oxidant species. NADPH is used to regenerate GSH and thus, to reduce oxidant species.

because during synaptic activity many oxidant species are generated. Neuronal oxidized ascorbic acid (dehydroascorbic acid) can be released through glucose transporters, GLUT1 or GLUT3 (because dehydroascorbic acid is a substrate for GLUTs; Vera et al., 1993). Astrocytes uptake dehydroascorbic acid through GLUT1 and thus, they reduce dehydroascorbic acid to ascorbic acid through mechanisms that require GSH. GSH regeneration and PPP activation under synaptic transmission conditions should thus be important for astrocytes (Figure 7, Figure 10).

Astrocytic cells also express metabotropic glutamate receptors (mGluRs). Thus, during excitatory brain activation, glutamate is not only taken up by excitatory amino acid transporters, but glutamate activates mGluRs, which produce and increase intracellular Ca2+

Fig. 6. Neuron metabolism during synaptic activity. Electrical stimulation opens Na+ channels in neuronal cells. An increase in intracellular Na+ concentration is able to activate Na+K+--ATPase at its Na+-sensitive intracellular site (early phase). This activation produces a decrease in ATP/ADP ratio and thus, glycolysis activation by allosteric activation of PFK1 by ADP. Glycolysis activation is accompanied by Ca2+ increase and therefore, mitochondrial oxidative metabolism activation. In the late phase of synaptic activation, glycolysis activity is inhibited and lactate uptake is stimulated (see Figure 10 for details of inhibition of glucose utilization and stimulation of lactate transport in synaptically-active neurons). PPP is always present in neuronal cells because NADPH is required to maintain the redox balance in these cells. During synaptic activity, PPP becomes more important because synaptic activity produces oxidant species. NADPH is used to regenerate GSH and thus, to reduce oxidant

because during synaptic activity many oxidant species are generated. Neuronal oxidized ascorbic acid (dehydroascorbic acid) can be released through glucose transporters, GLUT1 or GLUT3 (because dehydroascorbic acid is a substrate for GLUTs; Vera et al., 1993). Astrocytes uptake dehydroascorbic acid through GLUT1 and thus, they reduce dehydroascorbic acid to ascorbic acid through mechanisms that require GSH. GSH regeneration and PPP activation under synaptic transmission conditions should thus be

Astrocytic cells also express metabotropic glutamate receptors (mGluRs). Thus, during excitatory brain activation, glutamate is not only taken up by excitatory amino acid transporters, but glutamate activates mGluRs, which produce and increase intracellular Ca2+

species.

important for astrocytes (Figure 7, Figure 10).

and the production of arachidonic acid and prostaglandins. These substances stimulate dilation or constriction of adjacent arterioles and permit that the correct nutrients and oxygen supply are present in order to sustain neurotransmission (Peppiat & Attwell, 2004).

#### **3.3 Neuron-glia metabolic coupling**

As we discuss above, the key to neuron-glia metabolic coupling is the excitatory neurotransmitter glutamate and a rise in extracellular K+. Glutamatergic synaptic activity is necessary to maintain a low glutamate concentration in the extracellular space for efficient and successful synaptic transmission to occur and to prevent excitotoxicity. Glutamate uptake in astrocytes occurs via excitatory amino acid transporters. Within the cell, glutamate is then converted to glutamine by glutamine synthetase (EC 6.3.1.2). Glutamine is released into the extracellular space and is taken up by adjacent neurons that synthesize glutamate from glutamine in a reaction catalyzed by glutaminase (EC 3.5.1.2). This recycling of glutamate is named the glutamate–glutamine cycle (Figure 10, Sibson et al., 1997).

Fig. 7. Metabolic activation of astrocytes. Synaptic activity produces an increase in extracellular K+, which stimulates Na+K+-ATPase by binding of its extracellular K+-sensitive site. The excitatory neurotransmitter glutamate is taken up by astrocytes through excitatory amino acid transporters. This kind of transport produces an increase in intracellular Na+, which stimulates Na+K+-ATPase by binding of its intracellular Na+-sensitive site. Na+K+- ATPase activation produces a decrease in ATP/ADP ratio and, thus glycolysis and glycogenolysis activation. In addition, glucose is oxidized by PPP to produce NADPH and to maintain the redox balance, reducing glutathione and ascorbic acid (Asc). Ascorbic acid released by astrocytes is taken up by neurons to protect themselves from oxidant species (ascorbic acid is oxidized in neurons). Oxidized ascorbic acid (dehydroascorbic acid, Asc+) is released from neurons and taken up by astrocytes through GLUT1. Finally, in astrocytes, glutamate is able to bind ionotropic receptors, which are predominantly calcium channels. This Ca2+ increase cooperates with TCA activation and produces arachidonic acid (AA) and prostaglandin (PG) which stimulate the constriction and dilation of capillaries, respectively.

Brain Energy Metabolism in Health and Disease 347

Fig. 8. Intracellular ascorbic acid only inhibits transport of 2-NBDG in cells expressing GLUT3. A: Time course of uptake of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4 yl)amino]-2-deoxyglucose). 2-NBDG transport in cortical neurons in the presence (closed circles) or absence (open circles) of intracellular ascorbic acid. B: Time course of uptake of 2- NBDG transport in neuronal cells treated with shRNA to knock down GLUT3 expression in the presence (triangles) or absence (circles) of intracellular ascorbic acid. The data represent the mean ± SD of four experiments (14 control cells and 10 shRNA-treated cells). The images represent one control experiment and one experiment with shRNA-treated cells, in the presence or absence of ascorbic acid. Adapted from Beltran et al. (2011) with permission of

Neurodegenerative disease is a broad term for a range of conditions which primarily affect neurons in the brain. Progression of neurodegenerative diseases is accompanied by loss of neuronal cell structure and function and even cell death. These diseases have many similarities at a sub-cellular level including atypical protein assemblies, failure of normal protein degradation pathways, induced cell death, impaired axonal transport and metabolic failures (Rubinsztein et al., 2006; De Vos et al., 2008; Bredesen et al., 2006; Lin & Beal, 2006). A better knowledge of how these failures arise may offer fresh hope for development of

Directly or indirectly, energy is necessary for many, if not all, cellular processes. It is thus possible to speculate that metabolic failure is an early event in neurodegenerative disease. Indeed, there are neurodegenerative diseases caused by a deficiency of metabolic enzymes. One of these is pyruvate dehydrogenase complex deficiency (Brown et al., 1994). This condition has similar characteristics to those of other neurodegenerative diseases. Several neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease and Parkinson's disease, show metabolic failure represented by altered patterns of expression of nutrient transporters, metabolic enzymes and molecular components of cellular respiration.

**4. Metabolic failure in neurodegenerative diseases** 

therapies that improve treatment of these diseases.

the publisher.

According to ANLSH, glutamate uptake also stimulates glucose uptake (Pellerin & Magistretti, 1994), glycolysis and lactate release in astrocytes (Pellerin & Magistretti, 1994; Demestre et al., 1997). In a similar way, glycolysis and glucose uptake is activated by an increase in the extracellular concentration of K+ (Pellerin & Magistretti, 1994; Gegelashvili et al., 2007). An increased extracellular K+ concentration stimulates 2-deoxyglucose phosphorylation (Hof et al., 1988) and lactate formation and release of lactate (Walz & Mukerji, 1988) in cultured astrocytes. Neurons take up the lactate released from astrocytes (Figure 10, Magistretti et al., 2000; Allaman et al., 2010). Lactate flow happens would be possible because there are a differential MCTs and LDH isoenzymes expression between neuronal and astroglial cells. However, use of neuronal lactate has been strongly debated in studies that support the idea that glucose is the main metabolic substrate for these cells (Chih & Roberts, 2003; Dienel, 2009; Gjedde, 2002; Hertz, 2004; Hertz et al., 2007; Mangia et al., 2003; Mangia et al., 2009).

Activation of glycolysis produces an increase in pyruvate and proton concentrations that are accompanied by a decrease in the NAD+/NADH ratio. Neuronal LDH1 should catalyze reduction of pyruvate to lactate. So, lactate utilization by neurons would only be possible if the consumption of glucose, either at the transport or the glycolysis stage, were inhibited. To explain this, at least four different (though coexistent) ideas have been proposed (Figure 10). Firstly, using real-time microscopy, glucose transport inhibition by glutamate has been demonstrated in neurons co-cultured with astrocytes (Porras et al., 2008). But because these experiments were performed with co-cultures, it is not possible to discard the possibility that astrocytic stimulation by glutamate may induce the release of substances which inhibit glucose uptake in neurons. In other words, it is possible that the effect of glutamate on neuronal glucose uptake may be indirect. The second mechanism is explained by the redox switch/redox coupling hypothesis (Cerdan et al., 2006). This idea embraces a mechanism in which two different pyruvate pools exist: one is thought to operate in exchange with extracellular lactate or pyruvate and the other, thought to be derived from glycolytic activity. This mechanism considers a lactate/pyruvate redox shuttle, which is able to transfer lactate from astrocytes to neurons. So, high cytosolic lactate inhibits neuronal glycolysis at the glyceraldehyde- 3-phosphate dehydrogenase (EC 1.2.1.12) step by competing with cytosolic NAD+, favoring oxidation of extracellular lactate. The third mechanism is our own theory. We have demonstrated that neuronal intracellular ascorbic acid inhibits glucose utilization in neurons (Castro et al., 2009) through GLUT3 inhibition (Figure 8, Beltrán et al., 2011). This mechanism is supported by the idea of ascorbic acid recycling in brain (see above, Figure 10, Astuya et al., 2005). Using primary cultures of cortical neurons treated with a specific shRNA (to block GLUT3 expression) and a fluorescent glucose analogue, we have shown that this transporter is important, using real-time experiments (Figure 7). At the same time, intracellular ascorbic acid is able to stimulate lactate transport in neurons and in cells that express GLUT3 (Figure 9, Castro et al., 2008). Because ascorbic acid is able to change metabolic substrate preferences, we have termed this mechanism the ascorbic acid metabolic switch (Castro et al., 2009). The fourth proposed mechanism relates to the inability of neuronal cells to overactivate glycolysis (Herrero-Mendez et al., 2009; Bolaños et al., 2010). This idea is supported by elegant experimental data that demonstrate that in neurons, it is not possible to produce the allosteric activator of PFK1 (and therefore glycolysis activator) fructose 2,6-bisphosphate. The enzyme that catalyzes fructose-2,6-bisphosphate in neurons, PFK3B, has a very short half-life as it is being constantly degraded (Herrero-Mendez et al., 2009). This mechanism does not exclude basal glycolytic activity however.

According to ANLSH, glutamate uptake also stimulates glucose uptake (Pellerin & Magistretti, 1994), glycolysis and lactate release in astrocytes (Pellerin & Magistretti, 1994; Demestre et al., 1997). In a similar way, glycolysis and glucose uptake is activated by an increase in the extracellular concentration of K+ (Pellerin & Magistretti, 1994; Gegelashvili et al., 2007). An increased extracellular K+ concentration stimulates 2-deoxyglucose phosphorylation (Hof et al., 1988) and lactate formation and release of lactate (Walz & Mukerji, 1988) in cultured astrocytes. Neurons take up the lactate released from astrocytes (Figure 10, Magistretti et al., 2000; Allaman et al., 2010). Lactate flow happens would be possible because there are a differential MCTs and LDH isoenzymes expression between neuronal and astroglial cells. However, use of neuronal lactate has been strongly debated in studies that support the idea that glucose is the main metabolic substrate for these cells (Chih & Roberts, 2003; Dienel, 2009; Gjedde, 2002; Hertz, 2004; Hertz et al., 2007; Mangia et

Activation of glycolysis produces an increase in pyruvate and proton concentrations that are accompanied by a decrease in the NAD+/NADH ratio. Neuronal LDH1 should catalyze reduction of pyruvate to lactate. So, lactate utilization by neurons would only be possible if the consumption of glucose, either at the transport or the glycolysis stage, were inhibited. To explain this, at least four different (though coexistent) ideas have been proposed (Figure 10). Firstly, using real-time microscopy, glucose transport inhibition by glutamate has been demonstrated in neurons co-cultured with astrocytes (Porras et al., 2008). But because these experiments were performed with co-cultures, it is not possible to discard the possibility that astrocytic stimulation by glutamate may induce the release of substances which inhibit glucose uptake in neurons. In other words, it is possible that the effect of glutamate on neuronal glucose uptake may be indirect. The second mechanism is explained by the redox switch/redox coupling hypothesis (Cerdan et al., 2006). This idea embraces a mechanism in which two different pyruvate pools exist: one is thought to operate in exchange with extracellular lactate or pyruvate and the other, thought to be derived from glycolytic activity. This mechanism considers a lactate/pyruvate redox shuttle, which is able to transfer lactate from astrocytes to neurons. So, high cytosolic lactate inhibits neuronal glycolysis at the glyceraldehyde- 3-phosphate dehydrogenase (EC 1.2.1.12) step by competing with cytosolic NAD+, favoring oxidation of extracellular lactate. The third mechanism is our own theory. We have demonstrated that neuronal intracellular ascorbic acid inhibits glucose utilization in neurons (Castro et al., 2009) through GLUT3 inhibition (Figure 8, Beltrán et al., 2011). This mechanism is supported by the idea of ascorbic acid recycling in brain (see above, Figure 10, Astuya et al., 2005). Using primary cultures of cortical neurons treated with a specific shRNA (to block GLUT3 expression) and a fluorescent glucose analogue, we have shown that this transporter is important, using real-time experiments (Figure 7). At the same time, intracellular ascorbic acid is able to stimulate lactate transport in neurons and in cells that express GLUT3 (Figure 9, Castro et al., 2008). Because ascorbic acid is able to change metabolic substrate preferences, we have termed this mechanism the ascorbic acid metabolic switch (Castro et al., 2009). The fourth proposed mechanism relates to the inability of neuronal cells to overactivate glycolysis (Herrero-Mendez et al., 2009; Bolaños et al., 2010). This idea is supported by elegant experimental data that demonstrate that in neurons, it is not possible to produce the allosteric activator of PFK1 (and therefore glycolysis activator) fructose 2,6-bisphosphate. The enzyme that catalyzes fructose-2,6-bisphosphate in neurons, PFK3B, has a very short half-life as it is being constantly degraded (Herrero-Mendez et al., 2009). This mechanism does not exclude

al., 2003; Mangia et al., 2009).

basal glycolytic activity however.

Fig. 8. Intracellular ascorbic acid only inhibits transport of 2-NBDG in cells expressing GLUT3. A: Time course of uptake of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4 yl)amino]-2-deoxyglucose). 2-NBDG transport in cortical neurons in the presence (closed circles) or absence (open circles) of intracellular ascorbic acid. B: Time course of uptake of 2- NBDG transport in neuronal cells treated with shRNA to knock down GLUT3 expression in the presence (triangles) or absence (circles) of intracellular ascorbic acid. The data represent the mean ± SD of four experiments (14 control cells and 10 shRNA-treated cells). The images represent one control experiment and one experiment with shRNA-treated cells, in the presence or absence of ascorbic acid. Adapted from Beltran et al. (2011) with permission of the publisher.

#### **4. Metabolic failure in neurodegenerative diseases**

Neurodegenerative disease is a broad term for a range of conditions which primarily affect neurons in the brain. Progression of neurodegenerative diseases is accompanied by loss of neuronal cell structure and function and even cell death. These diseases have many similarities at a sub-cellular level including atypical protein assemblies, failure of normal protein degradation pathways, induced cell death, impaired axonal transport and metabolic failures (Rubinsztein et al., 2006; De Vos et al., 2008; Bredesen et al., 2006; Lin & Beal, 2006). A better knowledge of how these failures arise may offer fresh hope for development of therapies that improve treatment of these diseases.

Directly or indirectly, energy is necessary for many, if not all, cellular processes. It is thus possible to speculate that metabolic failure is an early event in neurodegenerative disease. Indeed, there are neurodegenerative diseases caused by a deficiency of metabolic enzymes. One of these is pyruvate dehydrogenase complex deficiency (Brown et al., 1994). This condition has similar characteristics to those of other neurodegenerative diseases. Several neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease and Parkinson's disease, show metabolic failure represented by altered patterns of expression of nutrient transporters, metabolic enzymes and molecular components of cellular respiration.

Brain Energy Metabolism in Health and Disease 349

Fig. 10. Neuron-glia metabolic coupling. During glutamatergic synaptic activity, increases in extracellular glutamate and K+ concentration are produced. This metabolically activates these cells (see Figure 7 for details of astrocytic metabolic activation). Astrocytes uptake glutamate and convert it into glutamine. Glutamine is released and taken up by neuronal cells to glutamate resynthesis (glutamate-glutamine shuttle). Metabolic activation of astrocytes produces an increase in glycolysis, glycogenolysis activities and lactate production. Lactate is taken up by neurons to support their energetic needs (astrocyteneuron lactate shuttle). Lactate uptake and lactate oxidation in neurons is possible because these cells are not able to activate glycolysis through fructose 2,6-bisphosphate production (see Figure 6 for details of metabolism in synaptically-active neuronal cells). Glutamate also stimulates ascorbic acid release form astrocytes. Neuronal cells take up ascorbic acid through SVCT2. Intracellular ascorbic acid inhibits glucose transport through GLUT3 inhibition and stimulates lactate transport (ascorbic acid metabolic switch). Synaptic activity is accompanied by production of oxidant species. Thus ascorbic acid is oxidized to reduce that species. Dehydroascorbic acid (oxidized ascorbic acid, Asc+) is released by neurons and taken up by astrocytes through GLUT1. Astrocytes reduce dehydroascorbic acid to ascorbic acid via gluthathione-dependent reductases (ascorbic acid recycling). Glu: glutathione, Gluc:

glucose, Gln: glutamine, Lac: lactate, Pyr: pyruvate.

Fig. 9. Intracellular ascorbic acid stimulates lactate uptake in cells expressing GLUT3. Substrate dependence for the inhibition of 0.1 mM lactate transport (10 s, 20 oC) by intracellular ascorbic acid (cells were preloaded with ascorbic acid at the concentrations indicated) in cultured cortical astrocytes (gray bars) and cultured cortical astrocytes expressing GLUT3-EGFP (black bars). Cortical astrocytes were transfected through electroporation. The data represent the mean ± SD of three experiments.

Metabolic failure is also represented by altered activities of the enzymes involved in energy metabolism. Mitochondria are the main platform for oxidative metabolism. They participate in cell metabolism, they produce ATP and they are important regulators of cytosolic calcium, which in turn, is related to whether or not programmed cell death occurs. Neurodegenerative diseases trigger programmed cell death and thus the role of mitochondria is key, from beginning to end of the disease, from the initial failure in energy metabolism to the later onset of cell death.

#### **4.1 Alzheimer's disease**

Alzheimer's disease is the most common cause of dementia. It is characterized by progressive cognitive dysfunction. The Alzheimer's disease brain appears atrophied. The temporal lobe, parietal lobe, frontal cortex and cingulate gyrus show degeneration and loss of cellular mass (Wenk, 2003). Parkinson's disease is characterized by loss of neurons and synapses in the cerebral cortex and in some subcortical regions. Genetically, Alzheimer's disease is heterogeneous and complex, showing no simple mode of inheritance. The causes of most Alzheimer's disease cases are still unknown, though in 1-5% of cases, genetic differences have been described. There are two neuropathological changes that can be correlated with an Alzheimer's disease diagnosis. First of all, neurofibrillary tangles (NFTs) accumulate in neuronal cytosol. And secondly, extracellular amyloid deposits appear in the form of senile plaques containing amyloid peptide (Duyckaerts et al., 2008).

Fig. 9. Intracellular ascorbic acid stimulates lactate uptake in cells expressing GLUT3. Substrate dependence for the inhibition of 0.1 mM lactate transport (10 s, 20 oC) by intracellular ascorbic acid (cells were preloaded with ascorbic acid at the concentrations indicated) in cultured cortical astrocytes (gray bars) and cultured cortical astrocytes expressing GLUT3-EGFP (black bars). Cortical astrocytes were transfected through

Metabolic failure is also represented by altered activities of the enzymes involved in energy metabolism. Mitochondria are the main platform for oxidative metabolism. They participate in cell metabolism, they produce ATP and they are important regulators of cytosolic calcium, which in turn, is related to whether or not programmed cell death occurs. Neurodegenerative diseases trigger programmed cell death and thus the role of mitochondria is key, from beginning to end of the disease, from the initial failure in energy

Alzheimer's disease is the most common cause of dementia. It is characterized by progressive cognitive dysfunction. The Alzheimer's disease brain appears atrophied. The temporal lobe, parietal lobe, frontal cortex and cingulate gyrus show degeneration and loss of cellular mass (Wenk, 2003). Parkinson's disease is characterized by loss of neurons and synapses in the cerebral cortex and in some subcortical regions. Genetically, Alzheimer's disease is heterogeneous and complex, showing no simple mode of inheritance. The causes of most Alzheimer's disease cases are still unknown, though in 1-5% of cases, genetic differences have been described. There are two neuropathological changes that can be correlated with an Alzheimer's disease diagnosis. First of all, neurofibrillary tangles (NFTs) accumulate in neuronal cytosol. And secondly, extracellular amyloid deposits appear in the

electroporation. The data represent the mean ± SD of three experiments.

form of senile plaques containing amyloid peptide (Duyckaerts et al., 2008).

metabolism to the later onset of cell death.

**4.1 Alzheimer's disease** 

Fig. 10. Neuron-glia metabolic coupling. During glutamatergic synaptic activity, increases in extracellular glutamate and K+ concentration are produced. This metabolically activates these cells (see Figure 7 for details of astrocytic metabolic activation). Astrocytes uptake glutamate and convert it into glutamine. Glutamine is released and taken up by neuronal cells to glutamate resynthesis (glutamate-glutamine shuttle). Metabolic activation of astrocytes produces an increase in glycolysis, glycogenolysis activities and lactate production. Lactate is taken up by neurons to support their energetic needs (astrocyteneuron lactate shuttle). Lactate uptake and lactate oxidation in neurons is possible because these cells are not able to activate glycolysis through fructose 2,6-bisphosphate production (see Figure 6 for details of metabolism in synaptically-active neuronal cells). Glutamate also stimulates ascorbic acid release form astrocytes. Neuronal cells take up ascorbic acid through SVCT2. Intracellular ascorbic acid inhibits glucose transport through GLUT3 inhibition and stimulates lactate transport (ascorbic acid metabolic switch). Synaptic activity is accompanied by production of oxidant species. Thus ascorbic acid is oxidized to reduce that species. Dehydroascorbic acid (oxidized ascorbic acid, Asc+) is released by neurons and taken up by astrocytes through GLUT1. Astrocytes reduce dehydroascorbic acid to ascorbic acid via gluthathione-dependent reductases (ascorbic acid recycling). Glu: glutathione, Gluc: glucose, Gln: glutamine, Lac: lactate, Pyr: pyruvate.

Brain Energy Metabolism in Health and Disease 351

complex, microtubules and endosomal compartments. Wild-type (normal) huntingtin has an important role in the intracellular transport of vesicles, organelles and traffic of proteins to the cell surface (Caviston et al., 2009). Expansion of a glutamine stretch within the Htt protein to more than 40 repeats appears to confer a dominant toxic property that is deleterious to neurons and detrimental to normal Htt biological activities. The precise

Huntington's disease is characterized by a failure in brain energy metabolism (Table 1). Defects in energy metabolism may even extend to presymptomatic subjects. Positron emission tomography studies have demonstrated marked reductions in glucose metabolism in the basal ganglia (Mazziotta et al., 1987), and in the cerebral cortex of symptomatic Huntington's disease patients (Leenders et al., 1986; Kuwert et al., 1993). However, Oláh and colleagues (2008) described an increase in several glycolytic enzymes and ATP production in brain from Huntington's disease animals. By contrast, the same authors described a marked decrease in glyceraldehyde-3-phosphate dehydrogenase activity. An impairment in enzyme activity of the TCA cycle (Lim et al., 2008) and oxidative phosphorylation has also been described. A key role for proliferator-activated receptor gamma coactivator-1 has been proposed in the control of energy metabolism in the early stages of Huntington's disease pathogenesis. Proliferatoractivated receptor gamma coactivator-1 is a transcriptional coactivator that regulates several metabolic processes, including mitochondrial biogenesis and oxidative phosphorylation (Finck & Kelly, 2006). Although the origin of metabolic impairment is unknown, it is proposed that a systematic downregulation of persoxisome proliferator-activated receptor- plays a critical role in the deregulation of energy homeostasis observed in Huntington's disease (Chiang et al., 2010). Metabolic failure in Huntington's disease not only occurs in brain. Htt is a ubiquitous protein and thus energetic failure may also be observed in peripheral tissues. Huntington's disease patients do show progressive weight loss and development of diabetes (Aziz et al., 2010). These symptoms may be related to an impairment of insulin secretion (Smith et al.,

Finally, oxidative damage has been shown (Mazziotta et al., 1987), as well as impaired SOD activity (Mazziotta et al., 1987) and impaired ascorbic acid homeostasis (Rebec et al., 1994) in Huntington's disease animal models. Our group has been making steady progress with respect to the function of ascorbic acid in neuronal metabolism (Castro et al., 2008; Castro et al., 2009; Beltrán et al., 2011, see above). Ascorbic acid, the reduced form of vitamin C,

Parkinson's disease is the most common neurodegenerative movement disorder of central nervous system. Clinical symptoms are due to the progressive degeneration of dopaminergic neurons in the substantia nigra and other monoaminergic neurons in the brainstem (Braak et al., 2003). The pathology of this disease is characterized by the accumulation of -synuclein into inclusions called Lewy bodies in neurons. Although Parkinson's disease is a sporadic condition of uncertain ethiology, there is some evidence that mitochondrial dysfunction considerably contributes to the pathogenesis of this disorder (Schapira, 2008). Mutations in several genes are found in Parkinson's disease. Point mutations, duplications and triplications in the -synuclein gene and mutations in the leucine-rich repeat kinase 2 (LRRK2). Mutations have also been described in genes coding

modulates neuronal metabolism between resting state and brain activation periods.

actions of the mutant huntingtin are still not clearly understood.

2009).

**4.3 Parkinson's disease** 

Type 2 diabetes mellitus appears to be a significant risk factor for Alzheimer's disease (Bosco et al., 2011). Alzheimer's disease is characterized by a significant and pre-symptomatic reduction of brain glucose utilization (Table 1, Ferreira et al., 2010). Indeed, it has been proposed that early diagnosis of Alzheimer's disease will be rendered possible through a combination of imaging modalities (such as *magnetic resonance imaging,* MRI and *positron emission tomography,* PET; Mosconi et al., 2005). There is a correlation between impairment of glucose cerebral metabolism, decreased GLUT expression and cerebral spinal fluid tau protein levels. (Liu et al., 2008; Cevarolo et al., 2008). Indeed, patients show decreased GLUT3 levels (Liu et al., 2008, Harr et al., 1995). A decrease in glycolytic enzyme levels and activity, PFK1 and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) has also been described. Decreased glycolysis could be related to an increase in glycogen synthesis. Pharmacological glycogen synthase kinase 3 inhibition improves behavioral dysfunction and decreases tau phosphorylation (Onishi et al., 2011). A spatial correlation between brain glycolysis without oxidative mitochondrial metabolism and amyloid deposition has been described (Vlassenko et al., 2010). Aggregate forms of amyloid can modify glucose metabolism and oxidative stress in astrocytes, affecting neuronal cells and probably contributing to neuronal death (Allaman et al., 2010). Indeed, reduction of GLUT1 and MCT expression, as well as the retraction of of astrocyte endfeet occurs in Alzheimer's disease producing uncoupling of the neurovascular unit (Merlini et al., 2011). The activity of PPP is increased in Alzheimer's disease. Increased oxidative stress markers in Alzheimer's disease samples correlated with enhanced PPP activity suggesting that PPP activity could play a role in the response against brain pro-oxidant activity in Alzheimer's disease. Finally, mitochondrial dysfunction (Schapira et al., 2006), decreased expression of oxidative phosphorylation and reduced activity of cytochrome oxidase were all observed in Alzheimer's disease.

#### **4.2 Huntington's disease**

Huntington's disease is a progressive, autosomal dominant, neurodegenerative disorder. It can affect individuals of all ages, from infancy to old age and develops over a period of 15- 20 years. Motor dysfunction and cognitive abnormalities are common symptoms. The disease is caused by an expanded polyglutamine (polyQ) stretch in the corresponding causal gene. Huntington's disease represents one of a growing number of polyQ repeat diseases that cause region-specific neuronal degeneration, including spinobulbar muscular atrophy and spinocerebellar ataxias (Pennuto et al., 2009). In Huntington's disease, polyQ expansion affects the *Huntingtin* gene, resulting in major cell loss in the striatum, a region of the basal ganglia that integrates cortical information for behavioral output. Huntington's disease is characterized by widespread neurodegeneration with preferential deterioration of medium-sized spiny neurons (MSSNs) in the striatum (Penney & Young, 1998). The major excitatory input to MSSNs comes from the cortex (corticostriatal pathway) and the thalamus. Huntington's disease also causes dysfunction and subsequent death of neurons in other brain regions, including the cortex.

The *Huntingtin g*ene codes for a protein called the huntingtin protein (Htt). It is a soluble 384 kDa protein, essential for embriogenesis, and ubiquitously expressed in moderate amounts in the nervous system as well as in other systems (Cattaneo et al., 2001). Htt is associated with various intracellular organelles, including the nucleus, endoplasmic reticulum, Golgi

Type 2 diabetes mellitus appears to be a significant risk factor for Alzheimer's disease (Bosco et al., 2011). Alzheimer's disease is characterized by a significant and pre-symptomatic reduction of brain glucose utilization (Table 1, Ferreira et al., 2010). Indeed, it has been proposed that early diagnosis of Alzheimer's disease will be rendered possible through a combination of imaging modalities (such as *magnetic resonance imaging,* MRI and *positron emission tomography,* PET; Mosconi et al., 2005). There is a correlation between impairment of glucose cerebral metabolism, decreased GLUT expression and cerebral spinal fluid tau protein levels. (Liu et al., 2008; Cevarolo et al., 2008). Indeed, patients show decreased GLUT3 levels (Liu et al., 2008, Harr et al., 1995). A decrease in glycolytic enzyme levels and activity, PFK1 and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) has also been described. Decreased glycolysis could be related to an increase in glycogen synthesis. Pharmacological glycogen synthase kinase 3 inhibition improves behavioral dysfunction and decreases tau phosphorylation (Onishi et al., 2011). A spatial correlation between brain glycolysis without oxidative mitochondrial metabolism and amyloid deposition has been described (Vlassenko et al., 2010). Aggregate forms of amyloid can modify glucose metabolism and oxidative stress in astrocytes, affecting neuronal cells and probably contributing to neuronal death (Allaman et al., 2010). Indeed, reduction of GLUT1 and MCT expression, as well as the retraction of of astrocyte endfeet occurs in Alzheimer's disease producing uncoupling of the neurovascular unit (Merlini et al., 2011). The activity of PPP is increased in Alzheimer's disease. Increased oxidative stress markers in Alzheimer's disease samples correlated with enhanced PPP activity suggesting that PPP activity could play a role in the response against brain pro-oxidant activity in Alzheimer's disease. Finally, mitochondrial dysfunction (Schapira et al., 2006), decreased expression of oxidative phosphorylation and reduced activity of cytochrome oxidase were all observed in

Huntington's disease is a progressive, autosomal dominant, neurodegenerative disorder. It can affect individuals of all ages, from infancy to old age and develops over a period of 15- 20 years. Motor dysfunction and cognitive abnormalities are common symptoms. The disease is caused by an expanded polyglutamine (polyQ) stretch in the corresponding causal gene. Huntington's disease represents one of a growing number of polyQ repeat diseases that cause region-specific neuronal degeneration, including spinobulbar muscular atrophy and spinocerebellar ataxias (Pennuto et al., 2009). In Huntington's disease, polyQ expansion affects the *Huntingtin* gene, resulting in major cell loss in the striatum, a region of the basal ganglia that integrates cortical information for behavioral output. Huntington's disease is characterized by widespread neurodegeneration with preferential deterioration of medium-sized spiny neurons (MSSNs) in the striatum (Penney & Young, 1998). The major excitatory input to MSSNs comes from the cortex (corticostriatal pathway) and the thalamus. Huntington's disease also causes dysfunction and subsequent death of neurons in

The *Huntingtin g*ene codes for a protein called the huntingtin protein (Htt). It is a soluble 384 kDa protein, essential for embriogenesis, and ubiquitously expressed in moderate amounts in the nervous system as well as in other systems (Cattaneo et al., 2001). Htt is associated with various intracellular organelles, including the nucleus, endoplasmic reticulum, Golgi

Alzheimer's disease.

**4.2 Huntington's disease** 

other brain regions, including the cortex.

complex, microtubules and endosomal compartments. Wild-type (normal) huntingtin has an important role in the intracellular transport of vesicles, organelles and traffic of proteins to the cell surface (Caviston et al., 2009). Expansion of a glutamine stretch within the Htt protein to more than 40 repeats appears to confer a dominant toxic property that is deleterious to neurons and detrimental to normal Htt biological activities. The precise actions of the mutant huntingtin are still not clearly understood.

Huntington's disease is characterized by a failure in brain energy metabolism (Table 1). Defects in energy metabolism may even extend to presymptomatic subjects. Positron emission tomography studies have demonstrated marked reductions in glucose metabolism in the basal ganglia (Mazziotta et al., 1987), and in the cerebral cortex of symptomatic Huntington's disease patients (Leenders et al., 1986; Kuwert et al., 1993). However, Oláh and colleagues (2008) described an increase in several glycolytic enzymes and ATP production in brain from Huntington's disease animals. By contrast, the same authors described a marked decrease in glyceraldehyde-3-phosphate dehydrogenase activity. An impairment in enzyme activity of the TCA cycle (Lim et al., 2008) and oxidative phosphorylation has also been described. A key role for proliferator-activated receptor gamma coactivator-1 has been proposed in the control of energy metabolism in the early stages of Huntington's disease pathogenesis. Proliferatoractivated receptor gamma coactivator-1 is a transcriptional coactivator that regulates several metabolic processes, including mitochondrial biogenesis and oxidative phosphorylation (Finck & Kelly, 2006). Although the origin of metabolic impairment is unknown, it is proposed that a systematic downregulation of persoxisome proliferator-activated receptor- plays a critical role in the deregulation of energy homeostasis observed in Huntington's disease (Chiang et al., 2010). Metabolic failure in Huntington's disease not only occurs in brain. Htt is a ubiquitous protein and thus energetic failure may also be observed in peripheral tissues. Huntington's disease patients do show progressive weight loss and development of diabetes (Aziz et al., 2010). These symptoms may be related to an impairment of insulin secretion (Smith et al., 2009).

Finally, oxidative damage has been shown (Mazziotta et al., 1987), as well as impaired SOD activity (Mazziotta et al., 1987) and impaired ascorbic acid homeostasis (Rebec et al., 1994) in Huntington's disease animal models. Our group has been making steady progress with respect to the function of ascorbic acid in neuronal metabolism (Castro et al., 2008; Castro et al., 2009; Beltrán et al., 2011, see above). Ascorbic acid, the reduced form of vitamin C, modulates neuronal metabolism between resting state and brain activation periods.

#### **4.3 Parkinson's disease**

Parkinson's disease is the most common neurodegenerative movement disorder of central nervous system. Clinical symptoms are due to the progressive degeneration of dopaminergic neurons in the substantia nigra and other monoaminergic neurons in the brainstem (Braak et al., 2003). The pathology of this disease is characterized by the accumulation of -synuclein into inclusions called Lewy bodies in neurons. Although Parkinson's disease is a sporadic condition of uncertain ethiology, there is some evidence that mitochondrial dysfunction considerably contributes to the pathogenesis of this disorder (Schapira, 2008). Mutations in several genes are found in Parkinson's disease. Point mutations, duplications and triplications in the -synuclein gene and mutations in the leucine-rich repeat kinase 2 (LRRK2). Mutations have also been described in genes coding

Brain Energy Metabolism in Health and Disease 353

for Parkin, DJ-1, PINK1 and ATP13A2 (Biskup et al., 2008). The exact role of -synuclein remains unknown, but it is thought to participate in maintenance of vesicle pools. Parkin directs ubiquitination of -synuclein (Haywood & Staveley, 2004). Ubiquitin tagged synuclein is directed to proteosome and degraded. The parkin protein thus promotes Parkinson's disease by enhancing the failure in normal protein degradation. Mutations in PINK1 and DJ1 genes are related to mitochondrial dysfunction in Parkinson's disease. Oxidative stress also occurs in Parkinson's disease. Dopamine metabolism produces oxidant species. In this way, the -synuclein mutation contributes to oxidative stress because in the presence of mutated -synuclein, dopamine remains in the cytosol instead of being loaded

A hypometabolism of glucose has been described in Parkinson's disease patients (Huang et al., 2007; Yong et al., 2007; Perneczy et al., 2008; Hosokai et al., 2009; Lee et al., 2008; Lee et al., 2010; Bohnen et al., 2011; Borghammer et al., 2011). However, there is only limited molecular evidence to support this feature. Defects in oxidative phophorylation with a decreased activity of complex I have been reported (Navarro et al., 2009; Hattingen et al., 2009). This evidence leads us to speculate that in Parkinson's disease, mitochondrial dysfunction by redox imbalance precedes metabolic failure. However, a role for glycogen synthase kinase 3-3, an enzyme participating in glycogen metabolism (see below) has been proposed in Parkinson's disease pathogenesis (as for Alzheimer's disease pathogenesis,

The metabolic cost of brain activity is high. Neuronal activity accounts for 80% of brain energy consumption. Glucose is an essential energy source for the adult human brain. It can be used to obtain energy and to produce metabolic intermediaries for biosynthesis of compound of biological interest. Glucose can also be stored in brain, in the form of glycogen. Glucose oxidation via glycolysis occurs in all brain cells. However, the ability of neuronal cells to activate this metabolic pathway is poor. Astrocytic glycolysis and glycogenolysis is activated in response to synaptic activation with the subsequent formation of lactate. Thus, during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The energetic coupling between neuronal and astroglial cells is essential to meet energy brain needs in an efficient way. Astrocytes interact with each other and with endothelial cells from the blood brain barrier and smooth muscle cells surrounding arterioles. Under fasting conditions, during intense exercise and in suckling infants, ketone bodies are a significant source of energetic fuel for brain. Metabolic failure has been described in several neurodegenerative diseases. These diseases present atypical protein assemblies, failures in normal protein degradation pathways, induced cell death, impaired axonal transport and metabolic failures. Almost all cellular processes need energy. It is thus possible that metabolic failure is an early event in these pathologies. This hypothesis is supported by evidence that shows metabolic failure in pre-symptomatic patients suffering

We gratefully acknowledge the helpful suggestions of T. Valencia and S. Brauchi. This work

from diseases such as Alzheimer's disease and Huntington's disease.

was supported by Chilean grant FONDECYT 1110571.

into synaptic vesicles (Henchcliffe, 2008).

Nagao et al., 2008; Garcia-Gorotiaga et al., 2009).

**5. Conclusions** 

**6. Acknowledgements** 


Table 1. Metabolic failures in neurodegenerative diseases.

Browen et al., 1979

Villain, 2010.

1994;

Gamberino & Brennan,

Gourfinkel-An et al., 2002; Gu et al., 1996; Hult et al., 2011; Kuwert et al., 1990; Kuwert et al., 1993;

Leenders et al., 1986; Lim et al., 2007; Mann et al., 1990; Martin et al., 1992; Mazziotta et al., 1987;

Bohnen et al., 2011; Borghammer et al., 2011 Brownell et al., 2003; Feng et al., 2008; Hattingen et al., 2009; Hou et al., 2010; Hyu Lee et al., 2009; Lee et al., 2010; Lyoo et al., 2008; Perneczky et al., 2008; Schapira et al., 1989.

Ferreira et al., 2010; Harr et al., 1995; Hooijimans et al., 2007; Horwood & Davies, 1994; Kalaria & Harik,1989;; Langbaum et al., 2009; Liu et al., 2008; Merlini et al., 2011; Mooradian et al., 1997;; Nicholson et al., 2010; Ojaimi et al., 1999; Parker et al., 1994; Schapira et al., 2006; Shima et al., 2011; Simpson et al., 1994; Sims et al., 1983; Sorbi et al., 1983;

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Table 1. Metabolic failures in neurodegenerative diseases.

Alzheimer's Disease (AD)

Huntington's disease (HD)

Parkinson's disease (PD) for Parkin, DJ-1, PINK1 and ATP13A2 (Biskup et al., 2008). The exact role of -synuclein remains unknown, but it is thought to participate in maintenance of vesicle pools. Parkin directs ubiquitination of -synuclein (Haywood & Staveley, 2004). Ubiquitin tagged synuclein is directed to proteosome and degraded. The parkin protein thus promotes Parkinson's disease by enhancing the failure in normal protein degradation. Mutations in PINK1 and DJ1 genes are related to mitochondrial dysfunction in Parkinson's disease. Oxidative stress also occurs in Parkinson's disease. Dopamine metabolism produces oxidant species. In this way, the -synuclein mutation contributes to oxidative stress because in the presence of mutated -synuclein, dopamine remains in the cytosol instead of being loaded into synaptic vesicles (Henchcliffe, 2008).

A hypometabolism of glucose has been described in Parkinson's disease patients (Huang et al., 2007; Yong et al., 2007; Perneczy et al., 2008; Hosokai et al., 2009; Lee et al., 2008; Lee et al., 2010; Bohnen et al., 2011; Borghammer et al., 2011). However, there is only limited molecular evidence to support this feature. Defects in oxidative phophorylation with a decreased activity of complex I have been reported (Navarro et al., 2009; Hattingen et al., 2009). This evidence leads us to speculate that in Parkinson's disease, mitochondrial dysfunction by redox imbalance precedes metabolic failure. However, a role for glycogen synthase kinase 3-3, an enzyme participating in glycogen metabolism (see below) has been proposed in Parkinson's disease pathogenesis (as for Alzheimer's disease pathogenesis, Nagao et al., 2008; Garcia-Gorotiaga et al., 2009).
