**3. Pathways related to energy metabolism in BBB and brain: Lactate shuttle: NALS or ALNS?**

94 Dehydrogenases

a prompt BBB response.

surrounding the neurons.

**Figure 2.** Effect of flow on BBB glucose metabolism. Comparative analysis of the expression level of key enzymes regulating the glycolytic and TCA pathways strongly supported the gene array data (Panel A). Note that the lactate production/glucose consumption ratio measured in the flow-exposed in vitro BBB modules was 1. Complete anaerobic metabolism would produce 2 lactates/glucose (ratio = 2) thus, indicating that at least 50% of the glucose consumed underwent aerobic metabolism (Panel **B**).

The BBB's ability in maintaining an optimal bioenergetics level at all time is thus crucial to meet the energy demand required by its multiple proprietary functions under normal as well as pathological conditions (e.g., cerebral ischemia). Energetic pathways (such as glycolysis and the tricarboxylic acid cycle -TCA cycle) work in an integrated yet independent manner in the BBB endothelium to respond to physiological (e.g., increased CNS demand in response to changes in neuronal activity) or pathological events that require

The brain on the other hand possesses an incredibly more complex physiology than the BBB vasculature. It is the control center of neuronal as well as hormonal signaling. It is crucial for various functions such as homeostasis, behavior, perception and processing of information, motor control and memory formation. From a physiological stand point, brain functions depend on the ability of neurons to transmit and respond to electrochemical signals. This complex crosstalk is controlled by a wide variety of biochemical and metabolic processes which involve interactions between neurotransmitters and receptors that take place at the synapses. Crucial to this function is the necessity to sustain the bioenergetic demand required to maintain optimal neuronal activity (e.g., generation of action potentials, release of neurotransmitters, restoration of the membrane polarization following an action potential etc). In this respect, the BBB endothelium and glial cells play a major role in brain metabolism, by controlling the influx and distribution of nutrients (e.g., glucose and lactate shuttles as described later) as well as the chemical composition of the extracellular fluids Before we begin describing the role of DHOs in energy metabolism, it is imperative to understand the various metabolic pathways in the brain involved in energy production. Glucose is one of the primary fuel sources available to the brain. 25% of the total glucose intake is utilized by the brain despite accounting for 2% of the total body mass (24).

Glucose enters the glycolytic pathway to produce pyruvate along with net production of 2ATP and 2NADH (reduced form of nicotinamide adenine dinucleotide) (see **Figure 3**). Ten intermediate reactions occur until pyruvate is formed in the last step. ATP is first used up in the first part of glycolysis (until formation of glyceraldehyde phosphate-GAP) and is produced later during the second half. The total net ATP gain for each glycolytic cycle is 2 ATP molecules. Another important step of glycolysis involving glyceraldehyde phosphate dehydrogenase (GAPDH), is the conversion of GAP to 1,3-bisphosphate glycerate (1,3- BPG) along with NADH formation. Part of the 1,3-BPG thus formed can be further converted into 2,3-biphosphate glycerate (2,3-BPG) which can bind to hemoglobin enhancing its deoxygenation. The remaining 1,3-BPG undergoes further conversion along the glycolytic pathways and is finally converted into pyruvate.

Pyruvate thus formed can either enter the citric acid chain (or Kreb's cycle or Tricyclic acid cycle-TCA) or get converted to lactate which represents the end product of glycolysis. Pyruvate to lactate conversion is the last step of anaerobic form of respiration which occurs via lactate dehydrogenase and results in 2 molecules of ATP production.

Conversely, pyruvate can be further converted into acetyl co-A and then enter the TCA cycle. This intermediate reaction is crucial in linking glycolysis to the TCA. In this step, pyruvate dehydrogenase (PDH) decarboxylates pyruvate to its acetyl form along with addition of co-enzyme A. Acetyl co-A then combines with oxaloacetate (4C) in presence of water molecule to form citrate (6C). The citrate thus formed cycles through TCA forming oxaloacetate in the last step, which can re-enter the cycle reacting with a new acetyl co-A (see **Figure 3**). The various steps in the TCA cycle involve various oxidation reactions involving various dehydrogenases such as isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase. Each of these reactions lead to formation of NADH (except for FADH2 i.e. reduced flavin adenine dinucleotide formation during conversion of succinate to fumarate). These reducing equivalents can then enter the electron transport chain and result in ATP formation or can be used by the cell to counteract the oxidative stress caused by reactive oxygen species (ROS) and free radicals. A complete cycling of two molecules of acetyl co-A (from a single molecule of glucose) results in the production of approximately 10 NADH and 2 FADH2. The complete metabolic conversion of a glucose molecule into water and CO2 (glycolysis and TCA cycles combined) results in the approximate production of 38 ATP molecules (including ATP formed indirectly through NADH and FADH2 which produce 3 and 2 ATP molecules respectively).

Role and Function of Dehydrogenases in CNS and Blood-Brain Barrier Pathophysiology 97

various cell types in the brain, be converted back into pyruvate and be fully reutilized through complete aerobic respiration. Although this concept of lactate shuttle was proposed long ago, it received lot of resistance from the scientific community especially from those who believed that glucose is the sole substrate in the brain (30;31). However now, it is getting accepted that lactose is also used as a main substrate for energy production under normal conditions (32-34). The direction of lactate flow between various cell types in the brain as well as its relative contribution with respect to glucose to the overall energy production is still debatable and not yet fully understood. As far as the lactate flow between the different cell types in the brain there are two current schools of thoughts. One supporting the Astrocyte-lactate-neuron shuttle (ALNS) (33;35-37) and the second

Classically, glucose is transported from the cerebral blood flow into the neurons or astrocytes via glucose transporters such as Glut-1 and Glut-3 respectively (see **Figure 4**). This glucose is then metabolized to produce ATP through complete aerobic respiration (glycolysis and citric acid pathways). In the astrocytes, part of the glucose also gets converted into glycogen, as an energy reserve to be used under critical conditions of low

Astrocytes can withstand low oxygen tension for a longer period of time than neurons and have proven to be more resilient to hypoxic insults (7;41-43). Based on the concept of lactate shuttle, at the astrocytic level (under resting conditions), glucose can be converted to lactate. Lactate thus produced can shuttle first into the interstitial fluid (through monocarboxylate transporter –MCT-1 & 4). From there lactate can be influxed into neurons by the neuronal specific MCT-2 transporter and converted back into its pyruvate form ready to undertake complete aerobic metabolism (36;44;45). This route is known as the Astrocyte-lactate-neuron

Continuous glutamatergic activation of neurons results in a more exhaustive energy expenditure. As pyruvate utilization during the TCA cycle increases and its cytoplasmic levels decrease correspondingly, the condition becomes favorable for increasing both glucose and lactate utilization. By the late phase of activation, glutamate released is taken up by the astrocytes for recycling. Aerobic glycolysis is enhanced in the astrocytes with increased lactate production. The lactate thus formed helps sustain the energy demands of astrocytes as well as replenishing the neurons. During intense and prolonged neuronal stimulation which may occur under certain conditions, energy replenishment becomes crucially important. This is because the continuous glutamate reuptake by the Na+, glutamate co-transporter in astrocytes (GLutamate ASpartate Transporter – GLAST; glutamate transporter 1 –GLT1) must be paired with an equivalent intense activity of the Na+, K+ATPase to efflux the Na+ back in the extracellular space thus continuing the cycle. When the extracellular glucose levels become insufficient to sustain this level of activity then glycogen stored in the astrocyte is mobilized to provide the extra glycosyl units necessary to support the cellular activity. Thus sustained activation of the neurons results in conversion

advocating a Neuron-Lactate-Astrocyte-shuttle (NLAS) (38-40).

oxygen supply.

shuttle (ALNS shuttle).

**Figure 3.** Enzymatic pathways of glycolysis and Kreb's cycle. Note the DHOs involved in the different metabolic pathways. (glycolysis and citric acid cycles).

It has been an age old concept that glucose is the major energy source to maintain brain functions and that lactate does not provide further metabolic use for the neuronal activities (25-28). However, it is now accepted that lactate can be produced in the brain under aerobic conditions, this is known as aerobic glycolysis. Aerobic glycolysis under normal conditions contributes to about 10% of the total energy production in the brain, which can increase under ischemic conditions(29). Lactate thus formed can then be shuttled between the various cell types in the brain, be converted back into pyruvate and be fully reutilized through complete aerobic respiration. Although this concept of lactate shuttle was proposed long ago, it received lot of resistance from the scientific community especially from those who believed that glucose is the sole substrate in the brain (30;31). However now, it is getting accepted that lactose is also used as a main substrate for energy production under normal conditions (32-34). The direction of lactate flow between various cell types in the brain as well as its relative contribution with respect to glucose to the overall energy production is still debatable and not yet fully understood. As far as the lactate flow between the different cell types in the brain there are two current schools of thoughts. One supporting the Astrocyte-lactate-neuron shuttle (ALNS) (33;35-37) and the second advocating a Neuron-Lactate-Astrocyte-shuttle (NLAS) (38-40).

96 Dehydrogenases

**Figure 3.** Enzymatic pathways of glycolysis and Kreb's cycle. Note the DHOs involved in the different

It has been an age old concept that glucose is the major energy source to maintain brain functions and that lactate does not provide further metabolic use for the neuronal activities (25-28). However, it is now accepted that lactate can be produced in the brain under aerobic conditions, this is known as aerobic glycolysis. Aerobic glycolysis under normal conditions contributes to about 10% of the total energy production in the brain, which can increase under ischemic conditions(29). Lactate thus formed can then be shuttled between the

metabolic pathways. (glycolysis and citric acid cycles).

Classically, glucose is transported from the cerebral blood flow into the neurons or astrocytes via glucose transporters such as Glut-1 and Glut-3 respectively (see **Figure 4**). This glucose is then metabolized to produce ATP through complete aerobic respiration (glycolysis and citric acid pathways). In the astrocytes, part of the glucose also gets converted into glycogen, as an energy reserve to be used under critical conditions of low oxygen supply.

Astrocytes can withstand low oxygen tension for a longer period of time than neurons and have proven to be more resilient to hypoxic insults (7;41-43). Based on the concept of lactate shuttle, at the astrocytic level (under resting conditions), glucose can be converted to lactate. Lactate thus produced can shuttle first into the interstitial fluid (through monocarboxylate transporter –MCT-1 & 4). From there lactate can be influxed into neurons by the neuronal specific MCT-2 transporter and converted back into its pyruvate form ready to undertake complete aerobic metabolism (36;44;45). This route is known as the Astrocyte-lactate-neuron shuttle (ALNS shuttle).

Continuous glutamatergic activation of neurons results in a more exhaustive energy expenditure. As pyruvate utilization during the TCA cycle increases and its cytoplasmic levels decrease correspondingly, the condition becomes favorable for increasing both glucose and lactate utilization. By the late phase of activation, glutamate released is taken up by the astrocytes for recycling. Aerobic glycolysis is enhanced in the astrocytes with increased lactate production. The lactate thus formed helps sustain the energy demands of astrocytes as well as replenishing the neurons. During intense and prolonged neuronal stimulation which may occur under certain conditions, energy replenishment becomes crucially important. This is because the continuous glutamate reuptake by the Na+, glutamate co-transporter in astrocytes (GLutamate ASpartate Transporter – GLAST; glutamate transporter 1 –GLT1) must be paired with an equivalent intense activity of the Na+, K+ATPase to efflux the Na+ back in the extracellular space thus continuing the cycle. When the extracellular glucose levels become insufficient to sustain this level of activity then glycogen stored in the astrocyte is mobilized to provide the extra glycosyl units necessary to support the cellular activity. Thus sustained activation of the neurons results in conversion of the stored energy substrate glycogen to glucose and further lactate production for shuttling to the neurons. This concept of ALNS shuttle during activation has received opposition and few groups suggest that lactate is primarily produced by the neurons and is then transported from the neurons to the astrocytes (38;40).

Role and Function of Dehydrogenases in CNS and Blood-Brain Barrier Pathophysiology 99

In summary at the microcapillary level, the BBB acts as a functional interface which is charged with the critical task to fuel the brain with energy sources. Whether this is glucose or glucose-derived lactate the BBB is the main fuel distribution system to the brain and a safe for energy storage to which the brain can avail when normal fuel supplies are short.

Mutation in single gene leads to enzyme deficiency that results in type of genetic disorders termed as inborn error in metabolism (IEM) (46-50). IEM related deficiencies are generally autosomal recessive or X-linked. IEM linked enzyme deficiencies may be directly related to metabolic pathways of energy metabolism, purines/pyrimidine synthesis or degradation, amino acid synthesis, fatty acid oxidation, etc. Metabolic errors lead to accumulation of toxic or absence of essential products in the brain with neurological implications such as ataxiamotor control, encephalopathy, mental deficits, learning disabilities and mental retardation with structural anomalies. Some examples of DHOs involved include the pyruvate

Pyruvate dehydrogenase is a multi-enzyme complex which catalyzes the conversion of pyruvate (the end product of glycolysis) into acetyl-coA- a substrate that can enter citric acid cycle (for production of ATP and energy equivalents). PDH is a six subunit complex composed of E1-pyruvate dehydrogenase, E2-dihydrolipoyl transacetylase and E3 dihydrolipoyl dehydrogenase, E3BP- E3 binding protein and two regulatory subunits pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. Although several mutations in the PDH complex deficiency (such as point mutations, deletions, duplications) have been reported so far; deficiency in PDH E1-alpha subunit (abbreviated as PDHA1) is the most common type (51-53). PDH deficiencies due to mutations in other subunits of the PDH complex are comparatively rare. All mutations leading to PDH deficiency are X linked except, the one in regulatory units, which are autosomally recessive (54-57). Since PDH results in acetyl co-A formation, the most common clinical manifestation of PDH deficiency is severe lactic acidosis. Defects in energy metabolism can cause neurological deficits such as mental retardation, developmental delay as well as psychomotor retardation. Hypertonia/hypotonia, ataxia, motor dysfunction like spasticity are the more common symptoms observed (58). Structural anomalies (such as microcephaly, facial dysmorphism) and epilepsy (focal or generalized seizures- both have been reported) may develop *in utero*. Optic atrophy, nystagmus and strabismus are observed at ocular level whereas peripheral neuropathies such as in nerve conduction have also been reported.

Based on clinical case studies, PDH deficiencies have been classified into four typical

i. Neonatal encephalopathic pattern with facial dimorphic features and cerebral

ii. Leigh syndrome like presentation with symmetric necrotic lesions of basal ganglia,

developmental defects, prenatal brain lesions, affecting the females

**4. IEM** 

dehydrogenase complex.

neurological patterns (58-60):

more common in males

**4.1. Pyruvate dehydrogenase (PDH) deficiency** 

**Figure 4.** Schematic of neuroenergetics pathways: The astrocyte – neuron lactate shuttle.

In summary whether lactate flux moves from astrocytes to neurons or vice-versa the important point is that glucose is not the sole substrate utilized in the brain. Lactate plays an equally important role especially during activation, neurotransmission and pathological conditions such as under ischemic insults. This further emphasizes the still dismissed importance of lactate dehydrogenase which represents the key switch in the metabolic pathway of glucose in the generation of lactate.

In summary at the microcapillary level, the BBB acts as a functional interface which is charged with the critical task to fuel the brain with energy sources. Whether this is glucose or glucose-derived lactate the BBB is the main fuel distribution system to the brain and a safe for energy storage to which the brain can avail when normal fuel supplies are short.
