**2. Structural and functional complexity of blood brain barrier (BBB) and cerebral physiology – A need for high energy**

Role and Function of Dehydrogenases in CNS and Blood-Brain Barrier Pathophysiology 93

acotinase, isocitrate dehydrogenase, succinate dehydrogenase were upregulated. These results clearly emphasize how altered rheological conditions (e.g., hypoperfusion and ischemia) may impact the BBB bioenergetic metabolism and how the BBB is well equipped

**Figure 1.** Schematic representation of the brain microvasculature in relation to the brain. Note how the blood vessels start branching in small capillaries while the pia disappears and the endothelium acquires the peculiar characteristics of a tight barrier that regulate the exchange of substances between blood and brain. TJ between adjacent endothelial cells form a diffusion barrier that selectively excludes most blood-borne and xenobiotic substances from entering the brain. In contrast to lipid soluble substances including alcohols, anesthetics and barbiturates, the BBB is highly impermeant to polar molecules or water soluble electrolytes. However, the passage of certain water soluble, but biologically important substances, such as D-glucose or phenylamine are regulated by a variety of specific carrier-mediated transport systems. By contrast, larger vessels (arterioles, small arteries and venous) differ from capillaries by the presence of smooth muscle cells in their walls and a less stringent vascular bed.

to respond to such changes (see **Figure 2**).

The BBB is a dynamic interface between the peripheral blood and the brain which controls the influx and efflux of substrates and metabolites necessary for normal neuronal function (see **Figure 1**). The BBB is crucial in protecting the brain from harmful substances both endogenous and exogenous in nature. Any alteration in normal BBB functions can play a central role in the pathogenesis and progression of broad variety of CNS disorders such as multiple sclerosis, Alzheimer's disease, neoplasia, hypertension, dementia, epilepsy, infection and trauma (1-4).

At the cellular level, the BBB consists of microvascular endothelial cells (ECs) lining the brain microvessels together with closely associated astrocytic end-feet processes and pericytes (5-8). These associated cells play a major role in EC differentiation and acquisition of morphological and functional characteristics unique to the BBB. At the cellular level, the brain microcapillary endothelium is characterized by the presence of tight junctions (TJ), lack of fenestrations, and minimal pinocytotic vesicles (9;10). In particular, TJs between the cerebral endothelial cells form a diffusion barrier, which selectively excludes most of the blood-borne substances from entering the brain, protecting it from systemic influences mediated by substances which are primarily polar in nature (such as electrolytes). Transport of nutrients (as well as other biologically important substances) from the peripheral circulation into brain parenchyma requires translocation through the capillary endothelium by specialized carrier-mediated transport systems. On the other hand, potentially harmful substances that are lipid soluble are discharged back into the cerebral circulation. This is mediated by specialized active efflux systems belonging to the ATP-binding cassette transporters (ABC-transporter) superfamily (such as P-glycoprotein (P-gp) and Multidrug resistance Protein (MRP)) (11;12). These efflux pumps rely heavily on adenosine triphosphate (ATP) as fuel source. Apart from these ATP dependent pumps, energy independent transporters such as organic ion carriers add to the complexity of BBB transport functions (13). Simultaneously, intake of essential nutrients such as glucose, amino acids, peptides, choline occurs through carrier mediated mechanisms (13-17). Topographic membrane localization of these transporters is indicative of the polarity of the endothelial functions and differentiation that sets apart the BBB endothelium from other vascular beds. The BBB endothelial cytoplasm is richly endowed with enzymes(18) including adenosine triphosphatase, acid and alkaline phosphatases, Na+/K+/ATPase, monoamine oxidase, cytochrome p450s and various dehydrogenases (19-22). The BBB ECs are also characterized by very high density of mitochondria denoting high metabolic activity (23) to support all the specialized cellular activities bestowed upon these highly specialized cells. In addition, previous work from our group has shown that blood flow can modulate the bioenergetic behavior of the BBB endothelial cells favoring the expression of the key metabolic enzyme pyruvate dehydrogenase (switch controller from anaerobic to aerobic pathway) (19). Contrarily, the RNA level of lactate dehydrogenase (switch controller from aerobic to anaerobic pathway) showed decreased expression. In parallel, TCA dehydrogenases such as acotinase, isocitrate dehydrogenase, succinate dehydrogenase were upregulated. These results clearly emphasize how altered rheological conditions (e.g., hypoperfusion and ischemia) may impact the BBB bioenergetic metabolism and how the BBB is well equipped to respond to such changes (see **Figure 2**).

92 Dehydrogenases

infection and trauma (1-4).

**2. Structural and functional complexity of blood brain barrier (BBB) and** 

The BBB is a dynamic interface between the peripheral blood and the brain which controls the influx and efflux of substrates and metabolites necessary for normal neuronal function (see **Figure 1**). The BBB is crucial in protecting the brain from harmful substances both endogenous and exogenous in nature. Any alteration in normal BBB functions can play a central role in the pathogenesis and progression of broad variety of CNS disorders such as multiple sclerosis, Alzheimer's disease, neoplasia, hypertension, dementia, epilepsy,

At the cellular level, the BBB consists of microvascular endothelial cells (ECs) lining the brain microvessels together with closely associated astrocytic end-feet processes and pericytes (5-8). These associated cells play a major role in EC differentiation and acquisition of morphological and functional characteristics unique to the BBB. At the cellular level, the brain microcapillary endothelium is characterized by the presence of tight junctions (TJ), lack of fenestrations, and minimal pinocytotic vesicles (9;10). In particular, TJs between the cerebral endothelial cells form a diffusion barrier, which selectively excludes most of the blood-borne substances from entering the brain, protecting it from systemic influences mediated by substances which are primarily polar in nature (such as electrolytes). Transport of nutrients (as well as other biologically important substances) from the peripheral circulation into brain parenchyma requires translocation through the capillary endothelium by specialized carrier-mediated transport systems. On the other hand, potentially harmful substances that are lipid soluble are discharged back into the cerebral circulation. This is mediated by specialized active efflux systems belonging to the ATP-binding cassette transporters (ABC-transporter) superfamily (such as P-glycoprotein (P-gp) and Multidrug resistance Protein (MRP)) (11;12). These efflux pumps rely heavily on adenosine triphosphate (ATP) as fuel source. Apart from these ATP dependent pumps, energy independent transporters such as organic ion carriers add to the complexity of BBB transport functions (13). Simultaneously, intake of essential nutrients such as glucose, amino acids, peptides, choline occurs through carrier mediated mechanisms (13-17). Topographic membrane localization of these transporters is indicative of the polarity of the endothelial functions and differentiation that sets apart the BBB endothelium from other vascular beds. The BBB endothelial cytoplasm is richly endowed with enzymes(18) including adenosine triphosphatase, acid and alkaline phosphatases, Na+/K+/ATPase, monoamine oxidase, cytochrome p450s and various dehydrogenases (19-22). The BBB ECs are also characterized by very high density of mitochondria denoting high metabolic activity (23) to support all the specialized cellular activities bestowed upon these highly specialized cells. In addition, previous work from our group has shown that blood flow can modulate the bioenergetic behavior of the BBB endothelial cells favoring the expression of the key metabolic enzyme pyruvate dehydrogenase (switch controller from anaerobic to aerobic pathway) (19). Contrarily, the RNA level of lactate dehydrogenase (switch controller from aerobic to anaerobic pathway) showed decreased expression. In parallel, TCA dehydrogenases such as

**cerebral physiology – A need for high energy** 

**Figure 1.** Schematic representation of the brain microvasculature in relation to the brain. Note how the blood vessels start branching in small capillaries while the pia disappears and the endothelium acquires the peculiar characteristics of a tight barrier that regulate the exchange of substances between blood and brain. TJ between adjacent endothelial cells form a diffusion barrier that selectively excludes most blood-borne and xenobiotic substances from entering the brain. In contrast to lipid soluble substances including alcohols, anesthetics and barbiturates, the BBB is highly impermeant to polar molecules or water soluble electrolytes. However, the passage of certain water soluble, but biologically important substances, such as D-glucose or phenylamine are regulated by a variety of specific carrier-mediated transport systems. By contrast, larger vessels (arterioles, small arteries and venous) differ from capillaries by the presence of smooth muscle cells in their walls and a less stringent vascular bed.

Role and Function of Dehydrogenases in CNS and Blood-Brain Barrier Pathophysiology 95

**3. Pathways related to energy metabolism in BBB and brain: Lactate** 

intake is utilized by the brain despite accounting for 2% of the total body mass (24).

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

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

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

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

via lactate dehydrogenase and results in 2 molecules of ATP production.

**shuttle: NALS or ALNS?** 

pathways and is finally converted into pyruvate.

molecules respectively).

**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 a prompt BBB response.

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 surrounding the neurons.
