Biochemistry of the Nervous System Transport through BBB

Biochemistry of the Nervous System Transport through BBB Metabolism of Neurotransmitters Metabolism of CNS Biochemical Aspects of CNS diseases CSF chemical analysis

Biochemistry of the Nervous System • Transport of substances through the blood-brain barrier (BBB) • Metabolism of neurotransmitters Synthesis (precursors – role of vitamins) Release Effect Termination • Metabolism of CNS : Energy Metabolism of CNS Lipid Metabolism in CNS Myelin sheath • Biochemical aspects of CNS diseases Hypoglycemia Cerebral ischemia • CSF chemical analysis

Transport through Blood-Brain Barrier (BBB) Large number of compounds are transported through endothelial capillaries by facilitated diffusion 1 - Fuels Glucose: The principal fuel of the brain Transported through endothelial membranes by facilitated diffusion via GLUT-1 At blood glucose of 60 m/dl, glucose is reduced to below Km of GLUT-1 leading to appearance of symptoms of hypoglycemia 2 - Others: as ketone bodies by another transport system When blood levels of KB are elevated (during starvation) KBs are important fuels for brain during prolonged starvation Non-essential fatty acids (of diet or lipolysis) do not cross BBB Essential fatty acids (linoleic & linolenic) can pass BBB

Transport through Blood-Brain Barrier (BBB) 2 - Amino acids are transported by amino acid transporters Amino acids are used in brain for synthesis of: - Proteins of CNS - Neurotransmitters (requires certain vitamins as B 12, B 6 & B 1 …) Types of transported amino acids: 1 - Long neutral amino acids (by single amino acid transporters) Essential: Phenylalanine , Leucine, Isoleucine, Valine, Tryptophan, Methionine Non-essential: Tyrosine Semi-essential: Histidine 2 - Small neutral amino acids (entry is markedly restricted as their influx markedly change content of neurotransmitters) Nonessential: Alanine, Glycine, Proline 3 - Vitamins: transported by special transporters

Metabolism of Neurotransmitters Are chemicals released at synapses for transmission of nerve impulses Generally, each neuron synthesizes only those neurotransmitters that it uses for transmission through synapses So, the neuronal tracts are often identified by their neurotransmitters Structurally divided into two categories: categories - Small nitrogen-containing neurotransmitters - Neuropeptides: Neuropeptides Targeted in CNS as endorphins OR Released to circulation as GH & TSH Major small nitrogen containing neurotransmitters: • • Glutamate GABA Glycine Acetylcholine Dopamine Norepinephrine Serotonin Histamine In addition to: epinephrine, aspartate, nitric oxide

Metabolism of Neurotransmitters General features of neurotransmitters synthesis, release & termination 1 - Most are synthesized in presynaptic terminal from : Amino acids Intermediates of glycolysis Intermediates of TCA 2 - Once synthesized, they are stored in vesicles (by active uptake) 3 - Released in response to nerve impulse: 1 - Nerve impulse causes Ca 2+ influx (through Ca 2+ channels) to presynaptic terminal 2 - Exocytosis of neurotransmitters into synaptic cleft 3 - Neurotransmitter binds to receptors on postsynaptic membrane-----EFFECT 4 - Termination: by: Reuptake of the neurotransmitter into presynaptic terminal (or by glial cells) Or/ Enzymatic inactivation (in presynaptic terminal, postsynaptic terminal or in astrocyte)

Metabolism of Neurotransmitters

Neurotransmitters: Catecholamines & Serotonin Phenylalanine Tyrosine Tryptophan Tyrosine Hydroxylase Cu-dependent BH 4 DOPA Decarboxylase DOPA PLP Tryptophan Hydroxylase BH 4 Melanin melanocytes Dopamine Hydroxylase Cu 2+ Vit. C Norepinephrine Monoamine Oxidases MAO-A 5 HIAA MAO-A Serotonin VMA in Adrenal Medulla Methyl transferase (& few neurons) using SAM Vit. B 12 Epinephrine Folate In URINE Melatonin

Neurotransmitter: Histamine • Histamine is an excitatory neurotransmitter in CNS • Synthesized in CNS from the amino acid histidine by histidine decarboxylase (requires PLP) PLP • Antihistaminic drugs (for treatment of allergy) cause drowsiness BUT new generations of antihistaminics do not pass BBB & so do not cause CNS effects

Neurotransmitter: Acetylcholine Synthesis in CNS (in presynapses) Choline Acetyltransferase enzyme Acetyl Co. A + Choline Acetyl Choline: 1 - From diet 2 - From phosphatidylcholine (PC) in membrane lipids PC is synthesized from PE utilizing methyl groups of S-adenosyl methionine (SAM) Requires vitamins B 12 & B 6 Acetyl group From glucose oxidation (requires oxygen) oxygen is the major source (little FA oxidation in CNS) Thiamine (vit. B 1) B 1 Glucose Pyruvate Acetyl Co. A ATP Pyruvate Decarboxylase N. B. In thiamine deficiency & hypoxia: no ATP & no acetylcholine neurotransmitter

Neurotransmitter: Glutamate & GABA • • Glutamate is the main excitatory neurotransmitter in the CNS Neurons that respond to glutamate are referred to as glutaminergic neurons Sources of glutamate in nerve terminals: 1 - Synthesized from glucose through glucose metabolism in neurons (main source) source Glucose --- a Ketoglutarate (a KG) ------ glutamate (Requires PLP) 2 - From glutamine (of glial cells) by glutaminase 2 - From blood (few as no cross BBB) Mechanism of action of glutamate as a neurotransmitter: 1 - Synthesis from glucose metabolism & concentration in vesicles (in presynapses) 2 - Release by exocytosis to synaptic cleft 3 - Uptake by postsynaptic 4 - Binding to glutaminergic receptors in postsynapses 5 - Functional effect 6 - Termination: glutamate reuptake by astrocytes (glial cells). . REQUIRES ATP (ENERGY) In astrocytes, glutamate is converted to glutamine (REQUIRES ATP) ATP Glutamine is released from astrocytes & is taken up by neurons In neurons, glutamine is converted to glutamate by glutaminase

Neurotransmitter: Glutamate & GABA Is an inhibitory neurotransmitters in CNS In presynaptic neurons, GABA is synthesized from glutamate by glutamate decarboxylase (GAD) GAD by a reaction that requires PLP Then, GABA is released to synaptic cleft. It is recognized by receptors on postsynaptic neurons. Termination: GABA in synaptic cleft is uptaken by glial cells (as astrocytes) & converted to glutamate Glutamate is converted to glutamine by glutamine synthetase (requires ATP) ATP Fate of glutamine of astrocytes: 1 - A fraction of glutamine is released from astrocytes & is taken up by neurons. In neurons, glutamine is deaminated to glutamate by glutaminase 2 - Another fraction of glutamine is released to blood------to kidney --- ammonia Tiagabine is used as an antiepileptic (anticonvulsant) as it inhibits the reuptake of GABA `

Neurotransmitter: Glutamate & GABA Glutamate metabolism in hyperammonemia: • During hyperammonemia, hyperammonemia ammonia can diffuse into the brain from the blood to neurons. • The ammonia is able to inhibit the glutaminase in neurons, neurons thereby decreasing formation of glutamate in presynaptic neurons (not regenerated) This effect of ammonia might contribute to the lethargy associated with the hyperammonemia found in patients with hepatic disease. (hepatic encephalopathy)

Neurotransmitter: Glutamate & GABA Relation between glutamate synthesis & citric acid cycle: • • In neurons, synthesis of glutamate removes a ketoglutarate from the citric acid cycle ending in a decrease in regeneration of oxalacetate Regeneration of oxalacetate is necessary for oxidation of acetyl Co. A & this is performed by two major anaplerotic pathways: pathways 1 - Degradation of isoleucine & valine amino acids to butyric succinyl Co. A, which yields oxalacetate. This reaction requires vitamin B 12 (coenzyme for methylmalonyl Co. A mutase) 2 - Pyruvate carboxylation to oxalacetate (by pyruvate carboxylase, requires the vitamin biotin as a coenzyme).

Metabolism of CNS Glucose & Energy Metabolism Energy source of the brain • The mass of the brain is only 2% of the total body mass, yet its energy requirement is more than seven times than that of the other organs • Thus for brain metabolism, there is a high requirement for glucose and oxygen at steady rate. • The main source of energy is the generation of ATP by the aerobic metabolism of glucose Aerobic Glycolysis In Cytosol Mitochondria & Oxygen Glucose ---- Pyruvate ----- Acetyl Co. A ---- With oxalacetate in CAC ------- ATP

Metabolism of CNS Glucose & Energy Metabolism Glucose metabolism & neurotransmitters (in CNS) There is a relationship between the oxidation of glucose in glycolysis and the supply of precursors for the synthesis of neurotransmitters in neurons within CNS. Accordingly, deficiencies of either glucose or oxygen (hypoglycemia or hypoxia) affect brain function because they influence: 1 - ATP production for CNS neurons 2 - Supply of precursors for neurotransmitter synthesis. Glucose Metabolism & Neurotransmitter Synthesis

Metabolism of CNS Brain Lipids Synthesis & Oxidation Sources of lipids to CNS: • BBB significantly inhibits entry of certain fatty acids & lipids into CNS. So, all lipids found in CNS must be synthesized within CNS (e. g. non-essential fatty acids, cholesterol, sphingolipids, glycosphingolipids & cerebrosides) All these are needed for neurological functions & synthesis of myelin by glial cells (nonneuronal cells) • EXCEPTION is: Essential fatty acids (linoleic & linolenic FAs) can enter the brain Within CNS, these two FAs are elongated & desaturated to yield the very-long chain fatty acids required for synthesis of myelin sheath Fatty acid oxidation as a source of energy: • Intake of fatty acids (coming from diet and/or lipolysis of TG ) to CNS is insufficient to meet the energy demands of CNS (by FA oxidation) & hence the requirement for aerobic glucose metabolism • Recall that ketone bodies are sources of energy to brain during prolonged starvation as they can pass BBB easily….

Metabolism of CNS Brain Lipids Synthesis & Oxidation • Peroxisomal fatty acid oxidation is important in the brain as the brain contains very-long-chain fatty acids & branchedchain fatty acids as phytanic acid (of diet) Both are oxidized by a oxidation in the peroxisomes • Refsumes disease: disease a disorder that affects the peroxisomes – severely affects the brain due to inability to metabolize both very long chain & branched chain fatty acids

Myelin Synthesis Rapid rate of nerve conduction in PNS & CNS depends on the formation of myelin Myelin is a multilayered lipid (sphingolipids) & protein structure that is formed by the plasma membrane of glial cells to wrap around the axon. In PNS, myelin is synthesized by Schwan cells In CNS, myelin is synthesized by oligodendrocytes Multiple sclerosis (MS) Progressive demyelination of CNS neurons May be due to an event that triggers the formation of autoimmune antibodies directed against components of the nervous system (as viral or bacterial infection) Loss of myelin (insulator) in the white matter of the brain that interferes with nerve conduction along demyelinated area CNS compensates by stimulating the oligodendrocytes to remyelinate the damaged axon(& hence remission is activated) Remyelination is accompanied by slowing in conduction (speed is proportional to myelin thickness)

Clinical Manifestation Hypoglycemic Encephalopathy Clinical manifestations of hypoglycemia: Early clinical signs in hypoglycemia initiated by hypothalamic sensory nuclei as sweating, sweating palpitations, palpitations anxiety and hunger In late stages, these symptoms give way to serious manifestations of CNS disorders as confusion, confusion lethargy, lethargy seizures & coma

Biochemistry of Hypoglycemic Encephalopathy • As blood glucose falls below 45 mg/d. L the brain attempts to use internal substrates such as glutamate and TCA cycle intermediates as fuels for ATP production. Because the pool size of these substrates is quite small, they are quickly depleted • As the blood glucose drops from 45 to 36 mg/d. L NO EEG changes are observed Symptoms appear to arise from decreased synthesis of neurotransmitters in particular regions of the brain (hippocampal & cortical structures) • If blood glucose levels continue to fall below 18 mg/d. L EEG becomes isoelectric Neuronal cell death ensues that may be caused by glutamate excitotoxicity ? ? (as result of ATP depletion) SO, GLUTAMATE METABOLIDSM HAS TO BE UNDERSTOOD

Glutamate as a neurotransmitter Role of glutamate as a transmitter in CNS: Within the CNS, glutaminergic neurons are responsible for the mediation of many vital processes such as the encoding of information, the formation and retrieval of memories, spatial recognition and the maintenance of consciousness. Postsynaptic glutaminergic neurons perform their roles through: 1 - Ionotropic receptors that bind glutamate released from presynaptic neurons referred to as kainate, kainate 2 -amino-3 -hydroxy-5 -methyl-4 -isoxalone propionic acid (AMPA) AMPA and N-methyl-D-aspartate (NMDA) NMDA receptors. 2 - Metabotropic glutamate receptors that are members of the G-protein coupled receptor (GPCR) family.

Ionotropic Glutamate Receptors Non- NMDA KAINATE NMDA AMPA and Kainate receptors generally allow the passage of Na+ and K+ NMDA receptors allows the passage of both Na+ and Ca++ ions. (More permeable to Ca++ )

Glutamate Excitotoxicity Exciotoxicity is the pathological process by which nerve cells are damaged and killed by glutamate (and similar substances). This occurs when receptors for glutamate such as the NMDA & AMPA receptor are over activated (overstimulated). Excessive excitation of glutamate receptors has been associated with hypoglycemia & stroke (cases in which there is lack of glucose and/or oxygen ending in lack of energy production in CNS)

Glutamate Excitotoxicity occurs when the cellular energy reserves are depleted (as in hypoglycemia or stroke, stroke etc ) Failure of the energy-dependent reuptake pumps of glutamate Accumulation of glutamate in the synaptic cleft Overstimulation of the postsynaptic glutamate receptors Prolonged glutamate receptor activation leads to prolonged opening of the receptor ion channel and the influx of lethal amounts of Ca 2 ions, ions which can activate cytotoxic intracellular pathways in the postsynaptic neurons

Biochemistry of Cerebral Ischemia Cerebral ischemia • It is the potentially reversible altered state of brain physiology and biochemistry that occurs when substrate delivery is cut off or substantially reduced by vascular stenosis or occlusion Stroke • is defined as “an acute neurologic dysfunction of vascular origin with sudden (within seconds) or at least rapid (within hours) occurence of symptoms and signs corresponding to the involvement of focal areas in the brain ” (Goldstein et al, 1989)

Pathophysiology of Cerebral Ischemia ↓↓ ATP Induction Amplification Expression

Lack of oxygen supply to ischemic neurones The cell switches to anaerobic metabolism, producing lactic acid ATP depletion malfunctioning of membrane ion system Depolarisation of neurones Influx of calcium Induction Phase Release of neurotransmitters as glutamate (causing glutamate excitotoxicity) Amplification Accumulation of more intracellular Phase levels of Ca 2+ which causes additional release of glutamate (viscious cycle)

Expression Phase Ca 2+ 1 -overexcites cells and causes the generation of harmful chemicals like free radicals ( causing oxidative stress) 2 - Activation of calcium-dependent enzymes such as: calpain ( causing apoptosis) phospholipases (causing membrane breakdown) 3 - Calcium can also cause the release of more glutamate (glutamate excitotoxicity) excitotoxicity The cell's membrane is broken down by phospholipases Cell membrane becomes more permeable, and more ions and harmful chemicals flow into the cell. + Mitochondria membrane break down, releasing toxins and apoptotic factors into the cell

lactic acid is produced in excess in ischemia In cerebral ischemia, lack of oxygen switches metabolism of glucose to the anaerobic pathway & lactic acid production Lactic acid contribute to the pathophysiology of ischemia as: as 1 - It decreases p. H that may injure and inactivate mitochondria 2 - Lactic acid degradation of NADH (which is needed for ATP synthesis) may also interfere with adequate post-ischemic recovery of ATP levels. 3 - Lactic acid increase the amount of free-radical mediated injury Lactic acid in neurons acidosis promotes the pro-oxidant effect ↑ the rate of conversion of O 2 - to H 2 O 2 or to hydroxyperoxyl radical

Oxidative stress is caused by ischemia What is meant by ROS? Reactive oxygen species (ROS) are formed from partial reduction of molecular O 2 i. e. adding electrons to oxygen leading to the formation of superoxide, hydrogen peroxide & hydroxyl radical. Generally, ROS cause damage to DNA, protein and unsaturated lipids of the cells What is meant by oxidative stress A condition in which cells are subjected to excessive levels of ROS (free radicals) & they are unable to counterbalance their deleterious effects with antioxidants

Oxidative stress is caused by ischemia Cellular Effects of Reactive Oxygen Species (ROS) in CNS • Nitric oxide is over produced and turns to be a neurotoxic mediator as it reacts with superoxide anions to generate toxic peroxynitrite which leads to production of more potent neurotoxin such as hydroxyl radicals • Lipid peroxidation • Inactivation of enzymes • Nucleic acid (DNA & RNA) damage • Release of calcium ions from intracellular stores with more damage to neurons • Damage to cytoskeleton

Apoptosis & necrosis are caused by ischemia • Necrosis: Necrosis is commonly observed early after severe ischemic insults • Apoptosis: Apoptosis occurs with more mild insults and with longer survival periods • The mechanism of cell death involves calcium-induced calpainmediated proteolysis of brain tissue • Substrates for calpain include: – Cytoskeletal proteins – Membrane proteins – Regulatory and signaling proteins

Apoptosis Mitochondria break down, releasing toxins and apoptotic factors into the cell. The caspase-dependent apoptosis cascade is initiated, causing cells to "commit suicide. " Broughton et al. , 2009; Stroke

Caplains are cytosolic proteinases Whose irreversible proteolytic activity is against cytoskeleton and regulatory proteins Broughton et al. , 2009; Stroke

Broughton et al. , 2009; Stroke