Biochemistry of the Nervous System Transport through BBBMetabolism of NeurotransmittersMetabolism of CNS Biochemical Aspects of CNS diseasesCSF 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

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 B12, B6 & B1 …)

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

NeurotransmittersAre chemicals released at synapses for transmission of nerve impulsesGenerally, 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: - Small nitrogen-containing neurotransmitters - 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 Ca2+ influx (through Ca2+ 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 & SerotoninPhenylalanine Tyrosine

Tyrosine HydroxylaseCu-dependent BH4 DOPA MelaninDOPA Decarboxylase melanocytesPLP

Dopamine

Dopamine HydroxylaseCu2+ Monoamine Oxidases Vit . C MAO-A Norepinephrine VMA in AdrenalMedulla Methyl transferase (& few neurons) using SAMVit. B12 EpinephrineFolate

Tryptophan

TryptophanHydroxylase

BH4

MAO-A5 HIAA Serotonin

MelatoninIn URINE

Neurotransmitter: Histamine

• Histamine is an excitatory neurotransmitter in CNS• Synthesized in CNS from the amino acid histidine by histidine decarboxylase

(requires 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 enzymeAcetyl CoA + Choline Acetyl CholineCholine:1- From diet2- From phosphatidylcholine (PC) in membrane lipids PC is synthesized from PE utilizing methyl groups of S-adenosyl methionine (SAM) Requires vitamins B12 & B6

Acetyl groupFrom glucose oxidation (requires oxygen) is the major source (little FA oxidation in CNS) Thiamine (vit. B1)Glucose Pyruvate Acetyl CoA ATP Pyruvate DecarboxylaseN.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) 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 cleft3- Uptake by postsynaptic 4- Binding to glutaminergic receptors in postsynapses5- Functional effect 6- Termination: glutamate reuptake by astrocytes (glial cells) .. REQUIRES ATP (ENERGY) In astrocytes, glutamate is converted to glutamine (REQUIRES ATP) Glutamine is released from astrocytes & is taken up by neurons In neurons, glutamine is converted to glutamate by glutaminase

GABA Is an inhibitory neurotransmitters in CNSIn presynaptic neurons, GABA is synthesized from glutamate by glutamate decarboxylase (GAD) by a reaction that requires PLPThen, 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 glutamateGlutamate is converted to glutamine by glutamine synthetase (requires 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

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Neurotransmitter: Glutamate & GABA

Glutamate metabolism in hyperammonemia:• During hyperammonemia, ammonia can diffuse into the brain from

the blood to neurons.• The ammonia is able to inhibit the glutaminase in 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 CoA & this is performed by two major anaplerotic pathways:

1- Degradation of isoleucine & valine amino acids to butyric succinyl CoA, which yields oxalacetate. This reaction requires vitamin B12 (coenzyme for methylmalonyl CoA mutase) 2- Pyruvate carboxylation to oxalacetate (by pyruvate carboxylase, requires the vitamin biotin as a coenzyme).

Neurotransmitter: Glutamate & GABA

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 CoA ---- With oxalacetate in CAC ------- ATP

Metabolism of CNSGlucose & 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 CNSGlucose & Energy Metabolism

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 (non- neuronal 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 CNSBrain Lipids Synthesis & Oxidation

• Peroxisomal fatty acid oxidation is important in the brain as the brain contains very-long-chain fatty acids & branched-chain fatty acids as phytanic acid (of diet)

Both are oxidized by a oxidation in the peroxisomes • Refsumes disease: a disorder that affects the peroxisomes – severely affects the brain due to inability to metabolize both very long chain & branched chain fatty acids

Metabolism of CNSBrain Lipids Synthesis & Oxidation

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 cellsIn CNS, myelin is synthesized by oligodendrocytes

Multiple sclerosis (MS)Progressive demyelination of CNS neuronsMay 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 manifestations of hypoglycemia:

Early clinical signs in hypoglycemia initiated by hypothalamic sensory nuclei as sweating, palpitations, anxiety and hunger.

In late stages, these symptoms give way to serious manifestations of CNS disorders as confusion, lethargy, seizures & coma

Clinical Manifestation Hypoglycemic Encephalopathy

• As blood glucose falls below 45 mg/dL 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/dL 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/dL 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

Biochemistry of Hypoglycemic Encephalopathy

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, 2-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors.

2- Metabotropic glutamate receptors that are members of the G-protein coupled

receptor (GPCR) family.

Glutamate as a neurotransmitter

Ionotropic Glutamate Receptors

Non- NMDA

AMPAKAINATE

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)

occurs when the cellular energy reserves are depleted (as in hypoglycemia or 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 Ca2 ions, which can

activate cytotoxic intracellular pathways in the postsynaptic neurons

Glutamate Excitotoxicity

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

Induction Phase

Lack of oxygen supply to ischemic neuronesThe cell switches to anaerobic metabolism, producing lactic acid.

ATP depletion

malfunctioning of membrane ion system

Depolarisation of neurones

Influx of calcium

Release of neurotransmitters as glutamate (causing glutamate excitotoxicity)

Accumulation of more intracellular levels of Ca2+ which causes

additional release of glutamate (viscious cycle)

Amplification Phase

Expression Phase

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)

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

Ca2+

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: 1- It decreases pH 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 O2

- to H2O2 or to hydroxyperoxyl radical

What is meant by ROS?Reactive oxygen species (ROS) are formed from partial reduction of molecular O2 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 stressA 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

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: is commonly observed early after severe ischemic insults

• Apoptosis:occurs with more mild insults and with longer survival periods

• The mechanism of cell death involves calcium-induced calpain-mediated proteolysis of brain tissue

• Substrates for calpain include:– Cytoskeletal proteins– Membrane proteins– Regulatory and signaling proteins

Apoptosis

Broughton et al., 2009; Stroke

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 proteinasesWhose irreversible proteolytic activityis against cytoskeleton and regulatory proteins

Broughton et al., 2009; Stroke