Motherisk Round 2011. 11. 29

B.Y. Lee

Dopamine Transporter in ADHD & Alcohol intake

• Dopamine? - neurotransmitter? - reward system - related diseases

• ADHD? • Alcohol? (pathway with dopamine) • ADHD- alcohol? • Dopamine – ADHD – alcohol?

• Related reports - epigenetic, dopaminergic, … • Epigenetic project

• Vesicle release process: docking → priming → fusion

• Proteins involved in presynaptic exocytosis

• SNARE proteins: docking (membs close together before fusion) • Synaptotagmin (vesicle protein) - Ca2+ sensor triggering fusion

I. Small molecule NT ① acetylcholine ② Amino Acids • glutamate • GABA & glycine ③ Biogenic amines • dopamine, norepinephrine (noradrenalin), epinephrine (adrenalin), serotonin (5-hydroxy tryptamine, 5-HT), histamine ④ Purines: ATP II. Peptides • Substance P, CGRP(calcitonin gene related peptide), Enkephalin, Cholecystokinin(CCK), etc. • Opioid peptides • Substance P III. Gaseous NT – Nitrous oxide, Carbon monoxide

1) Acetylcholine R

① Nicotinic: Ionotropic cys-loop superfamily

② Muscarinic: Metabotropic family R (M1~M5)

2) Inhibitory amino acid R: Ionotropic (GABAA,C & Gly), metabotropic (GABAB)

3) Excitatory amino acid R: Glutamate : NMDA, Kainate, AMPA

4) Purine (ATP) R: Ionotropic (P2X) & metabotropic (P2Y), presyn adenosine-R

5) Biogenic amine R: metabotropic except 5-HT3 (Ionotropic cys-loop superfamily)

6) Neuropeptides R: metabotropic R, diffused extracellular NT, more sensitive

7) Gas NT R: NO, CO - no binding to R, enzyme activation, modification of

protein activity by nitrosylating

A. Release and degradation of the NT inside the axon terminal B. Increased NT release into the synapse C. Prevention of NT release into the synapse D. Inhibition of synthesis of the NT E. Reduced reuptake of the NT from the synapse F. Reduced degradation of the NT in the synapse G. Agonist (evoke same response as neurotransmitter) or antagonists (block response to neurotransmitter) can occupy the receptors H. Reduced biochemical response inside the dendrite

Synthesis Storage and release Receptor binding Degradation

Amine Compounds: Serotonin, Noradrenalin and Dopamine These are the brain chemicals that to malfunction when stress levels become more than a person can handle

Norepinephrine (NE) • noradrenergic neurons in the locus ceruleus and nucleus subceruleus • PNS: Smooth muscles, cardiac muscle and glands - Increase in blood pressure, constriction of arteries • CNS: General behavior Serotonin (5-HT) • serotonergic neurons in the raphe nuclei • Regulation of mood, behavior, appetite, and cerebral circulation. • SSRIs (serotonin-specific reuptake inhibitors): – Inhibit reuptake and destruction of serotonin, prolonging the action of NT – Used as an antidepressant – Reduces appetite, treatment for anxiety, treatment for migraine headaches Histamine • Histaminergic neurons in tuberomammillary n. of hypothalamus Epinephrine • Adrenergic neurons in rostral medulla Epinephrine • Adrenergic neurons in rostral medulla

Dopamine (DA) is a monoamine neurotransmitter that upon binding to a dopamine receptor (G-protein coupled) releases a variety of downstream signals. Where is dopamine formed/produced? Mainly in areas of the central and peripheral nervous systems, such as in the hypothalamus, the arcuate nucleus, and the caudad. What are the functions of dopamine? In the cardiovascular, renal, hormonal, and central nervous systems. To control processes as diverse as movement to drug addiction. Dopamine dendrites extend into various regions of the brain, controlling different functions through the stimulation of α and β adrenergic and dopaminergic receptors (D1 and D2) (Velasco et al., 1998).

DAT (The dopamine transporter) regulates the uptake of dopamine into neurons. Dopamine is thought to bind to DT via a separate binding domain that is constructed of multiple amino acid residues. These amino acid residues are not present in the primary structure of DT, but are thought to have interactions with the protein in its tertiary form. MAO (monoamine oxidase) breaks down free dopamine (not contained in a vesicle). MAO play a role in recycling the components of dopamine, but it also keeps the concentration of dopamine lower than the rate at which it is being produced.

• Dopaminergic neurons in the two main brainstem regions − substantia nigra pars compacta & ventral tegmental area (VTA) a. Nigrostriatal dopamine system: • Neurons in substantia nigra send fibers to corpus striatum. • Initiation of skeletal muscle movement. • Parkinson’s disease: degeneration of neurons in substantia nigra. b. Mesolimbic dopamine system: • Neurons originate in midbrain, send axons to limbic system. • Involved in behavior and reward. • Addictive drugs: promote activity in nucleus accumbens c. Meso-cortical pathway • negative symptoms (motivation and emotional response) d. Tubulo-infundibular pathway (endocrine related, prolactin)

• The mesolimbic dopamine path correspond to the parts of the limbic system, which are connected to emotions and is responsible for the ability to learn, the short-term memory, maintenance of moods and the alignment of thought and action.

• The mesolimbic system represents a modulation system, a filter and gating system for signals coming from the limbic system and for signals which maintain the basic functions of life and motivation (Koob 1992).

• The Nucleus accumbens, - the most important end point of the mesolimbic dopamine path. - a clearing centre for signals coming from the limbic system - the switching point, which anticipates the probability of reward-stimuli and steers the attention of the nerve system.

Mesolimbic Dopamine Pathway

M-receptor D-receptor

• Dopamine deficiency in the striatum or substantia nigra : Parkinson’s-like symptoms. - movement becomes slow and rigid, accompanied by muscle tremor. - L-DOPA have been created to supplement the quantity of dopamine in the brain (Katzenschlager et al., 2002). • An excessive amount of dopamine : Schizophrenia - characterized by altered behavior, and delusions. - Cocaine, block the return of dopamine into the brain, resulting in a build up of dopamine in the synapse, leading to drug-induced psychosis or schizophrenia. Tourett’s syndrome, ADHD

• The most commonly dignosed neuropsyciatric disorder in child hood

• ADHD wanes with age, symptoms can persist into adolescence and adulthood. • The incidence in children : 3% ~ >5% (Biederman 1998) • The diagnosis of ADHD : is currently based on DSM-IV criteria. • The core symptoms : impulsivity, inattention, motor restlessness • Heterogenous clinical symptoms, comorbidity (approx. 65%) with other disorders (conduct, mood, bipolar and anxiety disorders, Tourette’s) • Abnormal morphology and function of ADHD brains, detected primarily by magnetic resonance imaging, converge on the catecholamine-rich frontal cortex and subcortical neural networks

• On the basis of the following lines of evidence, the DAT is one of several lead candidates for investigating the pathophysiology of ADHD and anti-ADHD drug mechanisms: 1) dopamine transport inhibitors indirectly activate dopamine receptor subtypes; D4 and D5 dopamine receptors are implicated in ADHD, and dopamine receptor activity enhances attention and experiential salience and engenders stimulation 2) the DAT is a selective and principal target of the most widely used antihyperactivity medications (amphetamine and methylphenidate) 3) the DAT gene is associated with ADHD 4) in some studies, abnormal levels of the DAT have been detected in brains of ADHD subjects.

• Dopamine is released from both dendrites and axons and might activate receptors locally or remotely through volume transmission.

• The DAT limits the duration of synaptic activity and diffusion by sequestering dopamine into neurons (Cragg and Rice 2004).

• The DAT is present on cell bodies, dendrites, and axons but apparently is not localized in the immediate active zone of the synapse (Hersch et al 1997)

• Accordingly, the DAT might reduce dopamine overflow into perisynaptic regions but not robustly sequester dopamine within the synapse. • Overexpression of the DAT might lead to compensatory increases in dopamine release, a reduction in extracellular dopamine levels, or altered cellular distribution of the DAT.

Monoamine Neurotransmitter Transporters

Extracellular neurotransmitter concentrations are regulated, in part, by monoamine transporters that sequester dopamine (DAT), serotonin (SERT), and norepinephrine (NET) into neurons. The majority of specific genes implicated in ADHD encode components of catecholamine signaling systems. The critical role of monoamine transporters for normal brain function is underscored by the wide range of drugs that target monoamine transporters, including medications for depression, ADHD, smoking addiction, obsessive-compulsive disorder, and sleep disorders.

DAT is one of several genes implicated

ADHD

• DAT is a principal target of the most widely used antihyperactivity medications (amphetamine and methylphenidate).

• All current anti-ADHD drugs modulate DA and NE levels in brain regions expressing DAT and NET. The majority of these drugs interfere with transporter-mediated clearance of extracellular neurotransmitters.

• Interfere with DAT by brain-region- and drug-specific mechanisms indirectly activating DA and possibly NE-R subtypes raising extracellular DA levels in DAT-rich brain regions enhancing attention and experiential salience.

• Repeated use of certain transport inhibitors (cocaine…), can eventually result in compulsive drug-seeking behavior.

• At the molecular level, the 12-membrane–spanning DAT protein contains a large extracellular loop, with consensus sites for glycosylation that function to regulate DAT trafficking and stability (Li et al 2004). • Similarly, glycosylation of the NET controls stability, trafficking, and transport activity (Melikian et al 1996). Potential phosphorylation sites (serine, threonine, and tyrosine) might also acutely modulate DAT trafficking.

• A length or of sequence in the 3=-untranslated region of the DAT gene might affect DAT expression. • A fixed length–repeat sequence, by number of repeats from 3 to 11. 10 repeats sequences are the most common • Inheritance of this repeat length in both alleles is associated with ADHD in some studies but accounts for less than 4% of the variance. • Intragenic allelic interactions might distinguish haplotype functional diversity, and association studies might inadvertently group haplotypes of different function together on the basis of VNTR length.

The DAT Gene Variants

• DAT control of dopamine release is region specific. - In the striatum, dopamine clearance is a primary function of the DAT, whereas in the substantia nigra the DAT regulates extracellular dopamine levels by controlling both clearance and release.

• The complexity of region-specific DAT function is relevant to the pharmacologic effects of anti-ADHD drugs.

• Methylphenidate blocks dopamine transport, whereas amphetamine is a substrate for the DAT and presumably can trigger dopamine release in the substantia nigra.

• These positron emission tomography (PET) scans show that patient with ADHD had lower levels of dopamine transporters in the nucleus accumbens , a part of the brains reward center, than control subjects

Main Effects: • Alcohol mainly acts on GABA receptors (GABA receptors down regulate the NT release of neurons, essentially reducing neural activity) by keeping its Cl- receptors open longer than they normally would be.

• This causes GABA receptors to have an increased effect and reduces neural activity resulting in the calm, sleepy feeling.

• Alcohol also inhibits glutamate receptor function which causes slurred speech, memory deficits, and discoordintaion.

Secondary Effects: • Raises the endorphin levels - causes the analgesic affect (pain killing).

• Increases the amount of dopamine in the brain – contributes to the addictive qualities.

• Can affect the methionine– homocysteine cycle by disrupting the enzymes required for methionine metabolism, thus interfering with SAM dependent methylation reactions. • Alcohol and its breakdown products (i.e., metabolites such as acetaldehyde) also cause different site specific modifications in histones. • Changes in the acetylation and methylation patterns of specific histones resulting from chronic alcohol feeding and high blood alcohol levels can lead to persistently altered gene expression and thus may play a role in this “epigenetic memory.” • May reduce the absorption of folate, a key component of the methionine– homocysteine cycle, thereby influencing methylation capacity.

• During prenatal and early postnatal development, the epigenome is highly susceptible to environmental stimuli such as diet, drugs, environmental agents, and maternal behavior.

• Preconception, preimplantation, and gastrulation - Appear to be particularly sensitive to the teratogenic effects of alcohol, - The clinical variability of FASD may be related not only to the dose of alcohol but also to when alcohol exposure occurs in relation to developmental periods.

• Gastrulation appears to be most sensitive to teratogenic insults- a wide range of adverse effects (morphological and behavioral abnormalities)

• Both preconception and preimplantation alcohol exposure can cause adverse effects, even though the embryo is not directly exposed to alcohol at this stage because it is not yet implanted in the uterus and therefore not connected to or nourished by the maternal system, probably provides the most compelling evidence of a role for epigenetic mechanisms in the outcomes observed.

early stage, although

Paternal effect (preconceptional)

Maternal effect (preconceptional/

prenatal)

- How long ? - How severe ? - Epigenetic target ? - DAT ? - SERT ? - MecP2 ?

Thank you for your attention !