4. Dopamine formation and storage

Dopamine is generated in dopaminergic cells, primarily in VTA and substantia nigra.

• 4. Synthesis and storage of dopamine, norepinephrine, epinephrine
  • 4.1. Synthesis of dopamine, noradrenaline, adrenaline
  • 4.2. Brain regions where dopamine is produced
  • 4.3. Individual elements of dopamine synthesis
    • 4.3.1. Tyrosine hydroxylase (TH)
    • 4.3.2. Tetrahydrobiopterin (BH4, Sapropterin, INN)
    • 4.3.3. GTP cyclohydrolase 1 (GTPCH)
    • 4.3.4. Aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase)
    • 4.3.5. Pyridoxal phosphate (PLP, PALP, P5P, active vitamin B6)
    • 4.3.6. Oxygen concentration and dopamine synthesis
  • 4.4. Incorporation of dopamine into vesicles (VMAT)
    • 4.4.1. VMAT isoforms
    • 4.4.2. Regulation of VMAT
    • 4.4.3. Effect of VMAT2

4. Synthesis and storage of dopamine, norepinephrine, epinephrine¶

4.1. Synthesis of dopamine, noradrenaline, adrenaline¶

The formation of dopamine occurs in several steps:¹²

• L-phenylalanine from food
  is produced by phenylalanine hydroxylase consuming tetrahydrobiopterin (BH4), folic acid and oxygen
• To L-Tyrosine
  this is achieved by tyrosine hydroxylase (TH) consuming BH4 and calcium citrate
• To L-Dopa (dihydroxyphenylalanine)
  this is achieved by aromatic-L-amino acid decarboxylase (AADC, dopa decarboxylase) consuming pyridoxal phosphate (vitamin B6)
• To dopamine and CO2 (carbon dioxide)

Alternative synthetic pathways of dopamine include ²

• L-phenylalanine from food
  is produced by aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase) with consumption of pyridoxal phosphate (vitamin B6)
• To phenylethylalamine
  this becomes
• To tyramine

or

• L-Tyrosine
  is produced by aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase) with consumption of pyridoxal phosphate (vitamin B6)
• To tyramine

The resulting

• Tyramine
  is induced by CYP2D
• To dopamine

In dopaminergic cells, it remains so.
In noradrenergic cells, dopamine is converted to norepinephrine:

• Dopamine
  is produced by the enzyme dopamine-β-hydroxylase (DHB) with consumption of oxidized vitamin C
• To norepinephrine

In noradrenergic cells, this remains the case.
In adrenergic cells, norepinephrine is converted to epinephrine:

• Norepinephrine
  is produced by the enzyme phenylethanolamine N-methyltransferase (PNMTase) with consumption of S-adenosylmethionine (SAM), pyridoxal phosphate (vitamin B6) and vitamin B12
• To adrenaline.

4.2. Brain regions where dopamine is produced¶

The formation of dopamine in the brain occurs in the

• VTA
• Substantia nigra
• Nucleus arcuatus

4.3. Individual elements of dopamine synthesis¶

4.3.1. Tyrosine hydroxylase (TH)¶

TH is the rate-limiting enzyme of dopamine synthesis. TH uses BH4 and molecular oxygen to convert tyrosine into DOPA.⁵

What influences the activity of TH thus influences dopamine synthesis. Some mechanisms alter the activity of TyrH, while others simply bring the enzyme into proximity with other related proteins. Post-translational mechanisms of TH regulation include:⁵

• Phosphorylation by kinases
  • The activity and stability of tyrosine hydroxylase is affected by several kinases such as PKA, PKC, CaMPKII, PKG, MPK, and ERK, which phosphorylate it at serine sites 8 (rat only), 19, 31, and 40. A phosphorylation of tyrosine hydroxylase⁶
    • an Ser40 strongly increases TH activity (up to 10-fold)
    • an Ser31 increases the TH activity weakly
    • at Ser19 or Ser8 (rat only) does not affect TH activity
    • at Ser19 increases the rate of Ser40 phosphorylation, which increases TH activity.
  • Further, arginine sites 37 and 38 appear to be able to regulate TH. Deletion or replacement of arginine 37 and 38 by glycine or glutamate resulted in better BH4 affinity and thus increased TH activity.²
• Dephosphorylation by phosphatases
  • The phosphatases PP2A and PP2C reverse the phosphorylation of TH and can thus deactivate TH²
• Feedback inhibition
  • Catecholamines regulated TH. Catecholamines compete with BH4 for binding of the ferric ion at the catalytic site of tyrosine hydroxylase. Thus, high levels of catecholamines inhibit tyrosine hydroxylase and thus its own synthesis in the form of a feedback loop.²
  • Dopamine inhibits TH (even in the presence of GTPCH) as a negative feedback loop²
• Oxidation by nitrites
  • TH can be inactivated by nitration using reactive nitrogen species (peroxynitrite) or by S-thiolation on cysteine residues.²
• Incorporation into protein complexes.
  • Stability, activity, and probably intracellular localization of TH is also regulated by interactions with other proteins, such as²
    • 14-3-3
      • activates TH⁷
    • DJ-1
    • α-Synuclein
      • inhibits TH⁷
    • VMAT-2
    • AADC
    • GTPCH
    • DJ-1 (protein deglycase DJ-1, also called Parkinson disease protein 7, PARK7) regulates TH.
      • DJ-1 regulates TH transcription by altering the acetylation status of the TH promoter. DJ-1 increases TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Silencing of DJ-1 decreases acetylation of TH promoter-bound histones. Histone deacetylase inhibitors restore DJ-1 siRNA-induced suppression of TH. DJ-1 silencing thus causes decreased TH expression and decreased dopamine synthesis.⁸
      • The oxidation state of DJ-1 regulates its own activity and, at the same time, TH expression⁵
        • DJ-1 is a transcriptional regulator
        • DJ-1 plays a role in bypassing oxidative stress
        • Certain DJ-1 variants have been identified in Parkinson’s disease⁹
        • DJ-1 is a TH repressor and binds to the promoter of the TyrH gene binds
        • DJ-1 binds directly to TyrH itself (as well as to AADC) binds and activates TH like AADC

Salsolinol, a tetrahydroisoquinoline alkaloid, inhibits TH¹⁰
Salsolinol

• is found at up to 25 µg/g in chocolate, cocoa and bananas, but cannot cross the blood-brain barrier.
• is also formed endogenously in the organism

Cortisol inhibits TH.¹¹

Aspirin at low doses (2 mg/kg/day) increased TH and subsequently dopamine in the striatum in mice.¹²

4.3.2. Tetrahydrobiopterin (BH4, Sapropterin, INN)¶

Tetrahydrobiopterin (BH4) is generated from guanosine triphosphate by GTP cyclohydrolase 1 (GTPCH). GTPCH is the limiting factor of BH4 synthesis.¹³

• too high BH4 levels block GTPCH
  • this prevents GTPCH-TH interaction
  • therefore too much BH4 impairs the TH
• bH4 levels that are too low inhibit TH
  • BH4 is a cofactor for TH
• BH4 promotes tyrosine hydroxylase only from 10 to 25 and up to 100 microM. Lower or higher amounts inhibit it²
  BH4 deficiency or BH4 excess can thus impede dopamine synthesis. BH4 administration can improve ADHD symptoms that develop due to phenylketunorie.¹⁴

Genetic BH4 disorders such as

• autosomal recessive (AR) guanosine triphosphate cyclohydrolase deficiency (GTPCH deficiency)
• 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency

appear to co-occur with ADHD and other mental health disorders such as anxiety, depression, aggression, or oppositional defiant behavior.¹⁵

4.3.3. GTP cyclohydrolase 1 (GTPCH)¶

GTPCH interacts with tyrosine hydroxylase²

• cAMP administration increases GTPCH gene expression
• Phenylalanine induces GTPCH activity
• Phosphorylation of Ser81 increases GTPCH activity.
• BH4 inhibits GTPCH via the GTPCH feedback regulatory protein (GFRP) in the form of a feedback loop

GTPCH interacts with tyrosine hydroxylase²

• The interaction depends on the phosphorylation of TH as well as GTPCH.
• Interaction with TH prevented BH4-mediated inhibition of GTPCH
  • this increased GTPCH and TH activity
• GTPCH activity is stimulated by phosphorylated TH
• Dopamine synthesis is dependent on GTPCH
• In Drosophila with inactive GTPCH gene versions, BH4 does not increase TH activity
• TH activity depends on an interaction between TH and GTPCH

4.3.4. Aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase)¶

AADC is involved in the synthesis of catecholamines, indoleamines, and trace amines.¹⁶

• Decarboxylation of aromatic amino acids occurs in different cell types, so trace amines can theoretically be generated in them:¹⁷
  • Nerve cells
  • Glial cells
  • Blood vessels
  • Cells of the gastrointestinal tract
  • Kidney
  • Liver
  • Lungs
  • Stomach
    • Serotonergic AADC, especially in the pylorus¹⁸

In the brain, cells containing AADC were found in the following areas:¹⁹

• Raphe nuclei (serotonergic, very strong AADC)
• Pons ventral (serotonergic, very strong AADC)
• Medulla ventral (serotonergic, very strong AADC)
• Mesencephalic reticular formation (dopaminergic, moderate to strong AADC)
• Substantia nigra (dopaminergic, moderate to strong AADC)
• VTA (domapinergic, moderate to severe AADC)
• Locus coeruleus (noradrenergic, moderate to strong AADC)
• Subcoeruleus nuclei (noradrenergic, moderate to strong AADC)
• Median and ventrolateral parts of the intermediate reticular nucleus in the medulla oblongata (noradrenergic/adrenergic, moderate AADC)
• Forebrain: few nonaminergic AADC-positive neurons (D neurons); these were not detectable in other brain parts in humans.

AADC catalyzes the decarboxylation of various aromatic l-amino acids such as:

• L-DOPA
• L-Tyrosine
• L-tryptophan
• L-Histidine

AADC, for its part, is subject to regulatory influences:

• Transcriptional (slower, longer-lasting regulation by adjusting gene expression)²
  • Expression is influenced by
    • Different promoter usage
    • Different splicing
• Posttranslational (fast-acting, short-term mechanism)
  • Phosphorylation¹⁶
    • By PKA in the striatum and midbrain
    • AADC increased by 20 to 70
  • PH value changes²⁰
  • Denaturation²⁰
  • Neurotransmitter²
    • Antagonists enhance, agonists reduce AADC activity²¹
    • Dopamine via
      • Dopamine D1 to D4 receptors²¹
      • Dopamine antagonists increase AADC, eg.
        • Cis-flupenthixol
        • Haloperidol
      • Dopamine increase decreases AADC²
        • MOA Inhibitors
      • Destruction of dopaminergic cells decreased only dopaminergic 3,4-dihydroxyphenylalanine decarboxylase, not serotonergic 5-hydroxytryptophan decarboxylase activity, which actually increased in the process²²
      • In contrast, another study found that destruction of dopaminergic as well as serotonergic neurons both reduced dopaminergic as well as serotonergic AADC equally.²³
    • Serotonin via
      • 5-HT 1A receptor²¹
      • 5-HT 2A receptor²¹
    • Glutamate via
      • NMDA receptor²¹
    • Nicotinic acetylcholine receptors²¹

Different forms of AADC cause dopaminergic and serotonergic decarboxylase. The two forms showed different activity maxima depending on ph, temperature and substrate concentrations:²⁴

• Dopaminerg: 3,4-dihydroxyphenylalanine decarboxylase
  * AADC dopaminergic activity:
  * Only in soluble cell fractions²⁴
  * Distribution corresponded to lactic acid dehydrogenase²⁴
  * Pyridoxal-5-phosphate increases this 20-fold²⁵
  * Carboxyl scavengers inhibit them completely²⁴

• Serotonerg: 5-hydroxytryptophan decarboxylase
  * In soluble as well as particulate cell fractions²⁴
  * Pyridoxal-5-phosphate doubles these²⁴
  * Pyridoxal-P antagonists hardly affect them at all²⁴
  * Serotonergic AADC is potentiated by 3-isobutyl-1-methylxanthine.¹⁸

In ADHD, 50% decreased AADC activity was found in the medial and left PFC, but not in the striatum or midbrain.²⁶ Another study by the same research group with a smaller number of subjects found increased AADC activity in ADHD²⁷

AADC is also relevant in the synthesis of melatonin. Here, there might be a connection with the increased sleep problems in ADHD.

4.3.5. Pyridoxal phosphate (PLP, PALP, P5P, active vitamin B6)¶

Pyridoxal phosphate (PLP, PALP, P5P) is one of the most important cofactors in the animal organism. Pyridoxal phosphate is the metabolically active form of vitamin B6.
PLP is involved in various reactions of amino acids:

• Transamination
  catalyzed e.g. by
  • GABA transaminase
• Decarboxylation
  catalyzed e.g. by
  • AADC
  • Histidine decarboxylase
  • Ornithine decarboxylase
• Dehydration
• Glycogen degradation

4.3.6. Oxygen concentration and dopamine synthesis¶

The intracellular O2 concentration in brain tissue influences synthesis and stability of dopamine. The O2 concentration in brain tissue is normally 1-5%, thus significantly lower than the 20% in the atmosphere. Increased oxygen induces dopamine oxidation and thus ROS formation.²⁸ Hypoxia increased TH activity and thus dopamine synthesis.²⁹

SAM is formed from a reaction of the amino acid methionine with ATP (adenosine triphosphate).
SAM is converted to adenosine and homocysteine by the enzyme S-adenosylhomocysteine.
Homocysteine can be remethylated back to methionine or degraded to the amino acid cysteine.

Dopamine formation is increased by

• Exercise and sport³⁰
• (strategic) thinking (context: work addiction)

Some ADHD sufferers engage in excessive sports. The sport is “needed”. Sport increases stress resistance.

Erythropoietine, a cytokine secreted primarily from the kidney in the presence of decreased blood oxygen saturation,³¹ showed a protective effect against the death of dopaminergic cells in hypoxia and ischemia in animal experiments.³²³³
In ADHD sufferers, a correlation was found between elevated blood Erythropoietine levels and inattention and highly elevated Erythropoietine levels and hyperactivity.³⁴

4.4. Incorporation of dopamine into vesicles (VMAT)¶

Once formed, dopamine and other catecholamines are stored in vesicles where it is kept until it is released:³⁵

• small synaptic vesicles in neurons
• small and large dense nuclear vesicles in neurons and neuroendocrine cells.

The incorporation of dopamine into vesicles in dopaminergic cell occurs through VMAT2 incorporated into the vesicles.
VMAT2-KO mice have reduced dopamine levels in neurons. Dopamine cannot be released by electrical signals in these, but can be released by amphetamine.³⁶ Absent or over-inhibited VMAT2 causes excessive cellular dopamine levels, which are neurotoxic after oxidation. VMAT-Full-KO mice usually survive only a few days.

While vesicles were previously thought to harbor uniformly large amounts (a “quantum”) of neurotransmitters, several factors are now known to influence the amount of neurotransmitter in vesicles.³⁷

4.4.1. VMAT isoforms¶

2 VMAT isoforms³⁸

Both belong to the SLC18 family.

• VMAT1
  • Is expressed only in endocrine and paracrine cells associated with the stomach, intestine, and sympathetic nervous system
• VMAT2
  • Is expressed by monoaminergic neurons throughout the CNS (rat)
  • In dopamine neurons on both types of secretory vesicles
    • Small synaptic vesicles
    • Large dense core vesicles

4.4.2. Regulation of VMAT¶

Suppression of general VMAT function with reserpine resulted in marked parkinsonian-like behavioral effects in rats. These could be reversed by the dopamine prodrug L-DOPA³⁸
Since L-dopa is helpful in ADHD only in rare cases, VMAT might be involved in ADHD correspondingly infrequently.
Similarly, the selective VMAT2 antagonist tetrabenazine inhibits dopamine formation.

VMAT2 inhibitors inhibit self-administration of methamphetamine and amphetamine in rats³⁸

• (+)eCYY477, VMAT2 inhibitor (a dihydrotetrabenazine derivative)
• Lobelan
• GZ-793A (Lobalan analog)
  • also reduces dopamine release specifically in limbic terminal fields (e.g., nucleus accumbens shell).

VMAT2 depend on V-ATPase (vacuolar-type H+ ATPase) for their dopaminergic function. Disturbances in the function of V-ATPase or the proton gradient it creates impair the incorporation of dopamine into the vesicles by VMAT2.

The incorporation of dopamine into vesicles by VMAT2 also depends on the amount of cytosolic dopamine concentration. L-dopa increases dopamine within the cell. Increased L-DOPA causes an increase in vesicle size without a change in vesicle number. The increased vesicle size represents a larger releasable dopamine pool³⁸

VMAT2 can be regulated by:³⁸

• the cytosolic chloride concentration
  • up to a certain maximum, a higher cytosolic chloride concentration promotes dopamine incorporation by VMAT2
• acidification by V-ATPase is a critical mediator of dopamine increase by VMAT2
• post-translational modifications
• Amount of dopamine in cytoplasm appears to directly affect VMAT2
• Changes in the expression level due to
  • Transcription factors
  • Protein kinases
    • Protein Kinase A (PKA)
      • indirectly influences VMAT2 expression:
        • regulates N-terminal glycosylation
        • directly phosphorylates cAMP response element-binding protein (CREB) for trafficking and transcriptional activation
  • heterotrimeric G proteins
    • depending on an intralumenal loop
  • Binding partner interactions
  • Early childhood (stress) experiences³⁹
  • Activity
    • VMAT2 expression can be up- or down-regulated in response to activity
  • medicinal
    • Dopamine reuptake inhibitors increase VMAT2
      • Methylphenidate
      • Cocaine
    • Dopamine release promoting drugs decreases VMAT2
      • Amphetamine
        • causes emptying of the vesicular dopamine pool into the cytoplasm
        • causes deacidification of secretory vesicles
        • typical for DAT-reversing drugs
        • different from DAT inhibitors such as methylphenidate
      • Methamphetamine
    • Apomorphine
      • D2 agonist
        • D2 agonists increase VMAT2 activity, D2 antagonists appear to inhibit VMAT2
      • D1 partial agonist
    • Reserpine
      • inhibits VMAT2

4.4.3. Effect of VMAT2¶

Increased VMAT2 expression increased³⁸

• Amount of dopamine in vesicles
• Dopamine release quantity from vesicles
• Frequency of release events from vesicles

VMAT2 forms complexes with TH and AADC enzymes and thus also directly affects dopamine synthesis.