Partial biopterin deficiency disturbs postnatal development of the dopaminergic system in the brain

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.     

Molecular Biology of Catecholamine-Related Enzymes in Relation to Parkinson's Disease

1. Catecholamine (dopamine, norepinephrine, and epinephrine) biosynthesis is regulated by tyrosine hydroxylase (TH). TH activity is regulated by the concentration of the cofactor tetrahydrobiopterin (BH4), whose level is regulated by GTP cyclohydrolase I (GCH) activity. Thus, GCH activity indirectly regulates TH activity and catecholamine levels.2. TH activity in the nigrostriatal dopaminergic neurons is most sensitive to the decrease in BH4.3. Mutations of GCH result in reductions in GCH activity, BH4, TH activity, and dopamine, causing either recessively inherited GCH deficiency or dominantly inherited hereditary progressive dystonia [HPD; Segawa's disease; also called dopa-responsive dystonia (DRD)].4. In juvenile parkinsonism and Parkinson's disease, which have dopamine deficiency in the basal ganglia as HPD/DRD, the GCH gene may be normal, and the molecular mechanism of the dopamine deficiency in the basal ganglia is different from that in HPD/DRD.     

AGC1‐malate aspartate shuttle activity is critical for dopamine handling in the nigrostriatal pathway

The mitochondrial transporter of aspartate‐glutamate Aralar/AGC1 is a regulatory component of the malate‐aspartate shuttle. Aralar deficiency in mouse and human causes a shutdown of brain shuttle activity and global cerebral hypomyelination. A lack of neurofilament‐labeled processes is detected in the cerebral cortex, but whether different types of neurons are differentially affected by Aralar deficiency is still unknown. We have now found that Aralar‐knockout (Aralar‐KO) post‐natal mice show hyperactivity, anxiety‐like behavior, and hyperreactivity with a decrease of dopamine (DA) in terminal‐rich regions. The striatum is the brain region most affected in terms of size, amino acid and monoamine content. We find a decline in vesicular monoamine transporter‐2 (VMAT2) levels associated with increased DA metabolism through MAO activity (DOPAC/DA ratio) in Aralar‐KO striatum. However, no decrease in DA or in the number of nigral tyrosine hydroxylase‐positive cells was detected in Aralar‐KO brainstem. Adult Aralar‐hemizygous mice presented also increased DOPAC/DA ratio in striatum and enhanced sensitivity to amphetamine. Our results suggest that Aralar deficiency causes a fall in GSH/GSSG ratio and VMAT2 in striatum that might be related to a failure to produce mitochondrial NADH and to an increase of reactive oxygen species (ROS) in the cytosol. The results indicate that the nigrostriatal dopaminergic system is a target of Aralar deficiency.     

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.     

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.     

Genetically rescued tetrahydrobiopterin-depleted mice survive with hyperphenylalaninemia and region-specific monoaminergic abnormalities

One of the possibly mutated genes in DOPA-responsive dystonia (DRD, Segawa's disease) is the gene encoding GTP cyclohydrolase I, which is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. Based on our findings on 6-pyruvoyltetrahydropterin synthase (PTS) gene-disrupted (Pts(-/-)) mice, we suggested that the amount of tyrosine hydroxylase (TH) protein in dopaminergic nerve terminals is regulated by the intracellular concentration of BH4. In this present work, we rescued Pts(-/-) mice by transgenic introduction of human PTS cDNA under the control of the dopamine beta-hydroxylase promoter to examine regional differences in the sensitivity of dopaminergic neurons to BH4-insufficiency. The DPS-rescued (Pts(-/-), DPS) mice showed severe hyperphenylalaninemia. Human PTS was efficiently expressed in noradrenergic regions but only in a small number of dopaminergic neurons. Biopterin and dopamine contents, and TH activity in the striatum were poorly restored compared with those in the midbrain. TH-immunoreactivity in the lateral region of the striatum was far weaker than that in the medial region or in the nucleus accumbens. We concluded that dopaminergic nerve terminals projecting to the lateral region of the striatum are the most sensitive to BH4-insufficiency. Biochemical and pathological changes in DPS-rescued mice were similar to those in human malignant hyperphenylalaninemia and DRD.     

Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GCH), 6-pyruvoyltetrahydropterin synthase (PTS), and sepiapterin reductase (SPD). GCH is the rate-limiting enzyme. BH4 is a cofactor for three pteridine-requiring monooxygenases that hydroxylate aromatic L-amino acids, i.e., tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and phenylalanine hydroxylase (PAH), as well as for nitric oxide synthase (NOS). The intracellular concentrations of BH4, which are mainly determined by GCH activity, may regulate the activity of TH (an enzyme-synthesizing catecholamines from tyrosine), TPH (an enzyme-synthesizing serotonin and melatonin from tryptophan), PAH (an enzyme required for complete degradation of phenylalanine to tyrosine, finally to CO2 + H2O), and also the activity of NOS (an enzyme forming NO from arginine), Dominantly inherited hereditary progressive dystonia (HPD), also termed DOPA-responsive dystonia (DRD) or Segawa's disease, is a dopamine deficiency in the nigrostriatal dopamine neurons, and is caused by mutations of one allele of the GCH gene. GCH activity and BH4 concentrations in HPD/DRD are estimated to be 2-20% of the normal value. By contrast, recessively inherited GCH deficiency is caused by mutations of both alleles of the GCH gene, and the GCH activity and BH4 concentrations are undetectable. The phenotypes of recessive GCH deficiency are severe and complex, such as hyperphenylalaninemia, muscle hypotonia, epilepsy, and fever episode, and may be caused by deficiencies of various neurotransmitters, including dopamine, norepinephrine, serotonin, and NO. The biosynthesis of dopamine, norepinephrine, epinephrine, serotonin, melatonin, and probably NO by individual pteridine-requiring enzymes may be differentially regulated by the intracellular concentration of BH4, which is mainly determined by GCH activity. Dopamine biosynthesis in different groups of dopamine neurons may be differentially regulated by TH activity, depending on intracellular BH4 concentrations and GCH activity. The nigrostriatal dopamine neurons may be most susceptible to a partial decrease in BH4, causing dopamine deficiency in the striatum and the HPD/DRD phenotype.     

Oral levodopa rescues retinal morphology and visual function in a murine model of human albinism

Albinism is a group of disorders characterized by pigment deficiency and abnormal retinal development. Despite being a common cause for visual impairment worldwide, there is a paucity of treatments and patients typically suffer lifelong visual disability. Residual plasticity of the developing retina in young children with albinism has been demonstrated, suggesting a post‐natal window for therapeutic rescue. L‐3, 4 dihydroxyphenylalanine (L‐DOPA), a key signalling molecule which is essential for normal retinal development, is known to be deficient in albinism. In this study, we demonstrate for the first time that post‐natal L‐DOPA supplementation can rescue retinal development, morphology and visual function in a murine model of human albinism, but only if administered from birth or 15 days post‐natal age.     

A brain-specific decrease of the tyrosine hydroxylase protein in sepiapterin reductase-null mice--as a mouse model for Parkinson's disease

Sepiapterin reductase (SPR) is an enzyme that acts in the third and final step of tetrahydrobiopterin (BH4) biosynthesis. The human Spr gene locates within the region of 2.5 MB mapped to PARK3, an autosomal dominant form of familial Parkinson’s diseases. In order to explore the role of SPR in the metabolism of BH4, we produced and analyzed Spr-deficient mice. Most of Spr-null mice survived beyond two weeks. Whereas the BH4 contents in the homozygous mutant mice were greatly decreased than those in wild-type and heterozygous mice, the substantial amounts of BH4 were remained even 17 days after delivery. Spr-null mice exhibited severe monoamine deficiencies and a tremor-like phenotype after weaning. The amount of TH protein in the brain of Spr-null mice was less than 10% of wild-type, while TH protein in the adrenal, phenylalanine hydroxylase protein in the liver, and nNOS in the brain were not altered. These data suggest an essential role of SPR in the biosynthesis of BH4, and that the SPR gene could be a candidate gene for PARK3.     

Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain

Estrogen plays an important role during differentiation of midbrain dopaminergic neurons. This is indicated by the presence of estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase within the dopaminergic cell groups. We have previously shown that estrogen regulates the plasticity of dopamine cells through the stimulation of neurite growth/arborization. In this study, we have analyzed the capability of estrogen to influence the activity of developing mouse dopamine neurons. The expression of tyrosine hydroxylase (TH) was assessed by competitive RT-PCR and Western blotting. The developmental expression of TH in the ventral midbrain was studied from embryonic day 15 until postnatal day 15 and revealed highest TH levels early postnatally. This profile coincides with the transient aromatase expression in this brain area. Using cultured midbrain cells, we found that estrogen increased TH mRNA/protein levels. The application of the estrogen receptor antagonist ICI 182,780 resulted in a complete inhibition of estrogen effects. To verify these data in vivo, fetuses were exposed in utero from E15 until birth to the aromatase inhibitor CGS 16949A or to CGS supplemented with estrogen. CGS caused a robust reduction in TH mRNA/protein levels in the midbrain, which could be restored by estrogen substitution. Taken together, our data strongly suggest that estrogen controls dopamine synthesis in the developing nigrostriatal dopaminergic system and support the concept that estrogen is implicated in the regulation of ontogenetic steps but also in the function of midbrain dopamine neurons.     

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.     

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR‐δ/AMPK pathway

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR‐δ) and the adenosine monophosphate (AMP)‐activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild‐type and PPAR‐δ‐deficient mice. Administration of telmisartan up‐regulated levels of PPAR‐δ and phospho‐AMPKα in cultured myotubes. However, PPAR‐δ gene deficiency completely abolished the telmisartan effect on phospho‐AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post‐exercise oxygen consumption, and increased slow‐twitch skeletal muscle fibres in wild‐type mice, but these effects were absent in PPAR‐δ‐deficient mice. The mechanism is involved in PPAR‐δ‐mediated stimulation of the AMPK pathway. Compared to the control mice, phospho‐AMPKα level in skeletal muscle was up‐regulated in mice treated with telmisartan. In contrast, phospho‐AMPKα expression in skeletal muscle was unchanged in PPAR‐δ‐deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR‐δ as a potential therapeutic target for the prevention of type 2 diabetes.     

Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase‐immunoreactive neurons in the sexually dimorphic nucleus in female mice

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase‐immunoreactive (TH‐ir) and kisspeptin‐immunoreactive (kisspeptin‐ir) neurons than males. In this study, we used proteomics analysis and gene‐deficient mice to identify proteins that regulate the number of TH‐ir and kisspeptin‐ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two‐dimensional fluorescence difference gel electrophoresis followed by MALDI‐TOF‐MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4‐knockout (CRMP4‐KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin‐ir neurons in the adult AVPV in either sex. However, the number of TH‐ir neurons was increased in the AVPV of adult female CRMP4‐KO mice as compared with the adult female wild‐type mice. During development, no significant difference in the number of TH‐ir neurons was detected between sexes or genotypes on embryonic day 15, but a female‐specific increase in TH‐ir neurons was observed in CRMP4‐KO mice on PD1, when the sex difference was not yet apparent in wild‐type mice. These results indicate that CRMP4 regulates the number of TH‐ir cell number in the female AVPV. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 502–517, 2013     

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators'' choice.We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein ^1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA ^6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).     

A murine model for human sepiapterin-reductase deficiency

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.     

Dopamine and Norepinephrine in Discrete Areas of the Copper-Deficient Rat Brain1

Abstract: Copper deficiency was induced in post-weaning rats by feeding the dams a low copper diet during gestation and lactation. In confirmation of an earlier study, both dopamine and norepinephrine concentrations in the total brain were approximately 30% lower in deficient than in control rats. Doparnine in the corpus striaturm was depressed nearly 60%, but the concentration of norepinephrine in the hypothalamus was unchanged. Tyrosine concentrations in the striatum, hypothalamus, and total brain were not affected by copper deficiency, suggesting a catalytic defect rather than lack of substrate. Copper repletion restored norepinephrine level in total brain but did not affect the low level of dopamine. The results suggest that copper deficiency depresses a catalytic function of the adrenergic pathways and, further, adversely affects a structural component of the dopaminergic system during development.     

Studies on the mechanisms of L-dopa-induced depletion of 5-hydroxytryptamine in the mouse brain

The administration of L-dopa to mice causes an increase in the brain concentrations of dopa and dopamine which is related temporally to a reduction in the brain concentration of 5HT. These effects occur concurrently with a reduction in the conversion of intravenously administered ³H-tryptophan to ³H-5HT without an alteration in the accumulation of ³H-tryptophan in the brain. The L-dopa-induced changes in the brain concentrations of dopa, dopamine and 5HT are not altered by pretreatment with drugs (imipramine, chlorimipramine, benztropine, cocaine) which inhibit the neuronal uptake of amines. Current evidence suggests that L-dopa is decarboxylated in 5HT neurons to dopamine, which displaces 5HT from intraneuronal storage sites.     

GTP cyclohydrolase I gene,tetrahydrobiopterin, and tysorine hydroxylase gene: Their relations to dystonia and parkinsonism

Catecholamine biosynthesis is regulated by tyrosine hydroxylase (TH) requiring tetrahydrobiopterin (BH4) as the cofactor. We found four (human TH type 1–4) and two isoforms (TH type 1 and 2) in humans and monkeys, while non-primate animals have a single TH corresponding to human TH type 1. BH4 is synthesized from GTP, and GTP cyclohydrolase I (GCH) is the first and regulatory enzyme. Mutations in GCH gene were found to cause both GCH deficiency with autosomal recessive trait and hereditary progressive dystonia with marked diurnal fluctuation (HPD) (Segawa's disease)/or DOPA-responsive dystonia (DRD) with autosomal dominant trait. When GCH activity is decreased to less than 20% of the normal value, the activity of TH in the nigrostriatal dopaminergic neurons may be first decreased resulting in decreases in TH activity and dopamine level, and in the symptoms of HPD/DRD. In contrast to HPD/DRD, juvenile parkinsonism (JP) have normal GCH activity. In Parkinson's disease (PD), GCH, TH, and dopamine in the striatum may decrease in parallel, as the secondary effects caused by cell death. Special issue dedicated to Dr. Kinya Kuriyama.     

Compensatory regulation of dopamine after ablation of the tyrosine hydroxylase gene in the nigrostriatal projection

The tyrosine hydroxylase (TH; EC 1.14.16.2) is a rate-limiting enzyme in the dopamine synthesis and important for the central dopaminergic system, which controls voluntary movements and reward-dependent behaviors. Here, to further explore the regulatory mechanism of dopamine levels by TH in adult mouse brains, we employed a genetic method to inactivate the Th gene in the nigrostriatal projection using the Cre-loxP system. Stereotaxic injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the substantia nigra pars compacta (SNc), where dopaminergic cell bodies locate, specifically inactivated the Th gene. Whereas the number of TH-expressing cells decreased to less than 40% in the SNc 2 weeks after the AAV-Cre injection, the striatal TH protein level decreased to 75%, 50%, and 39% at 2, 4, and 8 weeks, respectively, after the injection. Thus, unexpectedly, the reduction of TH protein in the striatum, where SNc dopaminergic axons innervate densely, was slower than in the SNc. Moreover, despite the essential requirement of TH for dopamine synthesis, the striatal dopamine contents were only moderately decreased, to 70% even 8 weeks after AAV-Cre injection. Concurrently, in vivo synthesis activity of l-dihydroxyphenylalanine, the dopamine precursor, per TH protein level was augmented, suggesting up-regulation of dopamine synthesis activity in the intact nigrostriatal axons. Collectively, our conditional Th gene targeting method demonstrates two regulatory mechanisms of TH in axon terminals for dopamine homeostasis in vivo: local regulation of TH protein amount independent of soma and trans-axonal regulation of apparent L-dihydroxyphenylalanine synthesis activity per TH protein.