Daigo Homma

Birth Regulates the Initiation of Sensory Map Formation through Serotonin Signaling

Although the mechanisms underlying the spatial pattern formation of sensory maps have been extensively investigated, those triggering sensory map formation during development are largely unknown. Here we show that the birth of pups instructively and selectively regulates the initiation of barrel formation in the somatosensory cortex by reducing serotonin concentration. We found that preterm birth accelerated barrel formation, whereas it did not affect either barreloid formation or barrel structural plasticity. We also found that serotonin was selectively reduced soon after birth and that the reduction of serotonin was triggered by birth. The reduction of serotonin was necessary and sufficient for the effect of birth on barrel formation. Interestingly, the regulatory mechanisms described here were also found to regulate eye-specific segregation in the visual system, suggesting that they are utilized in various brain regions. Our results shed light on roles of birth and serotonin in sensory map formation.

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 2.5MB mapped to PARK3, an autosomal dominant form of familial Parkinsons 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.

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.

The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine hydroxylase level in the brain

Tyrosine hydroxylase (TH) is a rate‐limiting enzyme for dopamine synthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. BH4 deficiency leads to the loss of TH protein in the brain, although the underlying mechanism is poorly understood. To give insight into the role of BH4 in the developmental regulation of TH protein level, in this study, we investigated the effects of acute and subchronic administrations of BH4 or dopa on the TH protein content in BH4‐deficient mice lacking sepiapterin reductase. We found that BH4 administration persistently elevated the BH4 and dopamine levels in the brain and fully restored the loss of TH protein caused by the BH4 deficiency in infants. On the other hand, dopa administration less persistently increased the dopamine content and only partially but significantly restored the TH protein level in infant BH4‐deficient mice. We also found that the effects of BH4 or dopa administration on the TH protein content were attenuated in young adulthood. Our data demonstrate that BH4 and catecholamines are required for the post‐natal augmentation of TH protein in the brain, and suggest that BH4 availability in early post‐natal period is critical for the developmental regulation of TH protein level.

GTP Cyclohydrolase Regulation: Implications for Brain Development and Function

Tetrahydrobiopterin (BH4) is essential for the biosynthesis of dopamine, noradrenaline, and serotonin, which serve as cofactors for tyrosine hydroxylase (TH) and tryptophan hydroxylase. GTP cyclohydrolase (GCH) is the first and rate-limiting enzyme for BH4 biosynthesis. Genetic defects in an allele of the GCH gene can result in dopa-responsive dystonia due to partial BH4 deficiency. To explore the transcriptional control of the GCH gene, we analyzed the signaling pathway. Bacterial lipopolysaccharide (LPS) greatly enhanced the expression of GCH in RAW264 cells, and the induction of GCH by LPS was suppressed by treatment with either a MEK1/2 inhibitor or an inhibitor for the NF-κB pathway. Next, we analyzed two types of biopterin-deficient transgenic mice. We found that both mice exhibited motor disorders with slight differences. Dopamine and TH protein levels were markedly and concurrently increased from birth (P0) to P21 in wild-type mice, and these increases were abolished in both types of biopterin-deficient mice. Our results suggest that the developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to the high dependence of dopaminergic development on the availability of BH4.

Chapter Three - GTP Cyclohydrolase Regulation: Implications for Brain Development and Function

Tetrahydrobiopterin (BH4) is essential for the biosynthesis of dopamine, noradrenaline, and serotonin, which serve as cofactors for tyrosine hydroxylase (TH) and tryptophan hydroxylase. GTP cyclohydrolase (GCH) is the first and rate-limiting enzyme for BH4 biosynthesis. Genetic defects in an allele of the GCH gene can result in dopa-responsive dystonia due to partial BH4 deficiency. To explore the transcriptional control of the GCH gene, we analyzed the signaling pathway. Bacterial lipopolysaccharide (LPS) greatly enhanced the expression of GCH in RAW264 cells, and the induction of GCH by LPS was suppressed by treatment with either a MEK1/2 inhibitor or an inhibitor for the NF-κB pathway. Next, we analyzed two types of biopterin-deficient transgenic mice. We found that both mice exhibited motor disorders with slight differences. Dopamine and TH protein levels were markedly and concurrently increased from birth (P0) to P21 in wild-type mice, and these increases were abolished in both types of biopterin-deficient mice. Our results suggest that the developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to the high dependence of dopaminergic development on the availability of BH4.

GTP cyclohydrolase regulation: implications for brain development and function.

Tetrahydrobiopterin (BH4) is essential for the biosynthesis of dopamine, noradrenaline, and serotonin, which serve as cofactors for tyrosine hydroxylase (TH) and tryptophan hydroxylase. GTP cyclohydrolase (GCH) is the first and rate-limiting enzyme for BH4 biosynthesis. Genetic defects in an allele of the GCH gene can result in dopa-responsive dystonia due to partial BH4 deficiency. To explore the transcriptional control of the GCH gene, we analyzed the signaling pathway. Bacterial lipopolysaccharide (LPS) greatly enhanced the expression of GCH in RAW264 cells, and the induction of GCH by LPS was suppressed by treatment with either a MEK1/2 inhibitor or an inhibitor for the NF-κB pathway. Next, we analyzed two types of biopterin-deficient transgenic mice. We found that both mice exhibited motor disorders with slight differences. Dopamine and TH protein levels were markedly and concurrently increased from birth (P0) to P21 in wild-type mice, and these increases were abolished in both types of biopterin-deficient mice. Our results suggest that the developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to the high dependence of dopaminergic development on the availability of BH4.

Alterations in the monoamines and the synthesizing enzymes in the postnatal developmental period of the brain of Spr-/- mice

Method: Male 8-weeks old C57BL/6J mice were purchased from CLEA (Japan Inc.). They were housed under a standardized light/dark cycle at room temperature of 24 ± 1 ◦C and a humidity of 60 ± 10% with food and water ad libitum. CPu and SCN were obtained at 09:00, 13:00, 17:00, 21:00, 01:00, 05:00. The mRNA levels of clock genes and PD-related genes in CPu and SCN were measured by RT-PCR. Result: MPTP-treated mice exhibited a loss of TH-immunoreactive neurons in the SN and TH-immunoreactive fibers in CPu. The levels of DA, DOPAC and HVA in CPu of MPTP-treated mice dramatically decreased. 24-hr rhythms in body temperature and locomotor activity were changed in MPTP-induced PD model mouse. Furthermore, 24-hr rhythm of clock genes in CPu and SCN were altered in MPTP-induced PD model mouse. Conclusions: These findings indicate that the circadian clock system is altered in PD. On the other hand, such disruption in 24-hr rhythm of clock genes may be underlying the several non-motor symptoms such as sleep disturbances and depression in PD.

Regulation of dopamine synthesis by alteration in the TH protein level

Dopamine transporter (DAT) internalization is a mechanism underlying the decreased dopamine reuptake caused by addictive drugs like methamphetamine (METH). We found that Piccolo was overexpressed in the nucleus accumbens (NAc) of the mice repeatedly administrated with METH. Expression of Piccolo C2A domain attenuated METH-induced inhibition of dopamine uptake in PC12 cells expressing human DAT. Consistent with this, it slowed down the accelerated DAT internalization induced by METH, thus maintaining the presentation of plasmalemmal DAT. In immunostaining and structural modeling Piccolo C2A domain displays an unusual feature of sequestering membrane phosphatidylinositol 4,5-bisphosphate. Together, our results indicate that Piccolo upregulation induced by METH represents a homeostatic response in the NAc to excessive dopaminergic transmission. Piccolo C2A domain may act as a cytoskeletal regulator for plasmalemmal DAT internalization, which may underlie its contributions in behavioral plasticity.