Keiji Mori

Peripheral injection of risperidone, an atypical antipsychotic, alters the body weight gains of rats.

1. Risperidone is an atypical antipsychotic drug that possesses 5-hydroxytryptamine 5-HT2 receptor antagonism combined with milder dopamine D2 receptor antagonism. 2. Excessive bodyweight gain is one of the side-effects of antipsychotics. Risperidone treatment causes a greater increase in the body mass of patients than treatment with conventional antipsychotics, such as haloperidol. Therefore, the present study was undertaken in order to address the aetiology of the risperidone-induced bodyweight change in rats by examining the expression of leptin, an appetite-regulating hormone produced in white adipose tissue (WAT), and uncoupling protein (UCP)-1, a substance promoting energy expenditure in the brown adipose tissues (BAT). 3. Eight-week-old male rats were injected subcutaneously with risperidone (0.005, 0.05 or 0.5 mg/kg) twice daily for 21 days. Both bodyweight and food intake were monitored daily. On day 21, rats were decapitated and their serum leptin and prolactin concentrations were measured. Expression levels of leptin, Ucp1 and beta3-adrenoceptor (beta3-AR) genes in WAT and BAT were quantified using real-time polymerase chain reaction amplification. 4. Injection of 0.005 mg/kg risperidone into rats increased food intake and the rate of bodyweight gain, as well as the augmentation of leptin gene expression in WAT. Injection of 0.05 mg/kg risperidone increased food intake and leptin gene expression in WAT, but the rate of bodyweight gain was not affected. Injection of 0.5 mg/kg risperidone caused a reduction in bodyweight gain, as well as enhanced Ucp1 gene expression in BAT and serum prolactin concentrations. The serum leptin concentration and beta3-AR gene expression in WAT and BAT were not affected by injection of 0.5 mg/kg risperidone. 5. Although the changes in food intake observed in risperidone-injected rats were rationalized neither by serum leptin nor prolactin concentrations, the reduction in the rate of bodyweight gain following injection of 0.5 mg/kg can be explained, in part, by increased energy expenditure, as revealed by the remarkable increase in the UCP-1 mRNA expression level in BAT. The role of leptin in risperidone-induced alterations in bodyweight gain remain to be clarified.Ota et al . 1 recently addressed the important issue of developing an animal (rat) model of weight gain induced by novel ‘atypical’ antipsychotics Their study involved risperidone, which is known to induce weight gain in humans. 2 Most, but not all, novel antipsychotics induce weight gain, 3 although the weight gain induced by risperidone in humans is smaller in magnitude that that induced by olanzapine. 4 Olanzapine-induced weight gain has recently been modelled by us in rats 5 and it would be a considerable advantage for work in this area if risperidone-induced weight gain was also modelled effectively in rodents. Novel antipsychotic-induced weight gain has very serious clinical implications, being associated with enhanced morbidity and mortality, as well as reduced compliance. 6 However, the causes of the weight gain induced by many novel antipsychotics remain enigmatic. Many of the wide range of receptors at which novel antipsychotics act are implicated in the control of food intake (e.g. -adrenoceptors, various serotonergic and dopaminergic receptors and histamine H 1 receptors). However, it has, to date, proved impossible, on the basis of clinical studies, to determine the role of any one such receptor in antipsychotic-induced weight gain. 7 It is well documented that the weight gain induced by novel antipsychotics is associated with abnormalities in various hormonal systems, including leptin, 8 insulin 9 and prolactin. 7 Thus, it seems most likely that the development of animal models of novel antipsychotic-induced weight gain in which measures of food intake and bodyweight are supplemented by metabolic and endocrine measures, exactly as in the study by Ota et al ., 1 will prove a very powerful approach with which to advance this field. Indeed, in a recent major review of this area, Baptista et al . 7 highlighted the need to develop such models as a primary current ‘research perspective’. Thus, the type of empirical research reported by Ota et al . 1 is to be highly commended. It is, however, regrettable that examination of the work reported by Ota et al . 1 suggests that the conclusions they have drawn from their data relating to bodyweight and food intake are not supported by the statistical analysis presented and, thus, attempts to relate these findings to metabolic and endocrinological measures may be invalid. In their study, they administered risperidone for 21 days to four groups of rats treated with either vehicle or one of three doses of risperidone. Bodyweights and food intake were recorded daily. These data are reported as being analysed by repeated-measures

Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine to l-dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal actions of CA. Thus, TH is related to various neuro-psychiatric diseases such as TH deficiency, Parkinson’s disease (PD), and schizophrenia. Four isoforms of human TH (hTH1–hTH4) are produced from a single gene by alternative mRNA splicing in the N-terminal region, whereas two isoforms exist in monkeys and only a single protein exist in all non-primate mammals. A catalytic domain is located within the C-terminal two-thirds of molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain. The catalytic activity of TH is end product inhibited by CA, and the phosphorylation of Ser residues (Ser19, Ser31, and especially Ser40 of hTH1) in the N-terminus relieves the CA-mediated inhibition. Ota and Nakashima et al. have investigated the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability by producing various mutants of the N-terminus of hTH1. The expression of the following three enzymes, TH, GTP cyclohydrolase I, which synthesizes the tetrahydrobiopterin cofactor of TH, and aromatic-l-amino acid decarboxylase, which produces DA from l-dopa, were induced in the monkey striatum using harmless adeno-associated virus vectors, resulting in a remarkable improvement in the symptoms affecting PD model monkeys Muramatsu (Hum Gene Ther 13:345–354, 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.

Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme.

Abstract: Tyrosine hydroxylase (TH), which converts L‐tyrosine to L‐DOPA, is a rate‐limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N‐terminus of TH that determines the efficiency of dopamine inhibition, wild‐type and N‐terminal 35‐, 38‐, and 44‐amino acid‐deleted mutants (del‐35, del‐38, and del‐44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichla coli and purified as a tetrameric form by affinity and size‐exclusion chromatography. The fused‐form wild‐type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N‐terminus‐deleted forms were about one‐fourth of the wild‐type value, there was no difference in Michaeiis constants for L‐tyrosine or (6R)‐(L‐erythro‐1,1′,2′‐dihydroxypropyl)‐2‐amino‐4‐hydroxy‐5,6,7,8‐tetrahydropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N‐terminus‐deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N‐terminus made the enzyme more resistant to dopamine inhibition than the wild‐type or del‐35 TH form. Dopamine bound to the del‐38 more than to the del‐35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del‐38 than from the del‐35 TH form. These observations suggest that the amino acid sequence Gly36‐Arg37‐Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.

Phosphorylation of the N-terminal portion of tyrosine hydroxylase triggers proteasomal digestion of the enzyme.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminal region of TH affects this stability. TH molecules phosphorylated at their Ser31 and Ser40 were localized predominantly in the cytoplasm of PC12D cells. However, those molecules phosphorylated at Ser19 were found mainly in the nucleus, whereas they seemed to be negligible in the cytoplasm. The inhibition of proteasomes increased the quantity of TH molecules phosphorylated at their Ser19 and Ser40, although it did not increase that of TH molecules or that of TH phosphorylated at its Ser31. The inhibition of autophagy did not affect the amount of the TH molecule or that of its three phosphorylated forms. Deletion mutants of human TH type-1 lacking the N-terminal region containing the three phosphorylation sites possessed high stability of the enzyme in PC12D cells. These results suggest that the phosphorylation of the N-terminal portion of TH regulates the degradation of this enzyme by the ubiquitin-proteasome pathway.

Determination of tetrahydrobiopterin in murine locus coeruleus by HPLC with fluorescence detection

Tetrahydrobiopterin in the murine locus coeruleus was measured as its fully oxidized form, biopterin, using a HPLC coupled to a fluorescence detector, because tetrahydrobiopterin itself cannot be detected by such means. The differential oxidization method distinguished tetrahydrobiopterin-derived biopterin and dihydrobiopterin-derived biopterin. The protocol reported here is a rapid and sensitive method that facilitates the measurement of tissue and/or cellular tetrahydrobiopterin. Using this assay protocol, we were able to detect and quantify variations in the tetrahydrobiopterin content in the murine locus coeruleus.

Effect of peripherally administered lipopolysaccharide (LPS) on GTP cyclohydrolase I, tetrahydrobiopterin and norepinephrine in the locus coeruleus in mice.

Lipopolysaccharide (LPS), an endotoxin released from the outer membranes of Gram-negative bacteria, triggers cells to synthesize and release inflammatory cytokines that may progress to septic shock in vivo. We found that LPS enhances tetrahydrobiopterin (BH4) biosynthesis by inducing the biosynthetic enzyme GTP cyclohydrolase I (GCH) in vitro in the mouse neuroblastoma cell line N1E-115. Furthermore, we observed that gene expression of GCH in the locus coeruleus (LC) in mice was enhanced by peripheral administration of LPS, resulting in increased concentrations of BH4, and norepinephrine, and its metabolite 4-hydroxy-3-methoxyphenylglycol (MHPG). These results suggest that tyrosine hydroxylase (TH) activity is increased by increased content of BH4 due to enhanced mRNA expression of GCH in the LC resulting in the increase in norepinephrine in the LC during endotoxemia. LPS in blood may act as a stressor to increase norepinephrine biosynthesis in the mouse LC.

A possible pathophysiological role of tyrosine hydroxylase in Parkinson's disease suggested by postmortem brain biochemistry: a contribution for the special 70th birthday symposium in honor of Prof. Peter Riederer.

Postmortem brain biochemistry has revealed that the main symptom of movement disorder in Parkinson’s disease (PD) is caused by a deficiency in dopamine (DA) at the nerve terminals of degenerating nigro-striatal DA neurons in the striatum. Since tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of DA, TH may play an important role in the disease process of PD. DA regulated by TH activity is thought to interact with α-synuclein protein, which results in intracellular aggregates called Lewy bodies and causes apoptotic cell death during the aging process. Human TH has several isoforms produced by alternative mRNA splicing, which may affect activation by phosphorylation of serine residues in the N-terminus of TH. The activity and protein level of TH are decreased to cause DA deficiency in the striatum in PD. However, the homo-specific activity (activity/enzyme protein) of TH is increased. This increase in TH homo-specific activity suggests activation by increased phosphorylation at the N-terminus of the TH protein for a compensatory increase in DA synthesis. We recently found that phosphorylation of the N-terminal portion of TH triggers proteasomal degradation of the enzyme to increase TH turnover. We propose a hypothesis that this compensatory activation of TH by phosphorylation in the remaining DA neurons may contribute to a further decrease in TH protein and activity in DA neurons in PD, causing a vicious circle of decreasing TH activity, protein level and DA contents. Furthermore, increased TH homo-specific activity leading to an increase in DA may cause toxic reactive oxygen species in the neurons to promote neurodegeneration.

Resistance to excessive bodyweight gain in risperidone-injected rats.

1. The present study was carried out to explain the resistance of rats injected subcutaneously with risperidone, the atypical antipsychotic drug, for 21 consecutive days at 0.1 mg/kg per day (a dose equivalent to the one used for patients) to result in an excessive bodyweight despite the increase in diet‐uptake in rats against risperidone‐induced decrease in body temperature.

Expression of GTP cyclohydrolase I in murine locus ceruleus is enhanced by peripheral administration of lipopolysaccharide

Among the enzymes involved in the system for catecholamine biosynthesis, GTP cyclohydrolase I (GCH) contributes to the system as the first and rate-limiting enzyme for the de novo biosynthesis of tetrahydrobiopterin (BH4), which is the cofactor for tyrosine hydroxylase (TH). Therefore, we investigated whether the endotoxemia caused by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) can modulate BH4 production in the norepinephrine nuclei, i.e. the locus ceruleus (LC; A6) and central caudal pons (A5), in C3H/HeN mice and whether such a change in BH4, if any, can result in the modification of norepinephrine production in these nuclei. After a 5-microg i.p. injection of LPS, the protein expression of GCH and TH in both nuclei was examined by immunohistochemistry. The staining intensity of GCH-positive cells increased at 6 h, whereas no significant change in the staining intensity of TH-positive cells was detected. Next, we measured the contents of BH4, norepinephrine, and its metabolites 4-hydroxy-3-methoxyphenylglycol (MHPG) and DL-4-hydroxy-3-methoxymandelic acid (VMA) in these nuclei after LPS i.p. injection. The BH4 content increased to a statistically significant level at 2 and 4 h after the injection. The contents of MHPG and VMA also showed a time-course similar to that of BH4. These data can be rationalized to indicate that an increased supply of BH4 in the LC increased TH activity and resulted in an increase in norepinephrine production rate at the site. This is the first report that sheds light on BH4 as a molecule that intervenes during endotoxemia to increase norepinephrine production rate in the LC.

Positive charge intrinsic to Arg37-Arg38 is critical for dopamine inhibition of the catalytic activity of human tyrosine hydroxylase type 1

Tyrosine hydroxylase (TH), which converts L‐tyrosine to L‐3,4‐dihydroxyphenylalanine, is a rate‐limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N‐terminal sequence Arg37‐Arg38 of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg37‐Arg38 site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum. The prediction of the secondary structure of N‐terminal residues 1–60 by computer software specified the location of the Arg37‐Arg38 sequence in the turn intervening between the two α‐helices (residues 16–29 and residues 41–59). These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine.