Naoki Hattori

Expression, regulation and biological actions of growth hormone (GH) and ghrelin in the immune system

Immune and neuroendocrine systems have bidirectional communications. Growth hormone (GH) and an orexigenic hormone ghrelin are expressed in various immune cells such as T lymphocytes, B lymphocytes, monocytes and neutrophils. These immune cells also bear receptors for hormones: growth hormone receptor (GHR) for GH and growth hormone secretagogue receptor (GHS-R) for ghrelin. The expression of GH in immune cells is stimulated by ghrelin as in anterior pituitary cells, whereas the regulation of GH secretion in the immune system by other peptides seems to be different from that in the anterior pituitary gland. Cytokines and mitogens enhance GH secretion from immune cells. GH has several biological actions in the immune system: enhancing thymopoiesis and T cell development, modulating cytokine production, enhancing B cell development and antibody production, priming neutrophils and monocytes for superoxide anion secretion, enhancing neutrophil adhesion and monocyte migration and anti-apoptotic action. Biological actions of ghrelin include attenuation of septic shock and anti-inflammatory actions, modulating phagocytosis, and enhancing thymopoiesis. The effect of ghrelin may be direct or through GH production, and that of GH may be direct or through insulin like growth factor-I (IGF-I) production. Elucidation of the roles of GH and ghrelin in the immune system may shed light on the treatment and prevention of immunological disorders such as AIDS and organ damages due to obesity/ageing-related chronic inflammation.

Macroprolactinaemia: prevalence and aetiologies in a large group of hospital workers

Objectiveu2002 Macroprolactinaemia is one of the causes of hyperprolactinaemia and often leads to misdiagnosis and inappropriate treatment, but the aetiologies are unclear. The objective of the study was to determine the prevalence of macroprolactinaemia in a healthy population and to investigate the mechanisms underlying the development of macroprolactin.

Anti‐prolactin (PRL) autoantibodies suppress PRL bioactivity in patients with macroprolactinaemia

Objectiveu2002 Macroprolactinaemia, mainly caused by anti‐prolactin (PRL) autoantibodies, is frequently found in patients with hyperprolactinaemia. Characteristically, these patients lack clinical symptoms of hyperprolactinaemia, but the serum bioactive PRL concentrations in vitro measured by the Nb2 bioassay are usually high. In this study, we investigated the causes of the discrepancy and the true biological features of macroprolactin.

Macroprolactinaemia in patients with hyperprolactinaemia: composition of macroprolactin and stability during long-term follow-up.

Objectiveu2002 Macroprolactin, which comprises immunoglobulin G (IgG) or anti‐prolactin (PRL) autoantibody‐bound PRL, is one of the causes of hyperprolactinaemia. This study evaluated the composition and stability of macroprolactin and determined whether the condition was long lasting. We also investigated whether we could predict the composition of macroprolactin based on the ratio of polyethylene glycol (PEG)‐precipitable PRL.

The natural history of macroprolactinaemia

OBJECTIVEnMacroprolactinaemia is a condition in which serum prolactin (PRL) consists mainly of large molecular weight PRL (macroPRL). The aim of this study was to examine the natural history of macroprolactinaemia.nnnDESIGN AND PARTICIPANTSnSix hundred and fifty-four hospital workers participated in this study, including 27 subjects with macroprolactinaemia and 627 controls. MacroPRL and serum PRL concentrations were evaluated over a 4-year period. The ratio of macroPRL was examined by the polyethylene glycol (PEG) method and gel filtration chromatography. IgG-bound PRL and anti-PRL autoantibodies were examined by protein G and (125)I-PRL binding studies respectively.nnnRESULTSnOver the 4 years of the study, all 27 macroprolactinaemic subjects had persistent macroprolactinaemia without the development of raised free PRL, while none of the 627 controls developed macroprolactinaemia. The ratios of PEG-precipitable PRL and IgG-bound PRL did not significantly change, but (125)I-PRL binding ratios significantly increased. As a whole, total and free serum PRL concentrations did not significantly change in subjects with macroprolactinaemia over the 4-year period. However, hyperprolactinaemia developed in five of the 18 macroprolactinaemic subjects who were initially normoprolactinaemic along with an increase in anti-PRL autoantibody titres. One of the remaining nine macroprolactinaemic subjects who were initially hyperprolactinaemic showed a decrease in serum PRL concentrations, which occurred concomitantly with a decrease in the anti-PRL autoantibody titre.nnnCONCLUSIONSnMacroprolactinaemia may develop before middle age and is likely a chronic condition leading to hyperprolactinaemia.

Development of insulin antibodies and changes in titers over a long-term period in patients with type 2 diabetes.

BACKGROUNDnRecently, insulin analogs have become widely used for the treatment of diabetes. The aim of this study was to determine differences in the antigenicity of insulin analogs and long-term changes in titers in patients with type 2 diabetes.nnnMETHODSnInsulin antibodies were examined using polyethylene glycol and protein G methods in 381 patients with type 2 diabetes.nnnRESULTSnInsulin antibodies were detected in 48 of 118 patients (40.7%) who used insulin, and insulin glargine and aspart were more antigenic. Insulin antibodies were unexpectedly found in seven of 263 patients (2.7%) who had never used insulin. Serum insulin concentrations in patients with insulin antibodies were significantly higher than those without them. Two years after the initial evaluation, insulin antibodies were still positive in 92.7% of patients who used insulin; while, they disappeared in all patients who had never used insulin. A patient who stopped insulin injections 6years ago was found to be positive for insulin antibodies at the first evaluation as well as 2years later.nnnCONCLUSIONSnInsulin glargine and aspart induced insulin antibodies more frequently, and insulin antibodies remained in patients for a long time. Insulin antibodies should be suspected even in patients not currently on insulin therapy.

Macro TSH in patients with subclinical hypothyroidism

TSH is a sensitive indicator of thyroid function. In subclinical hypothyroidism, however, serum TSH concentrations are elevated despite normal thyroid hormone levels, and macro TSH is one of the causes. This study aimed to clarify the prevalence and nature of macro TSH in patients with subclinical hypothyroidism.

Intrastriatal injection of 6-hydroxydopamine induces impairments of spatial memory and causes excessive expression of ezrin in hippocampus in mice

s / Neuroscience Research 71S (2011) e108–e415 e189 P2-q21 Region specific expression of the defense enzymes MTH1, OGG1 and MUTYH against oxidative damage in nucleic acids in the substantia nigra of mouse Zijing Sheng , Yusaku Nakabeppu Div. Neurofunc. Genomics, MIB, Kyushu Univ., Fukuoka, Japan Oxidative DNA damage has been inferred to be involved in the neurodegenerative pathway of Parkinson’s disease. Dopaminergic neurons are believed to be particularly vulnerable to oxidative stress. To counteract oxidative damage to nucleic acids, human and rodents are equipped with three defense enzymes, MTH1, OGG1 and MUTYH. MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP to their monophosphate forms, thus sanitizing nucleotide pools. OGG1, an 8-oxoguanine DNA glycosylase, prevents buildup of 8-oxoguanine in both nuclear and mitochondrial genomes. MUTYH, an adenine DNA glycosylase, excises adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine. Our previous studies showed that MTH1-deficient mice exhibited a greater accumulation of 8-oxoguanine in substantia nigra after MPTP administration (Yamaguchi et al., 2006), indicating that MTH1 plays a protective role in the substantia nigra, however, expression of these enzymes has yet to be clarified in the substantia nigra. In the present study, the expression of MTH1, OGG1 and MUTYH was examined immunohistochemically in the mouse substantia nigra. We found that large number of neurons in the substantia nigra pars compacta (SNc) express MTH1, OGG1 or MUTYH, respectively, whereas only small number of neurons in the ventral tegmental area (VTA) express these enzymes. Double-immunodetection of tyrosine hydroxylase (TH) revealed that many TH-positive neurons in SNc, but not in VTA, co-express MTH1, OGG1 or MUTYH. In addition, only a few microglial cells were detected in SNc, and these cells also express substantial levels of MTH1, OGG1 or MUTYH. On the other hand, astroglial cells abundantly populated in substantia nigra pars reticulata but not in SNc, and these cells express very low levels of MTH1, OGG1 or MUTYH. These results strongly suggest that these defense enzymes contribute to the maintenance of genomic integrity in dopaminergic neurons and microglia in the substantia nigra. Research fund: KAKENHI 22221004,Global COE Program. doi:10.1016/j.neures.2011.07.814 P2-r01 Effect of serotonergic drugs on motor function in a hemiparkinsonian rat model Masatoshi Inden 1,2 , Kazuyuki Takata 2, Takashi Taniguchi 2, Yoshihisa Kitamura 2 1 Clin. Pharmacol. Lab., College of Pharm. Sci., Ritsumeikan Univ., Kusatsu, Japan 2 Dept. Neurobiol., Kyoto Pharm. Univ., Kyoto, Japan Parkinson’s disease (PD) is an age-related neurodegenerative disease that is characterized by relatively selective nigrostriatal dopamine (DA) neurodegeneration. l-Dihydroxyphenylalanine (l-DOPA) is considered the gold standard for the treatment of PD. However, long-term administration of l-DOPA can induce abnormal side effects. On the other hand, selective serotonin reuptake inhibitors (SSRIs) have gained tremendous popularity in the treatment of depression in PD. SSRIs are thought to influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic neural networks, which are complex and not yet fully understand. In this study, rats received a unilateral intranigral injection of either 6-hydroxydopamine (6-OHDA) or vehicle. In addition, they further received a bilateral intraventricular injection of either 5,7-dihydroxytryptamine (5,7-DHT) or vehicle. In 6-OHDA-lesioned rats, lDOPA-induced rotational behavior was significantly decreased when these rats further received a bilateral intraventricular injection of 5,7-DHT. We also demonstrated that pre-treatment with fluoxetine, significantly suppressed l-DOPA-induced ERK1/2 and histone H3 phosphorylation, as well as l-DOPA-induced rotational behavior in 6-OHDA-lesioned rats. These data indicate that l-DOPA-derived DA, released as a false transmitter from 5HT terminals, enhances levels of phosphorylated ERK and histone H3, and this results in abnormal behavior. Fluoxetine may attenuate these effects of a false transmitter via 5-HT receptor. In further support of this supposition, these effects of fluoxetine were abolished by pre-treatment with WAY 100135, a 5-HT1A antagonist. These results suggest that fluoxetine may influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic neuronal networks. doi:10.1016/j.neures.2011.07.815 P2-r02 Intrastriatal injection of 6-hydroxydopamine induces impairments of spatial memory and causes excessive expression of ezrin in hippocampus in mice Yosuke Matsumoto 1 , Hiroki Murakami 1, Naoki Hattori 2, Kanji Yoshimoto 3, Shinji Asano 1, Masatoshi Inden 2 1 Mol. Physiol. Lab., Col. of Pharm. Sci., Ritsumeikan Univ., Kusatsu, Japan 2 Clin. Pharmacol. Lab., Col. of Pharm. Sci., Ritsumeikan Univ., Kusatsu, Japan 3 Dept. Legal. Med., Kyoto Pref. Univ. Med., Kyoto, Japan It is widely recognized that motor symptoms are accompanied by cognitive deficits in Parkinson’s disease (PD) patients even at the early stages of the disease. Dopamine (DA) lesions, obtained through dorsal striatum 6-hyroxydopamine (6-OHDA) administrations, have been found to impair memory in the spatial version of the Morris water maze in rodents of PD model. Ezrin, radixin, and moesin are collectively known as ERM proteins. Although ERM proteins have important implications in cell-shape determination and relevant signaling pathway, they have not been studied in the hippocampus in association with visuo-spatial memory impairments. The purpose of the present study is to examine whether the expression level of ERM proteins in the hippocampus is changed by an intrastriatal injection of 6-OHDA in mice. At 6 or 18 weeks after a microinjection of 6OHDA, we performed a water maze testing to examine the function of a visuo-spatial memory. The intrastriatal injection of 6-OHDA induced spatial memory impairments. After the water maze testing, we performed biochemical analyses to examine DA neurodegeneration. The microinjection of 6-OHDA reduced the striatal DA content by approximately 70%. We also performed western blotting of ERM proteins to determine the changes in their expression levels in the hippocampus. The protein level of ezrin was increased approximately 2.2-fold by intrastriatal injection of 6-OHDA whereas expression levels of other ERM proteins, moesin and radixin, were unaffected by intrastriatal injection of 6-OHDA. These results suggest that excessive ezrin may influence cognitive dysfunction in the pathological condition of PD. doi:10.1016/j.neures.2011.07.816 P2-r03 Decreased anxiety-like behavior in alpha-synuclein BAC transgenic mice recapitulates early non-motor symptoms in Parkinson disease Hodaka Yamakado 1 , Yasuhiro Moriwaki 2, Nobuyuki Yamasaki 3, Junko Kurisu 4, Tsuyoshi Miyakawa 5, Kengo Uemura 1, Haruhisa Inoue 6, Ryosuke Takahashi 1 1 Dept. of Neurol., Grad. Sch. of Med., Kyoto Univ., Kyoto 2 Dept. of Pharmacol., Keio Univ., Tokyo 3 Dept. of Psychiatry, Grad. Sch. of Med., Kyoto Univ., Kyoto 4 Lab. of Dev. Neurobiol., Grad. Sch. of Biostudies, Kyoto Univ., Kyoto 5 Div. of System Med. Sci., Inst. for Comp. Med. Sci., Fujita Health Univ., Toyoake, Aichi 6 CiRA, Kyoto Univ., Kyoto Alpha-synuclein (a-syn) is the main component of Lewy bodies, a pathological hallmark of Parkinson disease (PD), and its gene mutations (PARK1) as well as multiplications (PARK4) can cause familial forms of PD. Recently, asyn gene has also been identified as a genetic risk factor for idiopathic PD by genome-wide association studies. To make a mouse model for PARK4 as well as for idiopathic PD, we generated genome-based human a-syn BAC (bacterial artificial chromosome) transgenic mice (a-syn BAC tg mice) harboring the entire human a-syn gene as well as its gene expression regulatory regions. These mice are unique in that human a-syn is overexpressed in a physiological manner, as observed in human brains, both temporally and spatially. The a-syn BAC tg mice exhibited decreased anxiety-like behaviors and the biochemical analysis of tg mice brains showed an increase in DAT and SERT expression which may be responsible for these behavioral changes. At present increasing attention is being focused on the non-motor symptoms of PD which include autonomic failure, sleep abnormalities, and mood changes that often emerge prior to motor symptoms. The behavioral changes found in our a-syn BAC tg mice may reflect the non-motor symptoms in the preclinical stage of PD. Our a-syn BAC tg mice could be used as a valuable tool to evaluate a-syn gene dosage effects in vivo and a useful model for early therapeutic interventions against non-motor symptoms in the early stages of PD. doi:10.1016/j.neures.2011.07.817

918 Characterization of neuronal CI− pump

918 CHARACTERIZATION OF NEURONAL ClPUMP. XUN-TING ZENG, NAOKI HATTORI AND CHIYOKO INAGAKL mpartment omamacology, Kansai Medical University, Motiguchi, Osaka 570, Japan. ~. Chloride channel opening-induced hyperpolarizm, 0 Inhibitory postsynaptic potential is dependent on the inwardly directed Clgradient across the neuronal cell membrane, which suggests the presence of an outwardly directed active Cltransport system. Previously we reported ATP-dependent Cluptake activity as a candidate for such an active Cl‘ extrusion pump system. In this study, we purified the neuronal Clpump, and Immunologically exammed the localization of the Clpump among tissues The Clpump was solubilized using MEGA-10, and purified by HPLC with an amon exchanger column, Mono-Q, and polyacrylamide gel electrophoresis (PAGE). The purified 520 and 580 kDa proteins showed ATP-dependent 36CIuptake activity in reconstituted liposomes, and yielded 97, 71, 66, 55, 5 1 and 41 kDa peptides as major subunits m SDS-PAGE. The antibody against the purified Clpump was reactive to the 5 1 kDa subumt from cerebrum, cerebellum, splnal cord, and renal medulla and cortex, but such lmmunoreactivity was not found in the lung, intestme, stomach, liver, urinary bladder, adrenal gland and spleen In the braln, Clpump-like immunoreactivity was strongly detected on the cell membranes of cerebral cortical neurons, cerebellar Purkmle cells and spinal motoneurons. These findings suggest that the Clpump is located mainly on the plasma membrane which has Clextrusion activity.