Ichiro Kawahata

Two Distinct Mechanisms for Actin Capping Protein Regulation—Steric and Allosteric Inhibition

A crystallographic study reveals the structural basis for regulation by two different inhibitors of the actin capping protein, a critical factor controlling actin-driven cell motility.

Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitin–proteasome system in PC12D cells

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson’s disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin–proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-40Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin–proteasome system in PC12D cells.

Dopamine or biopterin deficiency potentiates phosphorylation at 40Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

SLC10A4 is a protease-activated transporter that transports bile acids.

SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. We performed multiple amino acid alignments and examined the relationships between the SLC10 proteins. The extracellular N-terminus of SLC10A4 was predicted to be relatively longer at the amino acid level than those of SLC10A1, SLC10A2 and SLC10A6. We examined the relationship between the N-terminus and transport activity of SLC10A4. Rat Slc10a4 is predominantly expressed in rat cholinergic neurons; therefore, TE671 cells expressing the acetylcholine receptor and acetylcholinesterase were used. After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. Substrates were added to the cells, and their uptake was quantified by liquid chromatography tandem mass spectrometry. Lithocholic acid (LCA) and taurocholic acid (TCA) uptake and cell death effects of LCA were increased by thrombin treatment. After RNA interference treatment for SLC10A4, bile acid uptake was also quantified. In consequence, increases in the LCA and TCA uptake did not occur. Therefore, SLC10A4 may have low activity but becomes activated by proteases, including thrombin, following cleavage. We have demonstrated that SLC10A4 appears to be a protease-activated transporter and transports bile acids.

Ginsenoside Rg1-induced antidepressant effects involve the protection of astrocyte gap junctions within the prefrontal cortex

ABSTRACT Ginsenoside Rg1 (Rg1) exhibits antidepressant‐like activity by increasing neurogenesis and dendritic spine density without discernible side effects. However, the molecular mechanisms underlying Rg1 antidepressant activity remain poorly understood. As the dysfunction of gap junctions between astrocytes in the prefrontal cortex (PFC) is implicated in major depression disorder, the aim of this study was to investigate the effects of Rg1 on astrocyte gap junctions in the PFC. Rats exposed to chronic unpredictable stress (CUS) were administered Rg1 (5, 10, and 20 mg/kg) for 28 days and analyzed for depressive symptoms using the sucrose preference and forced swimming tests. Functional and morphological changes of gap junction channels in the PFC were evaluated using dye transfer and electron microscopy, respectively. The expression of connexin 43 (Cx43) was analyzed by western blotting. Rg1 markedly alleviated depression‐like behavior in rats. Long‐term Rg1 treatment of CUS‐exposed rats also significantly prevented the decrease in dye diffusion and improved the ultrastructure of astrocyte gap junctions in the PFC, indicating beneficial effects on the functional activity of gap junction channels in the brain. In addition, Rg1 upregulated Cx43 expression in the PFC reduced by CUS exposure, which significantly correlated with its antidepressant‐like effects. The results demonstrate that Rg1‐induced antidepressant effects are might be mediated, in part, by protecting astrocyte gap junctions within the prefrontal cortex. HIGHLIGHTSGinsenoside Rg1 alleviated depression in a model of chronic unpredictable stress.Rg1 improved the function of astrocyte gap junctions in the prefrontal cortex (PFC).Rg1 upregulated Cx43 expression in the PFC of depressed rats.The increase in Cx43 expression correlated with antidepressant‐like effects of Rg1.

Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells

Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3β4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 μM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT protein expression, and simultaneous enhancement of nicotine-elevated adrenaline secretion also took place. These findings thus suggest that imidacloprid may facilitate the physiological functions of adrenal glands in mammals.

Oral sinensetin, but not nobiletin alone, prevents MK801-induced impairment of memory formation in mice, like nobiletin-rich chinpi, a kampo medicine

Our pilot clinical study suggested that nobiletin‐rich Citrus reticulata peel, also known as nchinpi, has the potential to be beneficial as a kampo medicine for Alzheimers disease. The nchinpi extract has a high content of polymethoxyflavones, including nobiletin and sinensetin. Our previous pharmacological study using cultured hippocampal neurons showed that the nchinpi extract more potently facilitated cAMP‐response element (CRE)‐mediated transcription associated with long‐term memory formation than did nobiletin alone, via a mechanism in which nobiletin and four other polymethoxyflavones, one being sinensetin, a potently CRE‐mediated transcription‐facilitating substance, cooperated. It remained unclear, however, whether nchinpi could be more effective in vivo in preventing memory deficits than could nobiletin alone. Here, we compared the impacts of the respective samples on N‐methyl‐D‐aspartate receptor antagonism‐induced learning and memory disability.

Fermented Citrus reticulata (ponkan) fruit squeezed draff that contains a large amount of 4′-demethylnobiletin prevents MK801-induced memory impairment

A previous study reported biotransformation of a citrus peel polymethoxyflavone, nobiletin, by Aspergillus enabling production of 4′-demethylnobiletin, and the product’s antimutagenic activity. However, the effects of fermented citrus peel on the basal forebrain–hippocampal system remain unidentified. Citrus reticulata (ponkan) fruit squeezed draffs are generated as mass waste in beverage factories. In this study using PC12D cells and cultured central nervous system neurons, we therefore examined whether Aspergillus kawachii-fermented citrus fruit squeezed draff could affect cAMP response element (CRE)- and choline acetyltransferase gene (ChAT) promoter region-mediated transcriptional activities relevant to memory formation and cholinergic function. Our current fermentation yielded approximately 80% nobiletin bioconversion, and a sample of hot-water extract of the fermented fruit squeezed draff was stronger than that of the unfermented one in facilitating CRE-mediated transcription in cultured hippocampal neurons as well as in PC12D cells. A sample of 0–80% ethanol-eluted fraction of Diaion HP-20 column-adsorbed components of the preparation obtained by the fermentation concentration-dependently and more strongly facilitated CRE-mediated transcription than did the fraction of the unfermented one in both cell culture systems. In a separate study, this polymethoxyflavone-rich fraction of the fermented fruit squeezed draff showed a potent ability to facilitate CRE-mediated and ChAT transcription in a co-culture of hippocampal neurons and basal forebrain neurons. Repeated oral gavage of mice with the fermented fraction sample prevented MK801-impaired memory formation in mice. These findings suggest that the 4′-demethylnobiletin-rich fraction prepared from the Aspergillus-fermented ponkan squeezed draff has a potential anti-dementia effect.

Long-term up-regulation of the level of phosphorylated tyrosine hydroxylase by depletion of dopamine or biopterin

O3-G2-5 Genetic inactivation of the habenulo-interpeduncular projection enhances the conditioned fear response in zebrafish Masakazu Agetsuma1, Hidenori Aizawa1, Tazu Aoki1, Mikako Takahoko1, Ryoko Nakayama1, Toshiyuki Shiraki1, Midori Goto1, Koichi Kawakami2, Shin-ichi Higashijima3, Hitoshi Okamoto1 1 RIKEN BSI, Japan; 2 National Institute of Genetics, Japan; 3 Okazaki Institute for Integrative Bioscience, Japan

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.