Publications by authors named "Masatoshi Inden"

66 Publications

Kaempferol Has Potent Protective and Antifibrillogenic Effects for α-Synuclein Neurotoxicity In Vitro.

Int J Mol Sci 2021 Oct 25;22(21). Epub 2021 Oct 25.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan.

Aggregation of α-synuclein (α-Syn) is implicated in the pathogenesis of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Therefore, the removal of α-Syn aggregation could lead to the development of many new therapeutic agents for neurodegenerative diseases. In the present study, we succeeded in generating a new α-Syn stably expressing cell line using a piggyBac transposon system to investigate the neuroprotective effect of the flavonoid kaempferol on α-Syn toxicity. We found that kaempferol provided significant protection against α-Syn-related neurotoxicity. Furthermore, kaempferol induced autophagy through an increase in the biogenesis of lysosomes by inducing the expression of transcription factor EB and reducing the accumulation of α-Syn; thus, kaempferol prevented neuronal cell death. Moreover, kaempferol directly blocked the amyloid fibril formation of α-Syn. These results support the therapeutic potential of kaempferol in diseases such as synucleinopathies that are characterized by α-Syn aggregates.
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http://dx.doi.org/10.3390/ijms222111484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8584179PMC
October 2021

DNA methyltransferase- and histone deacetylase-mediated epigenetic alterations induced by low-level methylmercury exposure disrupt neuronal development.

Arch Toxicol 2021 04 16;95(4):1227-1239. Epub 2021 Jan 16.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Department Biomedical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi Gifu city, Gifu, 501-1196, Japan.

Methylmercury (MeHg) is a chemical substance that causes adverse effects on fetal development. However, the molecular mechanisms by which environmental MeHg affects fetal development have not been clarified. Recently, it has been suggested that the toxic effects of chemicals on fetal development are related alterations in epigenetics, such as DNA methylation and histone modification. In order to analyze the epigenetic effects of low-level MeHg exposure on neuronal development, we evaluated neuronal development both in vivo and in vitro. Pregnant mice (C57BL/6J) were orally administrated 3 mg/kg of MeHg once daily from embryonic day 12-14. Fetuses were removed on embryonic day 19 and brain tissues were collected. LUHMES cells were treated with 1 nM of MeHg for 6 days and collected on the last day of treatment. In both in vivo and in vitro samples, MeHg significantly suppressed neurite outgrowth. Decreased acetylated histone H3 (AcH3) levels and increased histone deacetylase (HDAC) 3 and HDAC6 levels were observed in response to MeHg treatment in both in vivo and in vitro experiments. In addition, increased DNA methylation and DNA methyltransferase 1 (DNMT1) levels were observed in both in vivo and in vitro experiments. The inhibition of neurite outgrowth resulting from MeHg exposure was restored by co-treatment with DNMT inhibitor or HDAC inhibitors. Our results suggest that neurological effects such as reduced neurite outgrowth due to low-level MeHg exposure result from epigenetic changes, including a decrease in AcH3 via increased HDAC levels and an increase in DNA methylation via increased DNMT1 levels.
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http://dx.doi.org/10.1007/s00204-021-02984-7DOI Listing
April 2021

The neuroprotective effects of activated α7 nicotinic acetylcholine receptor against mutant copper-zinc superoxide dismutase 1-mediated toxicity.

Sci Rep 2020 12 17;10(1):22157. Epub 2020 Dec 17.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, 1-1-1, Gifu, 501-1196, Japan.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Although many drugs have entered clinical trials, few have shown effectiveness in the treatment of ALS. Other studies have shown that the stimulation of α7 nicotinic acetylcholine receptor (nAChR) can have neuroprotective effects in some models of neurodegenerative disease, as well as prevent glutamate-induced motor neuronal death. However, the effect of α7 nAChR agonists on ALS-associated mutant copper-zinc superoxide dismutase 1 (SOD1) aggregates in motor neurons remains unclear. In the present study, we examined whether α7 nAChR activation had a neuroprotective effect against SOD1-induced toxicity in a cellular model for ALS. We found that α7 nAChR activation by PNU282987, a selective agonist of α7 nAChR, exhibited significant neuroprotective effects against SOD1-induced toxicity via the reduction of intracellular protein aggregates. This reduction also correlated with the activation of autophagy through the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, the activation of α7 nAChRs was found to increase the biogenesis of lysosomes by inducing translocation of the transcription factor EB (TFEB) into the nucleus. These results support the therapeutic potential of α7 nAChR activation in diseases that are characterized by SOD1 aggregates, such as ALS.
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http://dx.doi.org/10.1038/s41598-020-79189-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746719PMC
December 2020

Moesin is involved in microglial activation accompanying morphological changes and reorganization of the actin cytoskeleton.

J Physiol Sci 2020 Oct 31;70(1):52. Epub 2020 Oct 31.

Department of Molecular Physiology, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, 525-8577, Japan.

Moesin is a member of the ezrin, radixin and moesin (ERM) proteins that are involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and proteins located on the plasma membranes as well as through regulation of small GTPase activities. Microglia, immune cells in the central nervous system, show dynamic reorganization of the actin cytoskeleton in their process elongation and retraction as well as phagocytosis and migration. In microglia, moesin is the predominant ERM protein. Here, we show that microglial activation after systemic lipopolysaccharide application is partly inhibited in moesin knockout (Msn-KO) mice. We prepared primary microglia from wild-type and Msn-KO mice, and studied them to compare their phenotypes accompanying morphological changes and reorganization of the actin cytoskeleton induced by UDP-stimulated phagocytosis and ADP-stimulated migration. The Msn-KO microglia showed higher phagocytotic activity in the absence of UDP, which was not further increased by the treatment with UDP. They also exhibited decreased ADP-stimulated migration activities compared with the wild-type microglia. However, the Msn-KO microglia retained their ability to secrete tumor necrosis factor α and nitric oxide in response to lipopolysaccharide.
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http://dx.doi.org/10.1186/s12576-020-00779-6DOI Listing
October 2020

Neuroprotective effects of 5-aminolevulinic acid against neurodegeneration in rat models of Parkinson's disease and stroke.

J Pharmacol Sci 2020 Nov 1;144(3):183-187. Epub 2020 Aug 1.

Laboratory of Pharmacology and Neurobiology, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga, 525-8577, Japan. Electronic address:

Oxidative stress is associated with the progression of the neurodegenerative diseases Parkinson's disease (PD) and cerebral ischemia. Recently, 5-aminolevulinic acid (5-ALA), an intermediate in the porphyrin synthesis pathway, was reported to exert antioxidative effects on macrophages and cardiomyocytes. Here, we demonstrated the neuroprotective effects of 5-ALA using rat models of PD and ischemia as well as in vitro in SH-SY5Y cells. 5-ALA partially prevented neurodegeneration in each condition. These results suggest that 5-ALA has a potential for promising therapeutic agent to protect against neurodegeneration exacerbated by oxidative stress.
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http://dx.doi.org/10.1016/j.jphs.2020.07.012DOI Listing
November 2020

The Novel -Dihydroperoxide 12AC3O Suppresses High Phosphate-Induced Calcification via Antioxidant Effects in p53LMAco1 Smooth Muscle Cells.

Int J Mol Sci 2020 Jun 29;21(13). Epub 2020 Jun 29.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, 1-1-1 Gifu 501-1196, Japan.

The excessive intake of phosphate (Pi), or chronic kidney disease (CKD), can cause hyperphosphatemia and eventually lead to ectopic calcification, resulting in cerebrovascular diseases. It has been reported that reactive oxygen species (ROS), induced by high concentrations of Pi loading, play a key role in vascular calcification. Therefore, ROS suppression may be a useful treatment strategy for vascular calcification. 12AC3O is a newly synthesized -dihydroperoxide (DHP) that has potent antioxidant effects. In the present study, we investigated whether 12AC3O inhibited vascular calcification via its antioxidative capacity. To examine whether 12AC3O prevents vascular calcification under high Pi conditions, we performed Alizarin red and von Kossa staining, using the mouse aortic smooth muscle cell line p53LMAco1. Additionally, the effect of 12AC3O against oxidative stress, induced by high concentrations of Pi loading, was investigated using redox- sensitive dyes. Further, the direct trapping effect of 12AC3O on reactive oxygen species (ROS) was investigated by ESR analysis. Although high concentrations of Pi loading exacerbated vascular smooth muscle calcification, calcium deposition was suppressed by the treatment of both antioxidants and 12AC3O, suggesting that the suppression of ROS may be a candidate therapeutic approach for treating vascular calcification induced by high concentrations of Pi loading. Importantly, 12AC3O also attenuated oxidative stress. Furthermore, 12AC3O directly trapped superoxide anion and hydroxyl radical. These results suggest that ROS are closely involved in high concentrations of Pi-induced vascular calcification and that 12AC3O inhibits vascular calcification by directly trapping ROS.
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http://dx.doi.org/10.3390/ijms21134628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369805PMC
June 2020

MicroRNA-5572 Is a Novel MicroRNA-Regulating in Sporadic Amyotrophic Lateral Sclerosis.

Int J Mol Sci 2020 Jun 24;21(12). Epub 2020 Jun 24.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi Gifu city, Gifu 501-1196, Japan.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease caused by the loss of motor neurons. Although the pathogenesis of sporadic ALS (sALS) remains unclear, it has recently been suggested that disorders of microRNA (miRNA) may be involved in neurodegenerative conditions. The purpose of this study was to investigate miRNA levels in sALS and the target genes of miRNA. Microarray and real-time RT-PCR analyses revealed significantly-decreased levels of miR-139-5p and significantly increased levels of miR-5572 in the spinal cords of sALS patients compared with those in controls. We then focused on miR-5572, which has not been reported in ALS, and determined its target gene. By using TargetScan, we predicted as the candidate target gene of miR-5572. In a previous study, we found decreased levels in the spinal cords of sALS patients. We revealed that was regulated by miR-5572. Taken together, these results demonstrate that the level of novel miRNA miR-5572 is increased in sALS and that is one of the target genes regulated by miR-5572.
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http://dx.doi.org/10.3390/ijms21124482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350020PMC
June 2020

Characteristics and Therapeutic Potential of Dental Pulp Stem Cells on Neurodegenerative Diseases.

Front Neurosci 2020 7;14:407. Epub 2020 May 7.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan.

To evaluate the therapeutic potential of stem cells for neurodegenerative diseases, emphasis should be placed on clarifying the characteristics of the various types of stem cells. Among stem cells, dental pulp stem cells (DPSCs) are a cell population that is rich in cell proliferation and multipotency. It has been reported that transplantation of DPSCs has protective effects against models of neurodegenerative diseases. The protective effects are not only through differentiation into the target cell type for the disease but are also related to trophic factors released from DPSCs. Recently, it has been reported that serum-free culture supernatant of dental pulp stem cell-conditioned medium (DPCM) contains various trophic factors and cytokines and that DPCM is effective for models of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis (ALS). Moreover, the use of stem cells from human exfoliated deciduous teeth (SHEDs) has been considered. SHEDs are derived from deciduous teeth that have been disposed of as medical waste. SHEDs have higher differentiation capacity and proliferation ability than DPSCs. In addition, the serum-free culture supernatant of SHEDs (SHED-CM) contains more trophic factors, cytokines, and biometals than DPCM and also promotes neuroprotection. The neuroprotective effect of DPSCs, including those from deciduous teeth, will be used as the seeds of therapeutic drugs for neurodegenerative diseases. SHEDs will be used for further cell therapy of neurodegenerative diseases in the future. In this paper, we focused on the characteristics of DPSCs and their potential for neurodegenerative diseases.
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http://dx.doi.org/10.3389/fnins.2020.00407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222959PMC
May 2020

Partial reduced Pi transport function of PiT-2 might not be sufficient to induce brain calcification of idiopathic basal ganglia calcification.

Sci Rep 2019 11 21;9(1):17288. Epub 2019 Nov 21.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan.

Idiopathic basal ganglia calcification (IBGC) is a rare intractable disease characterized by abnormal mineral deposits, including mostly calcium in the basal ganglia, thalamus, and cerebellum. SLC20A2 is encoding the phosphate transporter PiT-2 and was identified in 2012 as the causative gene of familial IBGC. In this study, we investigated functionally two novel SLC20A2 variants (c.680C > T, c.1487G > A) and two SLC20A2 variants (c.82G > A, c.358G > C) previously reported from patients with IBGC. We evaluated the function of variant PiT-2 using stable cell lines. While inorganic phosphate (Pi) transport activity was abolished in the cells with c.82G > A, c.358G > C, and c.1487G > A variants, activity was maintained at 27.8% of the reference level in cells with the c.680C > T variant. Surprisingly, the c.680C > T variant had been discovered by chance in healthy members of an IBGC family, suggesting that partial preservation of Pi transport activity may avoid the onset of IBGC. In addition, we confirmed that PiT-2 variants could be translocated into the cell membrane to the same extent as PiT-2 wild type. In conclusion, we investigated the PiT-2 dysfunction of four SLC20A2 variants and suggested that a partial reduced Pi transport function of PiT-2 might not be sufficient to induce brain calcification of IBGC.
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http://dx.doi.org/10.1038/s41598-019-53401-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872723PMC
November 2019

p-Coumaric Acid Has Protective Effects against Mutant Copper-Zinc Superoxide Dismutase 1 via the Activation of Autophagy in N2a Cells.

Int J Mol Sci 2019 Jun 16;20(12). Epub 2019 Jun 16.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons. In previous our study, an ethanol extract of Brazilian green propolis (EBGP) prevented mutant copper-zinc superoxide dismutase 1 (SOD1)-induced neurotoxicity. This paper aims to reveal the effects of p-coumaric acid (p-CA), an active ingredient contained in EBGP, against SOD1-induced neurotoxicity. We found that p-CA reduced the accumulation of SOD1 subcellular aggregation and prevented SOD1-associated neurotoxicity. Moreover, p-CA attenuated SOD1-induced oxidative stress and endoplasmic reticulum stress, which are significant features in ALS pathology. To examine the mechanism of neuroprotective effects, we focused on autophagy, and we found that p-CA induced autophagy. Additionally, the neuroprotective effects of p-CA were inhibited by chloroquine, an autophagy inhibiter. Therefore, these results obtained in this paper suggest that p-CA prevents SOD1-induced neurotoxicity through the activation of autophagy and provides a potential therapeutic approach for ALS.
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http://dx.doi.org/10.3390/ijms20122942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628046PMC
June 2019

Identification of reference genes for microRNAs of extracellular vesicles isolated from plasma samples of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods.

Am J Vet Res 2019 May;80(5):449-454

Objective: To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs.

Animals: 6 healthy Beagles.

Procedures: Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes.

Results: Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 10 vesicles/mL ± 4.2 × 10 vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable.

Conclusions And Clinical Relevance: The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.
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http://dx.doi.org/10.2460/ajvr.80.5.449DOI Listing
May 2019

Functional evaluation of PDGFB-variants in idiopathic basal ganglia calcification, using patient-derived iPS cells.

Sci Rep 2019 04 5;9(1):5698. Epub 2019 Apr 5.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan.

Causative genes in patients with idiopathic basal ganglia calcification (IBGC) (also called primary familial brain calcification (PFBC)) have been reported in the past several years. In this study, we surveyed the clinical and neuroimaging data of 70 sporadic patients and 16 families (86 unrelated probands in total) in Japan, and studied variants of PDGFB gene in the patients. Variant analyses of PDGFB showed four novel pathogenic variants, namely, two splice site variants (c.160 + 2T > A and c.457-1G > T), one deletion variant (c.33_34delCT), and one insertion variant (c.342_343insG). Moreover, we developed iPS cells (iPSCs) from three patients with PDGFB variants (c.160 + 2T > A, c.457-1G > T, and c.33_34 delCT) and induced endothelial cells. Enzyme-linked immunoassay analysis showed that the levels of PDGF-BB, a homodimer of PDGF-B, in the blood sera of patients with PDGFB variants were significantly decreased to 34.0% of that of the control levels. Those in the culture media of the endothelial cells derived from iPSCs of patients also significantly decreased to 58.6% of the control levels. As the endothelial cells developed from iPSCs of the patients showed a phenotype of the disease, further studies using IBGC-specific iPSCs will give us more information on the pathophysiology and the therapy of IBGC in the future.
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http://dx.doi.org/10.1038/s41598-019-42115-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450963PMC
April 2019

A Golgi-targeting fluorescent probe for labile Fe(ii) to reveal an abnormal cellular iron distribution induced by dysfunction of VPS35.

Chem Sci 2019 Feb 26;10(5):1514-1521. Epub 2018 Nov 26.

Laboratory of Pharmaceutical and Medicinal Chemistry , Gifu Pharmaceutical University , 1-25-4, Daigaku-Nishi , Gifu , 501-1196 , Japan . Email:

Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent metal transporter 1 (DMT1) acts as a primary transporter of Fe(ii) ions. The intracellular delivery of DMT1 toward the cellular membrane the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(ii) ions. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(ii) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox, for the Golgi-specific detection of Fe(ii) ions by integrating our original -oxide-based Fe(ii)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(ii), revealed that the intracellular distribution balance of Fe(ii) ions between the Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(ii) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(ii) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects subcellular iron homeostasis, promoting Fe(ii) leakage at the Golgi and lysosomal accumulation of Fe(ii) through missorting of DMT1.
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http://dx.doi.org/10.1039/c8sc04386hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357701PMC
February 2019

SLC20A2 variants cause dysfunctional phosphate transport activity in endothelial cells induced from Idiopathic Basal Ganglia Calcification patients-derived iPSCs.

Biochem Biophys Res Commun 2019 03 28;510(2):303-308. Epub 2019 Jan 28.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan. Electronic address:

Idiopathic Basal Ganglia Calcification (IBGC) is a rare neuropsychiatric illness also known as Fahr's disease or Primary Familial Brain Calcification (PFBC). IBGC is caused by SLC20A2 variants, which encodes the inorganic phosphate (Pi) transporter PiT-2, a transmembrane protein associated with Pi homeostasis. We have reported novel SLC20A2 variants in the Japanese population and established an induced pluripotent stem cells (iPSCs) from an IBGC patient carrying a SLC20A2 variant. To investigate the effect of these SLC20A2 variants identified in our previous study, we used Chinese hamster ovary (CHO) cells expressing these variant proteins using the Flp-In system (Flp-In CHO cells), and showed that variant SLC20A2 proteins significantly disrupted the Pi transport activity in Flp-In CHO cells. Endothelial cells (ECs) represent important target cells for elucidating the pathology of IBGC. Using patient-derived iPSCs in this study, we differentiated these cells into ECs and found no significant difference in their differentiation capacity into ECs compared with control iPSCs. However, the Pi transport activity of IBGC patient-derived iPS-ECs was significantly decreased compared with that of control iPS-ECs without changing the gene expression of the other SLC 20 family members. We confirmed that SLC20A2 variants caused the loss of function of the Pi transport activity in both Flp-In CHO cells and disease-specific iPSCs. This is the first report to show an in vitro model of iPSCs in IBGC with patient-identified SLC20A2 variants. These useful tools will help in elucidating IBGC pathogenesis and can be used for screening drug candidates.
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http://dx.doi.org/10.1016/j.bbrc.2019.01.096DOI Listing
March 2019

Effects of gem-dihydroperoxides against mutant copper‑zinc superoxide dismutase-mediated neurotoxicity.

Mol Cell Neurosci 2018 10 5;92:177-184. Epub 2018 Sep 5.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan. Electronic address:

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive muscle weakness, paralysis, and death. Although its neuropathology is well investigated, currently, effective treatments are unavailable. The mechanism of ALS involves the aggregation and accumulation of several mutant proteins, including mutant copper‑zinc superoxide dismutase (SOD1), TAR DNA binding protein 43 kDa (TDP-43) and fused in sarcoma (FUS) proteins. Previous reports have shown that excessive oxidative stress, associated with mitochondrial dysfunction and mutant protein accumulation, contributes to ALS pathology. The present study focuses on the promotion of SOD1 misfolding and aggregation by oxidative stress. Having recently synthesized novel organic gem-dihydroperoxides (DHPs) with high anti-oxidant activity, we now examined whether DHPs reduce the mutant SOD1-induced intracellular aggregates involved in oxidative stress. We found that, among DHPs, 12AC2O significantly inhibited mutant SOD1-induced cell death and reduced the intracellular mutant SOD1 aggregates. Moreover, immunofluorescence staining with redox-sensitive dyes showed that 12AC2O reduced the excessive level of intracellular mutant SOD1-induced reactive oxygen species (ROS). Additionally, ESR analysis showed that 12AC2O exerts a direct scavenging effect against the hydroxyl radical (OH) and the superoxide anion (O). These results suggest that 12AC2O is a very useful agent in combination with other agents against ALS.
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http://dx.doi.org/10.1016/j.mcn.2018.09.001DOI Listing
October 2018

Methylmercury causes epigenetic suppression of the tyrosine hydroxylase gene in an in vitro neuronal differentiation model.

Biochem Biophys Res Commun 2018 08 2;502(4):435-441. Epub 2018 Jun 2.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Department Biomedical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi Gifu City, Gifu, 501-1196, Japan. Electronic address:

Methylmercury (MeHg) is the causative substance of Minamata disease, which is associated with various neurological disorders such as sensory disturbance and ataxia. It has been suggested low-level dietary intake of MeHg from MeHg-containing fish during gestation adversely affects the fetus. In our study, we investigated the toxicological effects of MeHg exposure on neuronal differentiation focusing on epigenetics. We used human fetal brain-derived immortalized cells (LUHMES cells) as a human neuronal differentiation model. Cell viability, neuronal, and catecholamine markers in LUHMES cells were assessed after exposure to MeHg (0-1000 nM) for 6 days (from day 2 to day 8 of neuronal differentiation). Cell viability on day 8 was not affected by exposure to 1 nM MeHg for 6 days. mRNA levels of AADC, DBH, TUJ1, and SYN1 also were unaffected by MeHg exposure. In contrast, levels of TH, the rate-limiting enzyme for dopamine synthesis, were significantly decreased after MeHg exposure. Acetylated histone H3, acetylated histone H3 lysine 9, and tri-methyl histone H3 lysine 9 levels at the TH gene promoter were not altered by MeHg exposure. However, tri-methylation of histone H3 lysine 27 levels, related to transcriptional repression, were significantly increased at the TH gene promotor after MeHg exposure. In summary, MeHg exposure during neuronal differentiation led to epigenetic changes that repressed TH gene expression. This study provides useful insights into the toxicological mechanisms underlying the effects of developmental MeHg exposure during neuronal differentiation.
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http://dx.doi.org/10.1016/j.bbrc.2018.05.162DOI Listing
August 2018

Inorganic phosphorus (Pi) in CSF is a biomarker for SLC20A2-associated idiopathic basal ganglia calcification (IBGC1).

J Neurol Sci 2018 05 8;388:150-154. Epub 2018 Mar 8.

Department of Informative Clinical Medicine, Gifu University, Graduate School of Medicine, 1-1 Yanagido, Gifu, 501-1194, Japan.

Introduction: Idiopathic basal ganglia calcification (IBGC), also called Fahr's disease or recently primary familial brain calcification (PFBC), is characterized by abnormal deposits of minerals including calcium mainly and phosphate in the brain. Mutations in SLC20A2 (IBGC1 (merged with former IBGC2 and IBGC3)), which encodes PiT-2, a phosphate transporter, is the major cause of IBGC. Recently, Slc20a2-KO mice have been showed to have elevated levels of inorganic phosphorus (Pi) in cerebrospinal fluid (CSF); however, CSF Pi levels in patients with IBGC have not been fully examined.

Methods: We investigated the cases of 29 patients with IBGC including six patients with SLC20A2 mutation and three patients with PDGFB mutation, and 13 controls. The levels of sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and Pi in sera and CSF were determined by potentiometry and colorimetry. Moreover, clinical manifestations were investigated in the IBGC patients with high Pi levels in CSF.

Results: The study revealed that the average level of Pi in the CSF of the total group of patients with IBGC is significantly higher than that of the control group, and the levels of Pi in CSF of the IBGC patients with SLC20A2 mutations are significantly higher than those of the IBGC patients with PDGFB mutations, the other IBGC patients and controls.

Conclusion: Results of this study suggest that the levels of CSF Pi will be a good biomarker for IBGC1.
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http://dx.doi.org/10.1016/j.jns.2018.03.014DOI Listing
May 2018

Therapeutic Activities of DJ-1 and Its Binding Compounds Against Neurodegenerative Diseases.

Adv Exp Med Biol 2017 ;1037:187-202

Laboratory of Pharmacology and Neurobiology, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga, Japan.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Loss-of-function mutations in the gene encoding PARK7/DJ-1 were identified in familial PD. Wild-type DJ-1 acts as an oxidative stress sensor in neural cells. Previously, we identified binding compounds of DJ-1, including UCP0045037/compound A, UCP0054278/compound B, and compound-23 (comp-23), by in silico virtual screening. These compounds prevented oxidative stress-induced dopaminergic neuronal death and restored locomotion defects in animal models of PD. In addition, these binding partners reduced infarct size in cerebral ischemia in rats. The neuroprotective effects of these compounds are lost in DJ-1-knockdown cells and DJ-1-knockout animal. These results suggest that these compounds interact with endogenous DJ-1 and then produce antioxidant and neuroprotective responses in both animal models for PD and cerebral ischemia in rats. This raises the possibility that interaction partners of DJ-1, such as UCP0045037, UCP0054278, and comp-23, may represent a novel dopaminergic neuroprotective drug for the treatment of PD.
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http://dx.doi.org/10.1007/978-981-10-6583-5_12DOI Listing
April 2018

Induced pluripotent stem cells derived from a patient with familial idiopathic basal ganglia calcification (IBGC) caused by a mutation in SLC20A2 gene.

Stem Cell Res 2017 10 29;24:40-43. Epub 2017 Jul 29.

Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan; Drug-Discovery Cellular Basis Development Team, RIKEN BioResource Center, Kyoto, Japan. Electronic address:

Idiopathic basal ganglia calcification (IBGC), also known as Fahr disease or primary familial brain calcifications (PFBC), is a rare neurodegenerative disorder characterized by calcium deposits in basal ganglia and other brain regions, causing neuropsychiatric and motor symptoms. We established human induced pluripotent stem cells (iPSCs) from an IBGC patient. The established IBGC-iPSCs carried SLC20A2 c.1848G>A mutation (p.W616* of translated protein PiT2), and also showed typical iPSC morphology, pluripotency markers, normal karyotype, and the ability of in vitro differentiation into three-germ layers. The iPSC line will be useful for further elucidating the pathomechanism and/or drug development for IBGC.
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http://dx.doi.org/10.1016/j.scr.2017.07.028DOI Listing
October 2017

Neuroprotective effect of 5-aminolevulinic acid against low inorganic phosphate in neuroblastoma SH-SY5Y cells.

Sci Rep 2017 07 18;7(1):5768. Epub 2017 Jul 18.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan.

PiT-1 (encoded by SLC20A1) and PiT-2 (encoded by SLC20A2) are type-III sodium-dependent phosphate cotransporters (NaPiTs). Recently, SLC20A2 mutations have been found in patients with idiopathic basal ganglia calcification (IBGC), and were predicted to bring about an inability to transport Pi from the extracellular environment. Here we investigated the effect of low Pi loading on the human neuroblastoma SH-SY5Y and the human glioblastoma A172 cell lines. The results show a different sensitivity to low Pi loading and differential regulation of type-III NaPiTs in these cells. We also examined whether 5-aminolevulinic acid (5-ALA) inhibited low Pi loading-induced neurotoxicity in SH-SY5Y cells. Concomitant application of 5-ALA with low Pi loading markedly attenuated low Pi-induced cell death and mitochondrial dysfunction via the induction of HO-1 by p38 MAPK. The findings provide us with novel viewpoints to understand the pathophysiology of IBGC, and give a new insight into the clinical prevention and treatment of IBGC.
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http://dx.doi.org/10.1038/s41598-017-06406-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515920PMC
July 2017

Protective function of SLC30A10 induced via PERK-ATF4 pathway against 1-methyl-4-phenylpyridinium.

Biochem Biophys Res Commun 2017 09 5;490(4):1307-1313. Epub 2017 Jul 5.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Department Biomedical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi Gifu City, Gifu 501-1196, Japan. Electronic address:

Solute carrier family 30 member 10 (SLC30A10) has been known as manganese transporter. It has been suggested that neurodegenerative diseases are related with cellular stress such as oxidative stress or endoplasmic reticulum (ER) stress. However, it remains unknown whether SLC30A10 is actually involved in several intracellular stress. We found that the level of Slc30a10 was increased in midbrain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Therefore, we further investigated the role of SLC30A10 in the 1-methyl-4-phenylpiridium ion (MPP)-induced intracellular stress, and the molecular mechanism underlying SLC30A10 induction by MPP treatment. In human neuroblastoma cell line (SH-SY5Y) treated with MPP (1 mM), the SLC30A10 mRNA level was significantly increased, and in addition, the expression of CHOP, which is known as one of ER stress markers, was significantly increased by MPP. Interestingly, the level of SLC30A10 mRNA was significantly increased by tunicamycin as an ER stressor, suggesting that the induction of SLC30A10 by MPP was caused via ER stress. Considering that PKR-like endoplasmic reticulum kinase (PERK) pathway is activated under ER stress induced by MPP, we investigated whether the expression of SLC30A10 is increased through ATF4, which is major transcription factor in PERK pathway. The increase of SLC30A10 expression in MPP-treated cells was eliminated by ATF4 knockdown. And the protective role of SLC30A10 against MPP-induced ER stress was confirmed by measuring cell viability in SLC30A10 knockdown cells. In conclusion, SLC30A10 is thought to have protective role for MPP-induced toxicity via PERK-ATF4 pathway.
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http://dx.doi.org/10.1016/j.bbrc.2017.07.018DOI Listing
September 2017

The effects of Brazilian green propolis that contains flavonols against mutant copper-zinc superoxide dismutase-mediated toxicity.

Sci Rep 2017 06 6;7(1):2882. Epub 2017 Jun 6.

Lab. Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical Univ, Gifu, Japan.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. The purpose of this study was to clarify effects of brazilian green propolis and the active ingredient against ALS-associated mutant copper-zinc superoxide dismutase (SOD1)-mediated toxicity. Ethanol extract of brazilian green propolis (EBGP) protected N2a cells against mutant SOD1-induced neurotoxicity and reduced aggregated mutant SOD1 by induction of autophagy. Kaempferide and kaempferol, the active ingredients of EBGP, also inhibited mutant SOD1-induced cell death and reduced the intracellular mutant SOD1 aggregates. Both kaempferide and kaempferol significantly suppressed mutant SOD1-induced superoxide in mitochondria. Western blot analysis showed that kaempferol potentially induced autophagy via the AMP-activated protein kinase (AMPK) - the mammalian target of rapamycin (mTOR) pathway. These results suggest that EBGP containing the active ingredient against mutant SOD1-mediated toxicity is a promising medicine or health food for prevention and treatment of ALS.
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http://dx.doi.org/10.1038/s41598-017-03115-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460160PMC
June 2017

DJ-1/PARK7: A New Therapeutic Target for Neurodegenerative Disorders.

Biol Pharm Bull 2017 ;40(5):548-552

Laboratory of Pharmacology and Neurobiology, College of Pharmaceutical Sciences, Ritsumeikan University.

DJ-1, encoded in a causative gene of familial Parkinson's disease (PARK7), has multiple functions: it works as an antioxidant, in transcriptional regulation, as a molecular chaperone and in protein degradation. Three types of pathogenic mutants of DJ-1 (M26I, D149A and L166P) have been reported to disrupt proper structures and lead to a loss of function. DJ-1 receives oxidation at the cysteine residue, and the degree of oxidation at the C106 residue determines DJ-1 activity. In this decade, DJ-1 has been reported to suppress the progression of various neurodegenerative disorders in animal models. The administration of recombinant wild-type DJ-1 protein suppresses the neuronal loss associated with both Parkinson's disease and ischemic stroke in rats. Furthermore, in studies focused on DJ-1 as the therapeutic target, compounds that have the capacity of binding to DJ-1 at the C106 residue have been reported to exert therapeutic effects on various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and ischemic stroke. DJ-1 and DJ-1-targeting molecules/compounds will be useful therapeutic targets for various neurodegenerative disorders due to their various functions such as antioxidant capacity, chaperone function and as a proteolytic pathway.
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http://dx.doi.org/10.1248/bpb.b16-01006DOI Listing
February 2018

Enhanced alcohol-drinking behavior associated with active ghrelinergic and serotoninergic neurons in the lateral hypothalamus and amygdala.

Pharmacol Biochem Behav 2017 02 7;153:1-11. Epub 2016 Dec 7.

Department of Food Science and Biotechnology, Hiroshima Institute of Technology, Hiroshima, Japan.

Central ghrelin is required for the rewarding properties of drug abuse. We investigated whether alcohol affects ghrelinergic, dopaminergic, and serotoninergic neurons and growth hormone secretagogue receptor 1A (GHS-R1A) levels in the reward system of the brain. Alcohol-naïve C57BL/6J mice received 2g/kg ethanol (EtOH) intraperitoneally (i.p.). Plasma ghrelin levels decreased between 1 and 4h. We investigated the effects of EtOH administration on plasma ghrelin levels in two different animal models at 1, 3, and 10months of age. Plasma ghrelin levels decreased following the EtOH treatment in 1- and 3-month-old short-term (1-day) alcohol vapor-exposed (STA) mice. In contrast, EtOH administration increased plasma ghrelin levels in 1- and 3-month-old long-term (20-day) alcohol vapor-exposed (LTA) mice. In vivo ghrelin release in the lateral hypothalamus (LH) increased in STA and LTA mice after the i.p. administration of EtOH. EtOH increased in vivo dopamine (DA), but not serotonin (5-HT) release in the LH of STA mice, and increased in vivo DA and 5-HT release in the LH of LTA mice. GHS-R1A mRNA expression and GHS-R1A protein levels in the LH were increased in LTA mice. The number of GHS-R1A-immunoreactive cells was greater in the LH and amygdala of LTA mice. These results support the neurobiological correlation between the development of drinking behavior and activation of ghrelinergic and serotonergic neurons in the LH. The activation of ghrelinergic systems in the amygdala may also induce an increase in 5-HT release in the LH during long-term alcohol intake.
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http://dx.doi.org/10.1016/j.pbb.2016.12.001DOI Listing
February 2017

Fluorodopa is a Promising Fluorine-19 MRI Probe for Evaluating Striatal Dopaminergic Function in a Rat Model of Parkinson's Disease.

J Neurosci Res 2017 07 28;95(7):1485-1494. Epub 2016 Oct 28.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, 520-2192, Japan.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra projecting to the striatum. It has been estimated that approximately 80% of the striatal dopamine and 50% of nigral dopaminergic neurons are lost before the onset of typical motor symptoms, indicating that early diagnosis of PD using noninvasive imaging is feasible. Fluorine-19 ( F) magnetic resonance imaging (MRI) represents a highly sensitive, easily available, low-background, and cost-effective approach to evaluate dopaminergic function using non-radioactive fluorine-containing dopaminergic agents. The aim of this study was to find a potent F MRI probe to evaluate dopaminergic presynaptic function in the striatum. To select candidates for F MRI probes, we investigated the following eight non-radioactive fluorine-containing dopaminergic agents: fluorodopa (F-DOPA), F-tyrosine, haloperidol, GBR13069 duhydrochloride, GBR12909 duhydrochloride, 3-bis-(4-fluorophenyl) methoxytropane hydrochloride, flupenthixol, and fenfluramine. In F nuclear magnetic resonance measurements, F-tyrosine and F-DOPA displayed a relatively higher signal-to-noise ratio value in brain homogenates than in others. F-DOPA, but not F-tyrosine, induced the rotational behavior in a 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonian rat model. In addition, a significantly high amount of F-DOPA accumulated in the ipsilateral striatum of hemiparkinsonian rats after the injection. We performed F MRI in PC12 cells and isolated rat brain using a 7T MR scanner. Our findings suggest that F-DOPA is a promising F MRI probe for evaluating dopaminergic presynaptic function in the striatum of hemiparkinsonian rats. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jnr.23983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586037PMC
July 2017

Protective roles of SLC30A3 against endoplasmic reticulum stress via ERK1/2 activation.

Biochem Biophys Res Commun 2016 Oct 24;479(4):853-859. Epub 2016 Sep 24.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Department Biomedical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu City, Gifu, 501-1196, Japan. Electronic address:

Endoplasmic reticulum (ER) stress has been thought to be involved to neurodegenerative diseases such as Alzheimer's disease (AD) or Amyotrophic lateral sclerosis (ALS). The previous studies have shown that SLC30A3 level is decreased in prefrontal cortex of AD patients. In addition, we have shown that level of zinc (Zn) is increased in cerebrospinal fluid and SLC30A3 level is decreased in spinal cord of ALS patients. It was thought that both SLC30A3 and ER stress could be related to the cause of AD and ALS, however the relationship between ER stress and SLC30A3 has not been elucidated. Therefore we investigated that the role of SLC30A3 against ER stress. The level of SLC30A3 mRNA was significantly increased by tunicamycin treatment in human neuroblastoma cell line (SH-SY5Y) and human embryonic kidney cell line (HEK293). Cell viability under tunicamycin treatment was significantly decreased in SLC30A3 knockdown cells by siRNA in comparison with negative control (NC) cells. Cleaved caspase-3 level was significantly increased in SLC30A3 knockdown cells, not in NC cells. These results showed that SLC30A3 has a protective role to ER stress-induced toxicities. The previous study has shown that SLC30A3 protect cells from oxidative stress in ERK1/2 signal dependent manner, thus we determined the activity of ERK1/2 in SLC30A3 knockdown cells under ER stress condition. The level of ERK1/2 phosphorylation was significantly increased by tunicamycin treatment in NC cells, not in SLC30A3 knockdown cells. The ERK1/2 pathway is thought to have an association with defensive effects of SLC30A3 on cellular stress such as ER stress. In conclusion, this study suggested that SLC30A3 is supposed to play a protective role against ER stress, which is related to ERK1/2 activation.
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http://dx.doi.org/10.1016/j.bbrc.2016.09.119DOI Listing
October 2016

Effects of a DJ-1-Binding Compound on Spatial Learning and Memory Impairment in a Mouse Model of Alzheimer's Disease.

J Alzheimers Dis 2017 ;55(1):67-72

Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

Previously, DJ-1 modulator UCP0054278/comp-B was identified by virtual screening, where comp-B interacts with DJ-1 to produce antioxidant and neuroprotective responses in Parkinson's disease models. However, the effect of comp-B in an in vivo Alzheimer's disease (AD) model is yet undetermined. Thus, we examined the effect of comp-B on spatial learning, memory, and amyloid-β (Aβ) clearance in a transgenic mouse model of AD. We found that comp-B resolved the cognitive deficits, reduced insoluble Aβ42 levels, and prevented the degeneration of synaptic functions, thereby suggesting that comp-B may become a major compound for AD treatment.
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http://dx.doi.org/10.3233/JAD-160574DOI Listing
February 2018

The type III transporters (PiT-1 and PiT-2) are the major sodium-dependent phosphate transporters in the mice and human brains.

Brain Res 2016 Apr 26;1637:128-136. Epub 2016 Feb 26.

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan. Electronic address:

PiT-1/SLC20A1 and PiT-2/SLC20A2 are members of the mammalian type-III inorganic phosphate (Pi) transporters encoded by the SLC20 genes. The broad distribution of SLC20A1 and SLC20A2 mRNAs in mammalian tissues is compatible with housekeeping maintenance of intracellular Pi homeostasis by transporting Pi from intrastitial fluid for normal cellular functions. Recently, mutations of SLC20A2 have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr's disease. However, the localization of PiT-1 and PiT-2 in the normal brain has not been clarified yet. The aim of this study was to reveal the distribution of PiT-1 and PiT-2 in the mouse and human brains. As results, gene expressing analysis showed that SLC20A1 and SLC20A2 mRNAs were widely expressed throughout the mouse and human brains, although other Pi transporters encoded by SLC17 and SLC34 mRNAs were hardly detected. The region of cerebellum contained a higher level of SLC20A1 and SLC20A2 mRNAs than the other brain regions. Additionally, the cerebellum in the mouse brain contained higher levels of PiT-1 and PiT-2 than those in the other regions in the brain, respectively. The immunohistochemical studies showed that PiT-1 was recognized in neuron, astrocytes and vascular endothelial cells. Similarly to PiT-1 immunopositivity, PiT-2 was clearly recognized in these cells. These results suggest that SLC20 family plays a pivotal role in the maintenance of cellular Pi homeostasis particularly in the brain. The viewpoint is compatible with the finding that calcification in IBGC is recognized only in the brain. This provides us with a novel viewpoint to understand the basic pathophysiology of IBGC through type-III Pi transporters.
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http://dx.doi.org/10.1016/j.brainres.2016.02.032DOI Listing
April 2016

α4 nicotinic acetylcholine receptor modulated by galantamine on nigrostriatal terminals regulates dopamine receptor-mediated rotational behavior.

Neurochem Int 2016 Mar 18;94:74-81. Epub 2016 Feb 18.

Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto, Japan; Pharmacology and Neurobiology Laboratory, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, Japan. Electronic address:

Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that α4 nAChRs were highly expressed on striatal dopaminergic terminals, while no α7 nAChRs were detected. Pretreatment with the α4 nAChR antagonist dihydroxy-β-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the α7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the α4 nAChR agonist ABT-418 induced ipsilateral rotation, while the α7 nAChR agonist PNU282987 had no significant effect on rotational behavior. These results suggest that galantamine can enhance striatal dopamine release through allosteric modulation of α4 nAChRs on nigrostriatal dopaminergic terminals.
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http://dx.doi.org/10.1016/j.neuint.2016.02.008DOI Listing
March 2016
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