Noritaka Nakamichi

RAGE-mediated signaling contributes to intraneuronal transport of amyloid-β and neuronal dysfunction

Intracellular amyloid-β peptide (Aβ) has been implicated in neuronal death associated with Alzheimers disease. Although Aβ is predominantly secreted into the extracellular space, mechanisms of Aβ transport at the level of the neuronal cell membrane remain to be fully elucidated. We demonstrate that receptor for advanced glycation end products (RAGE) contributes to transport of Aβ from the cell surface to the intracellular space. Mouse cortical neurons exposed to extracellular human Aβ subsequently showed detectable peptide intracellularly in the cytosol and mitochondria by confocal microscope and immunogold electron microscopy. Pretreatment of cultured neurons from wild-type mice with neutralizing antibody to RAGE, and neurons from RAGE knockout mice displayed decreased uptake of Aβ and protection from Aβ-mediated mitochondrial dysfunction. Aβ activated p38 MAPK, but not SAPK/JNK, and then stimulated intracellular uptake of Aβ-RAGE complex. Similar intraneuronal co-localization of Aβ and RAGE was observed in the hippocampus of transgenic mice overexpressing mutant amyloid precursor protein. These findings indicate that RAGE contributes to mechanisms involved in the translocation of Aβ from the extracellular to the intracellular space, thereby enhancing Aβ cytotoxicity.

Chronic vitamin D3 treatment protects against neurotoxicity by glutamate in association with upregulation of vitamin D receptor mRNA expression in cultured rat cortical neurons

The vitamin D receptor (VDR) is believed to mediate different biologic actions of vitamin D3, an active metabolite of vitamin D, through regulation of gene expression after binding to specific DNA‐response element (VDRE) on target genes. To further understand roles of both vitamin D3 and VDR in the central nervous system, we examined VDRE binding in nuclear extracts prepared from discrete rat brain regions and cultured rat cortical neurons by electrophoretic mobility shift assay. The highest activity of VDRE binding was found in the cerebellum among other brain regions examined, but sequence specific by taking into consideration the efficient competition with excess unlabeled VDRE but not with mutated VDRE. On in situ hybridization analysis, cells stained for VDR mRNA were abundant in neuron‐enriched areas of cerebral cortex, hippocampus and cerebellar cortex in the mouse brain. Chronic treatment of vitamin D3 increased the expression of microtubule‐associated protein‐2, growth‐associated protein‐43 and synapsin‐1 in cultured rat cortical neurons, suggesting a trophic role of vitamin D3 in differentiation and maturation of neurons. Neuronal cell death by brief glutamate exposure was significantly protected in cultured cortical neurons chronically treated with vitamin D3. Parallel studies showed that VDR mRNA was significantly upregulated 12–24 hr after brief glutamate exposure in cultured neurons chronically treated with vitamin D3, but not in those with vehicle alone. Our results suggest that vitamin D3 may play a role in mechanisms relevant to protective properties against the neurotoxicity of glutamate through upregulation of VDR expression in cultured rat cortical neurons.

The Rewards of Nicotine: Regulation by Tissue Plasminogen Activator–Plasmin System through Protease Activated Receptor-1

Nicotine, a primary component of tobacco, is one of the most abused drugs worldwide. Approximately four million people die each year because of diseases associated with tobacco smoking. Mesolimbic dopaminergic neurons mediate the rewarding effects of abused drugs, including nicotine. Here we show that the tissue plasminogen activator (tPA)–plasmin system regulates nicotine-induced reward and dopamine release by activating protease activated receptor-1 (PAR1). In vivo microdialysis revealed that microinjection of either tPA or plasmin into the nucleus accumbens (NAc) significantly potentiated whereas plasminogen activator inhibitor-1 reduced the nicotine-induced dopamine release in the NAc in a dose-dependent manner. Nicotine-induced dopamine release was markedly diminished in tPA-deficient (tPA−/−)mice, and the defect of dopamine release in tPA−/− mice was restored by microinjection of either exogenous tPA or plasmin into the NAc. Nicotine increased tPA protein levels and promoted the release of tPA into the extracellular space in the NAc. Immunohistochemistry revealed that PAR1 immunoreactivity was localized to the nerve terminals positive for tyrosine hydroxylase in the NAc. Furthermore, we demonstrated that plasmin activated PAR1 and that nicotine-induced place preference and dopamine release were diminished in PAR1-deficient (PAR1−/−) mice. Targeting the tPA–plasmin–PAR1 system would provide new therapeutic approaches to the treatment of nicotine dependence.

Blockade by ferrous iron of Ca2+ influx through N-methyl-d-aspartate receptor channels in immature cultured rat cortical neurons

Rat cortical neurons cultured for 3 days in vitro were loaded with the fluorescent indicator fluo‐3 for assessment of intracellular free calcium ion (Ca2+) concentrations with the aid of a confocal laser‐scanning microscope. In the absence of added MgCl2, the addition of NMDA induced a rapid but sustained increase in the number of fluorescent neurons in a concentration‐dependent manner at a concentration range of 1–100 µm with the increase by KCl being transient. The addition of FeCl2, but not FeCl3, markedly inhibited the increase by NMDA in a reversible manner at concentrations of 10–200 µm, without affecting that by KCl. Extensive analyses revealed clear differentiation between inhibitions by ferrous iron and other channel blockers known to date. The inhibition by FeCl2 was completely prevented by the addition of two different iron chelators. Exposure to NMDA alone did not lead to cell death in immature cultured neurons, however, while further addition of FeCl2 invariably induced neuronal cell death 24 h after exposure. These results give support to our previous proposal that NMDA receptor complex may contain a novel site sensitive to blockade by ferrous iron in rat brain.

An increase in intracellular free calcium ions by nicotinic acetylcholine receptors in a single cultured rat cortical astrocyte

Neuronal nicotinic acetylcholine receptors (nAChRs) are composed of an assembly between at least seven alpha (α2–α7, α9) and three beta (β2–β4) subunits in mammals. The addition of 50 mM KCl or 1 mM nicotine immediately increased the number of cells with high fluorescence intensity in rat cortical astrocytes on fluo‐3 fluorescence measurement. Nicotine was effective at increasing the fluorescence intensity in astrocytes cultured for 2 days after replating, but not in those used 1 or 5 days after replating, without markedly affecting the cellular viability irrespective of the exposure period. Nicotine markedly increased the fluorescence intensity in a concentration‐dependent manner at a concentration range of 10–100 μM in cultured astrocytes when analyzed on a responsive single cell. In these responsive single cells, the increase by nicotine was significantly prevented by the heteromeric α4/β2 subtype antagonist dihydro‐β‐erythroidine and the homomeric α7 subtype antagonist methyllycaconitine, as well as by nifedipine and EGTA but not thapsigargin. Methyllycaconitine failed to inhibit further the increase by nicotine in the presence of nifedipine, however, whereas the expression of mRNA was seen for all mammalian neuronal nAChR subunits in cultured rat cortical astrocytes as well as neurons. These results suggest that nicotine may increase intracellular free Ca2+ through the influx of extracellular Ca2+ across L‐type voltage‐gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to the α4/β2 and/or α7 nAChR channels functionally expressed in cultured rat cortical astrocytes.

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons.

In cortical neurons cultured for 3 or 9 days in vitro (DIV), exposure to hydrogen peroxide (H2O2) led to a marked decrease in cell viability in a concentration‐dependent manner at a concentration range of 10 µm to 1 mm irrespective of the duration between 6 and 24 h. However, H2O2 was more potent in decreasing cellular viability in cortical neurons cultured for 9 DIV than in those for 3 DIV. Pyruvate was effective in preventing the neuronal cell death at 1 mm even when added 1–3 h after the addition of H2O2. Semi‐quantitative RT–PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9 DIV than in those for 3 DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9 DIV, without markedly affecting that in neurons cultured for 3 DIV. These results suggest that vulnerability to H2O2 may at least in part involve expression of particular MCT isoforms responsible for the bi‐directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.

Glutamate Inhibits Chondral Mineralization through Apoptotic Cell Death Mediated by Retrograde Operation of the Cystine/Glutamate Antiporter

Although we have previously demonstrated the functional significance of excitatory amino acid transporters as well as glutamate (Glu) receptors (GluRs) expressed by chondrocytes, little attention has been paid to the possible expression of the cystine/Glu antiporter responsible for the bi-directional transmembrane transport of Glu in chondrocytes to date. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, the chondral mineralization was significantly decreased in the presence of Glu at a high concentration. Apoptotic cells were detected within the late proliferating and prehypertrophic chondrocytic layers in metatarsals cultured in the presence of Glu. A group III metabotropic GluR (mGluR) antagonist partially, but significantly, prevented the inhibition of mineralization by Glu in metatarsals without affecting the number of apoptotic cells. Both decreased mineralization and apoptosis by Glu were significantly prevented by the addition of the cystine/Glu antiporter inhibitor homocysteic acid, as well as reduced glutathione (GSH) and cystine. Expression of mRNA for xCT and 4F2hc subunits, which are components of the cystine/Glu antiporter, was seen in both cultured mouse metatarsals and rat costal chondrocytes. In chondrocytes cultured with Glu, a significant decrease was seen in intracellular GSH levels, together with increases in the number of apoptotic cells and the level of intracellular reactive oxygen species. These results suggest that Glu could regulate chondrogenic differentiation toward mineralization through a mechanism associated with apoptosis mediated by the depletion of intracellular GSH after the retrograde operation of the cystine/Glu antiporter, in addition to the activation of group III mGluR, in chondrocytes.

Possible Protection by Notoginsenoside R1 against Glutamate Neurotoxicity Mediated by N-methyl-D-aspartate Receptors Composed of an NR1/NR2B Subunit Assembly

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. Wefound that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 μM Glu for 1 hr in a dose‐dependent manner at concentrations from 0.1 to 10 μM, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca2+ ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl‐2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N‐methyl‐D‐aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 μM NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 μM NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain.

Modulation of cellular proliferation and differentiation through GABAB receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain

In this study, we have attempted to evaluate the possible role of metabotropic GABAB receptors (GABABR) expressed by neural progenitor cells prepared from neocortex of embryonic Std‐ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABAAR, GABABR, and GABACR subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration‐dependent increases in the total area and proliferation activity of neurospheres at 10–300 µM, while the GABABR agonist baclofen at 100 µM significantly increased the size of neurospheres expressing both GABABR1 and GABABR2 subunits in a manner sensitive to a GABABR antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABABR1 subunit. In neurospheres of GABABR1‐null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABABR may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self‐replication in the developing mouse brain. J. Cell. Physiol. 216: 507–519, 2008.

Functional Expression of Carnitine/Organic Cation Transporter OCTN1/SLC22A4 in Mouse Small Intestine and Liver

Carnitine/organic cation transporter (OCTN1/SLC22A4) accepts various therapeutic agents as substrates in vitro and is expressed ubiquitously, although its function in most organs has not yet been examined. The purpose of the present study was to evaluate functional expression of OCTN1 in small intestine and liver, using octn1 gene knockout [octn1(−/−)] mice. After oral administration of [3H]ergothioneine ([3H]ERGO), a typical substrate of OCTN1, the amount of [3H]ERGO remaining in the small intestinal lumen was much higher in octn1(−/−) mice than in wild-type mice. In addition, uptake of [3H]ERGO by human embryonic kidney 293 cells heterologously expressing OCTN1 gene product and uptake of [3H]ERGO at the apical surface of intestinal everted sacs from wild-type mice were inhibited by OCTN1 substrates, tetraethylammonium and verapamil. Immunohistochemical analysis revealed that OCTN1 is localized on the apical surface of small intestine in mice and humans. These results suggest that OCTN1 is responsible for small intestinal absorption of [3H]ERGO. However, the plasma concentration of [3H]ERGO after oral administration was higher in octn1(−/−) mice than in wild-type mice, despite the lower absorption in octn1(−/−) mice. This was probably because of efficient hepatic uptake of [3H]ERGO, as revealed by integration plot analysis; the uptake clearance was close to the hepatic plasma flow rate. The uptake of [3H]ERGO by isolated hepatocytes was minimal, whereas [3H]ERGO uptake was observed in isolated nonparenchymal cells. This finding is consistent with immunostaining of OCTN1 in liver sinusoids. Thus, our results indicate that OCTN1 is functionally expressed in nonparenchymal liver cells.