Intracellular amyloid-␤ peptide (A␤) has been implicated in neuronal death associated with Alzheimer's disease. Although A␤ is predominantly secreted into the extracellular space, mechanisms of A␤ 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 A␤ from the cell surface to the intracellular space. Mouse cortical neurons exposed to extracellular human A␤ 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 A␤ and protection from A␤-mediated mitochondrial dysfunction. A␤ activated p38 MAPK, but not SAPK/JNK, and then stimulated intracellular uptake of A␤-RAGE complex. Similar intraneuronal co-localization of A␤ 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 A␤ from the extracellular to the intracellular space, thereby enhancing A␤ cytotoxicity.is a progressive neurodegenerative process characterized by senile plaques, neurofibrillary tangles, and neuronal loss (1, 2). Deposition of amyloid-␤ peptide (A␤), a 39-43-amino acid peptide derived from the transmembrane amyloid precursor protein (APP), is found in extracellular senile plaque cores and is associated with neurodegeneration in later stages of AD. In contrast, recent studies suggest that accumulation of intraneuronal A␤ may be an early event in the pathogenesis of AD (3-16). Addition of A␤ to human neuronal-like cells caused significant mitochondrial damage (17). Furthermore, our recent study revealed that binding of A␤ to A␤-binding alcohol dehydrogenase (ABAD) or cyclophilin D (10, 11) intracellularly triggered events leading to neuronal apoptosis through a mitochondrial pathway (12,13,18,19). However, mechanisms through which A␤ produced at the plasma membrane and released into the extracellular space reaches the intracellular milieu remain to be elucidated.Receptor for advanced glycation end products (RAGE) is a multiligand receptor of the Ig superfamily of cell surface molecules (20)(21)(22). RAGE acts as a counter-receptor for several quite distinct classes of ligands, such as AGEs, S100/calgranulins, HMG1 (high mobility group 1 or amphoterin), and the family of crossed ␤-sheet fibrils/macromolecular assemblies, which activate receptormediated signal transduction pathways. These ligand-receptor interactions are believed to exert pathogenic effects through sustained cellular perturbation in a range of chronic disorders, including the secondary complications of diabetes, inflammation, and neurodegenerative processes (23,24). RAGE, a cell surface binding site for A␤ (25), is expressed at higher levels in an A␤-rich environment (...

Several lines of evidence suggest that extracellular signal-regulated kinase1/2 (ERK1/2) and dopaminergic system is involved in learning and memory. However, it remains to be determined if the dopaminergic system and ERK1/2 pathway contribute to cognitive function in the prefrontal cortex (PFC). The amount of phosphorylated ERK1/2 was increased in the PFC immediately after exposure to novel objects in the training session of the novel object recognition test. An inhibitor of ERK kinase impaired long-term recognition memory 24 h after the training although short-term memory tested 1 h after the training was not affected by the treatment. The dopamine D1 receptor agonist increased ERK1/2 phosphorylation in the PFC in vivo as well as in cortical neurons in vitro. Microinjection of the dopamine D1 receptor antagonist into the PFC impaired long-term recognition memory whereas the D2 receptor antagonist had no effect. Immunohistochemistry revealed that exposure to novel objects resulted in an increase in c-Fos expression in the PFC. Microinjection of the protein synthesis inhibitor anisomycin into the PFC impaired the long-term recognition memory. These results suggest that the activation of ERK1/2 following the stimulation of dopamine D1 receptors is necessary for the protein synthesis-dependent long-term retention of recognition memory in the PFC.

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.

ABSTRACT: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 [

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 nicotineinduced dopamine release in the NAc in a dose-dependent manner. Nicotine-induced dopamine release was markedly diminished in tPA-deficient (tPA Ϫ/Ϫ )mice, and the defect of dopamine release in tPA Ϫ/Ϫ 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 Ϫ/Ϫ ) mice. Targeting the tPA-plasmin-PAR1 system would provide new therapeutic approaches to the treatment of nicotine dependence.