Kaori Noguchi

Exposure of rat hippocampal neurons to amyloid β peptide (25–35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase I/glycogen synthase kinase-3β

Exposure of rat hippocampal neurons to the peptide amyloid beta (A beta) (25-35) as well as A beta (1-40) peptides enhances phosphorylation of tau to a paired helical filament (PHF)-state through activation of tau protein kinase I (TPK I)/glycogen synthase kinase-3 beta (GSK-3 beta) [Busciglio, J., Lorenzo, A., Yeh, J. and Yankner, B.A., Neuron, 14 (1995) 879-888; Takashima, A., Ishiguro, K., Noguchi, K., Michel, G., Hoshi, M., Sato, K., Takahashi, M., Hoshino, T., Uchida, T. and Imahori, K., Neurosci. Meeting Abstr., 671 (1995) 17]. In order to examine the effects of A beta treatment on intracellular signaling mechanism, we have investigated the role of phosphatidyl inositol-3 (PI-3) kinase in the phosphorylation of tau. A beta (25-35) exposure induced an inactivation of PI-3 kinase and an activation of TPK I/GSK-3 beta in rat hippocampal culture. Wortmannin, an inhibitor of PI-3 kinase, also activated TPK I/GSK-3 beta, leading to an enhancement of tau phosphorylation and neuronal death in hippocampal culture. These results suggest that A beta (25-35) inhibition of PI-3 kinase results in the activation of TPK I/GSK-3 beta, the phosphorylation of tau, and resultant neuronal death in rat hippocampal neurons.

Characterization of human presenilin 1 using N‐terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing

The majority of cases of early‐onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane‐spanning domain. We produced 7 monoclonal antibodies that react with 3 non‐overlapping epitopes on the N‐terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full‐size PS1 at M r 47 000 and a more abundant M r 28 000 product in membrane extracts from human brain and human cell lines. PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild‐type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.

Characterization of tau phosphorylation in glycogen synthase kinase-3β and cyclin dependent kinase-5 activator (p23) transfected cells

One of the histopathological markers in Alzheimers disease is the accumulation of hyperphosphorylated tau in neurons called neurofibrillary tangles (NFT) composing paired helical filaments (PHF). Combined tau protein kinase II (TPK II), which consists of CDK5 and its activator (p23), and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate tau to the PHF-form in vitro. To investigate tau phosphorylation by these kinases in intact cells, the phosphorylation sites were examined in detail using well-characterized phosphorylation-dependent anti-tau antibodies after overexpressing the kinases in COS-7 cells with a human tau isoform. The overexpression of tau in COS-7 cells showed extensive phosphorylation at Ser-202 and Ser-404. The p23 overexpression induced a mobility shift of tau, but most of the phosphorylation sites overlapped the endogenous phosphorylation sites. GSK-3beta transfection showed the phosphorylation at Ser-199, Thr-231, Ser-396, and Ser-413. Triplicated transfection resulted in phosphorylation of tau at 8 observed sites (Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, Ser-404, and Ser-413).

Amyloid β peptide induces cytoplasmic accumulation of amyloid protein precursor via tau protein kinase I/glycogen synthase kinase-3β in rat hippocampal neurons ☆

Abstract Exogenous application of synthetic amyloid β protein (Aβ) is known to induce neurotoxic effects in rat hippocampal culture. We report here that Aβ (25–35) induces accumulation of amyloid precursor protein (APP) derivatives in the cytoplasm of neurons. At the same time, the level of the secreted form of APP released into the culture medium decreases. Tau protein kinase I/glycogen synthase kinase-3β (TPK I/GSK-3β) antisense oligonucleotide blocked APP accumulation and prevented neuronal death. These results provide evidence that APP accumulation after Aβ treatment is regulated by TPK I/GSK-3β. Aβ neurotoxicity is probably mediated via phosphorylation of tau by TPK I/GSK-3β, resulting in an impairment of axonal transport, and cytoplasmic accumulation of APP.

Different GTP-binding proteins mediate regulation of calcium channels by acetylcholine and noradrenaline in rat sympathetic neurons.

In dissociated neurons of rat superior cervical ganglion (SCG), noradrenaline (NA) and acetylcholine (ACh) suppressed Ca2+ currents elicited by depolarizations to 0 mV from -60 mV. With GTP-gamma-S in patch electrodes, ACh and NA caused persistent inhibition of Ca2+ currents. Pretreatment of SCG cells with pertussis toxin abolished the action of ACh but not of NA. The results suggest that ACh and NA reduce the Ca2+ currents in SCG cells through different G proteins.

Adrenergic and cholinergic inhibition of Ca2+ channels mediated by different GTP-binding proteins in rat sympathetic neurones.

Effects of acetylcholine (ACh) and noradrenaline (NA) on voltage-gated ion channels of sympathetic neurones acutely dissociated from rat superior cervical ganglion (SCG) were examined using the whole-cell voltage-clamp technique. Depolarizing voltage steps elicited two types of low- and high-voltage-activated (LVA and HVA) Ca2+ currents. Pressure applications of ACh and NA produced concentration-dependent inhibition of the HVA Ca2+ current without affecting the LVA Ca2+ current. The inhibitory action of ACh on the Ca2+ current was blocked by a muscarinic antagonist, atropine. The action of NA was suppressed by an α2-adrenergic antagonist, yohimbine, but not by an α1-adrenergic antagonist, prazosin. Delayed rectifying outward K+ currents and inward rectifying K+ current were not affected by either ACh or NA. Tetrodotoxin-sensitive and -insensitive Na+ currents also remained unaffected under actions of ACh and NA. When recorded with electrode containing guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S), the inhibitory actions of ACh and NA on Ca2+ currents became irreversible. After treatment of SCG neurones with pertussis toxin, the inhibitory action of ACh on the Ca2+ current was almost completely abolished, whereas the action of NA was only partially reduced. The results suggest that ACh and NA differentially inhibit the HVA Ca2+ current via different G proteins coupling muscarinic and α2-adrenergic receptors to Ca2+ channels in rat SCG neurones.

Molecular cloning and expression of the rat homologue of presenilin-1☆

The rat homologue of the presenilin-1 (PS-1) gene, which is responsible for early-onset familial Alzheimers disease linked to chromosome 14, was cloned and sequenced. The predicted amino acid sequence showed quite high homology among rat, mouse, and human PS-1. Especially, the amino acid sequences of the putative transmembrane domains were highly conserved among the three species. The expression ĺevel of the PS-1 gene increased during brain development and the number of transcripts of the PS-1 gene changed during brain development. We found one transcript of the PS-1 gene in embryonic day 12 (E12)-E15 rat brain and two transcripts in E18-adult rat brain. Therefore, PS-1 may play a role in neurogenesis.

Cloning of cDNA and expression of the gene encoding rat presenilin-2

We have cloned the rat homologue of the presenilin-2 (PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimers disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%). All the mutated codons in PS-2 and PS-1 in chromosome 1- or 14-linked familial Alzheimers disease patients were conserved in rat PS-2. The expression of PS-2 was weaker than that of PS-1. The alternatively spliced short form of PS-2 mRNA, which was detected in human tissues was not detected in various rat tissues. During brain development, the expression level of both PS-2 and PS-1 increased but decreased in the adult. No remarkable change was observed in neural differentiation of PC12 cells.

Chapter 39: Characteristics of the changes in intracellular calcium concentration on the activation of muscarinic receptors in hippocampal neurons

Publisher Summary Hippocampus has been regarded as a brain region crucial in the consolidation of memory. The synapses within the region exhibit a characteristic nature, the long term potentiation (LTP) of synaptic transmission efficiency, which has been studied as a model system for analysis of the basic processes underlying the synaptic plasticity. The increase in the intracellular concentration of Ca 2+ ([Ca 2+ ] i ) during the LTP-inducible stimulation was demonstrated, and this increase triggers the intracellular enzymatic cascade leading to the modulation of transmission efficiency. In this context, a subtype of glutamate receptor activatable by N-methyl-D-aspartic acid (NMDA) has attracted attention. But beside glutamatergic neurotransmission within the hippocampus, the region receives an intense cholinergic input from the septum. It has been reported in various cells and tissues that the activation of muscarinic acetylcholine receptors (mAChRs) produces the increase in [Ca 2+ ] i , through an activation of voltage-sensitive Ca 2+ channels or an activation of G-protein-coupled phosphatidylinositol turnover system. Thus, it is important to know whether mAChR coupled with Ca 2+ mobilization machinery are present in the hippocampus and to reveal their causal relationship to the plastic nature of hippocampal synapses. This chapter describes a heterogeneous expression of the mAChRs in the hippocampal neurons, variation in the pattern of [Ca 2+ ] i change during the stimulation of these receptors and topographical patterns of their distribution in this brain area.

Interleukin-6 as a Neurotrophic Factor for Promoting Survival of Septal Cholinergic Neurons and Mesencephalic Catecholaminergic Neurons from Postnatal Rats

Neuronal differentiation and survival are supported by several kinds of neurotrophic factors in the central nervous system (CNS) as in the peripheral nervous system (PNS). The well-known nerve frowth factor (NGF), target derived growth factor, is synthesized1–4 and acts on cholinergic neurons5–12 in the CNS; however the action of NGF on other neurons is limited. We have been studying such factors that act on neuronal survival by using primary cultures of postnatal rat brain.