Keiichi Kadoyama

Long-term potentiation-like facilitation through GABAA receptor blockade in the mouse dentate gyrus in vivo.

We have investigated the characteristics of a long-term potentiation (LTP)-like facilitation in vivo through GABAA receptor blockade in C57BL/6 and Alzheimer-model transgenic mice dentate gyrus. Bicuculline induced the ‘LTP-like’ facilitation in a dose-dependent manner. MK-801 inhibited the induction but not maintenance of ‘LTP-like’ facilitation through bicuculline. Tetanic LTP was further enhanced by bicuculline, but the ‘LTP-like’ facilitation was conversely attenuated by tetanus. In transgenic mice expressing mutant human tau as an Alzheimer-model, the ‘LTP-like’ facilitation through bicuculline was significantly greater than tetanic LTP. Bicuculline recovered tetanic LTP impaired in these transgenic mice. These results suggest that GABAA receptor blockade plays an important role in learning and memory, providing a clue for the clinical use of GABAA antagonists to improve cognitive disorders.

Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice

Hepatocyte growth factor (HGF) and its receptor, c‐Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron‐specific manner (HGF‐Tg) to deliver HGF into the brain without injury. HGF‐Tg mice displayed increased alternation rates in the Y‐maze test compared with age‐matched wild‐type (WT) controls. In the Morris water maze (MWM) test, HGF‐Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF‐Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short‐ and long‐term memory. Western blot analyses revealed that the levels of N‐methyl‐D‐aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF‐Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

Lipid A-activated inducible nitric oxide synthase expression via nuclear factor-κB in mouse choroid plexus cells.

Choroid plexus (CP) which is responsible for the inflammatory mediators including nitric oxide (NO) are thought to play a crucial role in the process of bacterial meningitis. The present study investigated the mechanisms regulating inducible nitric oxide synthase (iNOS) expression in the choroid plexus epithelium (CPe) in mice. Initially, the expression of iNOS in mouse CPe was strengthened by intracerebroventriclar (i.c.v.) administration of lipid A, which is part of a Gram-negative bacterial endotoxin located at one end of the lipopolysaccharide (LPS) molecule. Next, the expression of iNOS in the CP epithelial cell line ECPC-4 cells was increased from 24 to 48h after lipid A treatment, although mRNA and proteins of toll-like receptor (TLR)-2 and -4 expressed in ECPC-4 cells were not changed by lipid A. The expression of total nuclear factor κB (NFκB), an inflammatory transcriptional factor, in ECPC-4 cells was not changed for 72 h after lipid A treatment, while cytoplasmic NFκB was decreased and nuclear NFκB was increased from 1 to 2 h. In addition, the phosphorylation of inhibitor κB (IκB) was peaked at 10 min, and the level of IκB was attenuated from 10 to 45 min after lipid A treatment. Moreover, the RNA interference (RNAi) of NFκB suppressed the expression of iNOS induced by lipid A. We demonstrated that lipid A-induced iNOS expression in ECPC-4 cells was mainly regulated by the activation of NFκB-IκB intracellular signaling pathway. Thus, we propose that the CPe plays a pivotal role in innate immunity responses of the brain, that is, the signal pathway TLRs on the CPe following inflammatory stimulation such as meningitis is activated, leading to iNOS expression through NFκB.

Proteomic analysis of time-dependent changes in proteins expressed in mouse hippocampus during synaptic plasticity induced by GABAA receptor blockade.

Protein synthesis is required for long-lasting synaptic plasticity. We examined the time-dependent changes in protein expression that occurred in the hippocampus during synaptic plasticity using two-dimensional gel electrophoresis followed by mass spectrometry. The levels of 15 proteins were significantly changed in mouse hippocampus 8h after bicuculline application (1.0mg/kg, i.p.). Expression of 14 proteins (i.e., dihydropyrimidinase-related protein 2, α-tubulin isotype M-α-2, tubulin β-1 chain, tubulin β-2A chain, protein disulfide-isomerase ERp61 precursor, chaperonin-containing T complex polypeptide 1 β subunit, T complex polypeptide 1 [partial], creatine kinase B-type, cytosolic malate dehydrogenase [partial], vacuolar adenosine triphosphatase subunit A, and uncharacterized protein LOC433182) was increased and expression of one protein (i.e., actin γ, cytoplasmic 1) was decreased. Western blotting also validated the changes in dihydropyrimidinase-related protein 2, creatine kinase B-type, and vacuolar adenosine triphosphatase subunit A levels in mouse hippocampus 8h after bicuculline application. The identified proteins were effectors of cellular functions including neuronal differentiation, cytoskeletal dynamics, folding of proteins, stress response, energy metabolism, synapse formation, and unknown function. Taken together, these findings indicate that the identified proteins play an important role in synaptic plasticity in the hippocampus.

The influence of chronic ibuprofen treatment on proteins expressed in the mouse hippocampus.

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), treatment with which has been shown to delay the onset, slows the cognitive decline, and decreases the incidence of Alzheimer׳s disease (AD) in epidemiological and clinical studies. However, a comprehensive understanding of its mechanism of action remains unclear. To elucidate the prophylactic effect of ibuprofen on the onset of the learning and memory disturbances of AD, we performed proteomic analysis of the hippocampus of chronic ibuprofen-treated mice using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. Twenty-eight proteins and seven phosphoproteins were identified to be significantly changed in the hippocampus of chronic ibuprofen-treated mice: translationally controlled tumor protein, thioredoxin-dependent peroxide reductase, and peroxiredoxin 6 were increased, and glial fibrillary acidic protein, dihydropyrimidinase-related protein 2, EF-hand domain-containing protein D2, and 14-3-3ζ were decreased. These identified proteins and phosphoproteins could be classified as cytoskeletal, neuronal development, chaperone, metabolic, apoptosis, neurotransmitter release, ATP synthase, deubiquitination, proteasome, NOS inhibitor, adapter, vesicle transport, signal transduction, antioxidant enzyme, proton transport, synaptogenesis, and serine/threonine phosphatase types. Western blot analysis showed the changes in dihydropyrimidinase-related protein 2, heat shock protein 8, ubiquitin carboxyl-terminal hydrolase PGP9.5, and γ-enolase levels in the hippocampus of chronic ibuprofen-treated mice. These findings showed that the chronic treatment with ibuprofen changed the levels of some proteins and phosphoproteins in the hippocampus. We propose that these identified proteins and phosphoproteins play an important role in decreasing the incidence of AD, especially impaired learning and memory functions.

Changes in the expression of collapsin response mediator protein-2 during synaptic plasticity in the mouse hippocampus.

We have previously reported that nicotine application to the adult mouse causing long‐term potentiation‐like facilitation in vivo in the hippocampus can serve as a model of synaptic plasticity. The present study clarifies the involvement of collapsin response mediator protein‐2 (CRMP2) in synaptic plasticity. CRMP2 was detected in hippocampal neurons of adult mice. The levels of CRMP2 mRNA and protein were increased 2–24 hr and 4–24 hr, respectively, after application of nicotine (3 mg/kg, i.p.), finally returning to the basal level by 48 hr. Furthermore, the ratio of phosphorylated CRMP2 (pCRMP2) at Thr514 residue, an inactive form, to total CRMP2 levels was not changed during synaptic plasticity expressed by nicotine, indicating an enhanced level of non‐pCRMP2. This increase of CRMP2 was inhibited by blockade of nicotinic acetylcholine receptors (nAChRs) and required activation of both α4β2 and α7 nAChRs. Although the level of ubiquitinated CRMP2 was increased 8 hr after nicotine treatment, the ratio of ubiquitinated CRMP2 to total CRMP2 protein was similar for nicotine‐treated and nontreated mice. This study demonstrates that the expression of CRMP2 increases in hippocampal neurons during synaptic plasticity and that the increment is due mainly to mRNA expression. We propose that CRMP2, particularly non‐pCRMP2, could contribute to long‐lasting synaptic plasticity.

Loss of dopaminoreceptive neuron causes L-dopa resistant parkinsonism in tauopathy

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a family of inherited dementias caused by tauopathy. A mutation in exon 10 of the tau gene, N279K, causes a particular kindred of FTDP-17, which is predominant for parkinsonism. The disease initially presents as L-dopa resistant parkinsonism which then rapidly progresses. The final pathological features reveal disappearing dopamine (DA) neurons, but the causes remain poorly understood. We previously established a transgenic mouse with human N279K mutant tau as a model for FTDP-17, which showed cognitive dysfunctions caused by the mutant. Here we analyze L-dopa resistant parkinsonism by several behavioral tests, and focus on the distributions and accumulations of the mutant tau in the DA system by immunohistochemistry and Western blot. Interestingly, dopaminoreceptive (DAr) neurons in the striatum showed neurofibrils degeneration and apoptosis through caspase-3 activation by mutant tau accumulation. The DAr neuron loss in the caudoputamen, the target of the nigrostriatal system occurred before DA neuron loss in young symptomatic mice. Residual DA neurons in the mouse functioned in DA transportation, whereas dysregulation of intracellular DA compartmentalization implied an excess level of DA caused by DAr neuron loss. In the final stages, both DAr and DA neurons decreased equally, unlike Parkinsons disease. Therefore, DAr neurons were fundamentally vulnerable to the mutation indicating a critical role for the L-dopa resistant parkinsonism in tauopathy.

The influence of chronic nicotine treatment on proteins expressed in the mouse hippocampus and cortex

Chronic treatment with nicotine, the primary psychoactive substance in tobacco smoke, affects central nervous system functions, such as synaptic plasticity. Here, to clarify the effects of chronic nicotine treatment on the higher brain functions, proteomic analysis of the hippocampus and cortex of mice treated for 6 months with nicotine was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. There was significant change in the expression of 16 proteins and one phosphoprotein in the hippocampus (increased tubulin β-5, atp5b, MDH1, cytochrome b-c1 complex subunit 1, Hsc70, dynamin, profilin-2, 4-aminobutyrate aminotransferase, mitochondrial isoform 1 precursor, calpain small subunit 1, and vacuolar adenosine triphosphatase subunit B and decreased γ-actin, α-tubulin isotype M-α-2, putative β-actin, tubulin β-2A, NDUFA10, and G6PD) and 24 proteins and two phosphoproteins in the cortex (increased spectrin α chain, non-erythrocytic 1 isoform 1, tubulin β-5, γ-actin, creatine kinase B-type, LDH-B, secernin-1, UCH-L1, 14-3-3 γ, type II peroxiredoxin 1, PEBP-1, and unnamed protein product and decreased tubulin α-1C, α-internexin, γ-enolase, PDHE1-B, DPYL2, vacuolar adenosine triphosphatase subunit A, vacuolar adenosine triphosphatase subunit B, TCTP, NADH dehydrogenase Fe-S protein 1, protein disulfide-isomerase A3, hnRNP H2, γ-actin, atp5b, and unnamed protein product). Additionally, Western blotting validated the changes in dynamin, Hsc70, MDH1, NDUFA10, α-internexin, tubulin β-5 chain, and secernin-1. Thus, these findings indicate that chronic nicotine treatment changes the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that effect of smoking on higher brain functions could be mediated by alterations in expression levels of these proteins.

The expression changes of EphA3 receptor during synaptic plasticity in mouse hippocampus through activation of nicotinic acetylcholine receptor.

We have reported that systemic application of nicotinic agonists results in expression of a long-term potentiation-like facilitation, a model of synaptic plasticity, in the mouse hippocampus in vivo. Eph receptors and their ephrin ligands, are thought to participate in synaptic plasticity. The present study was conducted to clarify the involvement of EphA3 receptor in synaptic plasticity by investigating the time-dependent change of the expression levels of EphA3 receptor during long-term potentiation-like facilitation in the mouse hippocampus. EphA3 receptor mRNA and protein expression was found in adult mouse hippocampus. EphA3 receptor was localized in neuronal cells but not astrocytes or microglia of hippocampus. After intraperitoneal application of nicotine (3 mg/kg), the protein expression of EphA3 receptor in hippocampus increased during 2–24-h period, significantly increasing during 2–12-h period, and finally returned to the basal level in 72 h, although the mRNA expression of EphA3 receptor was not changed for 24 h. This enhanced expression of EphA3 receptor protein at 4 h was inhibited by pretreatment of mecamylamine (0.5 mg/kg, intraperitoneally), a nonselective nicotinic acetylcholine receptor antagonist. Our findings demonstrated that EphA3 receptor localized only in neuronal cells of the hippocampus was enhanced without transcriptional regulation during synaptic plasticity through activation of the nicotinic acetylcholine receptor. These results suggest that the enhancement of EphA3 receptor after synaptic plasticity may contribute to long-lasting synaptic plasticity through positive, feedforward mechanisms.

Proteomic Analysis of Hippocampus and Cortex in Streptozotocin-Induced Diabetic Model Mice Showing Dementia

Aim Diabetes with its associated hyperglycemia induces various type of peripheral damage and also impairs the central nervous system (CNS). This study is aimed at clarifying the precise mechanism of diabetes-induced dementia as an impairment of CNS. Methods The proteomic analysis of the hippocampus and cortex in streptozotocin- (STZ-) treated mouse diabetic model showing dementia was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (n = 3/group). Results Significant changes in the expression of 32 proteins and 7 phosphoproteins were observed in the hippocampus and cortex. These identified proteins and phosphoproteins could be functionally classified as cytoskeletal protein, oxidoreductase, protein deubiquitination, energy metabolism, GTPase activation, heme binding, hydrolase, iron storage, neurotransmitter release, protease inhibitor, transcription, glycolysis, antiapoptosis, calcium ion binding, heme metabolic process, protein degradation, vesicular transport, and unknown in the hippocampus or cortex. Additionally, Western blotting validated the changes in translationally controlled tumor protein, ATP-specific succinyl-CoA synthetase beta subunit, and gamma-enolase isoform 1. Conclusions These findings showed that STZ-induced diabetes changed the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that alterations in expression levels of these proteins play an important role in diabetes-induced dementia.