The neuroprotective effect of G-CSF against oAβ1-42 toxicity

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Abstracts / Neuroscience Research 68S (2010) e109–e222

people with memory dysfunction. However, molecular and cellular effects of TMS on neural tissue have not been well explored. We thus set out to study electrophysiological properties, such as long-term potentiation and neuronal excitability, in cortical and hippocampal slices obtained from 3xTg AD model mice (Oddo et al, 2003), in which genes for amyloid precursor protein, tau, and presenilin-1 have been modified to produce excessive extracellular amyloid deposits as well as in tracellular amyloid accumulation. A group of 3xTg was TMS treated once a day for 1 month chronically. The magnitude of LTP evoked at Schaffer collateral-CA1 pyramidal cell synapses was significantly larger in slices taken from TMS-treated 3xTg mice than in those from unstimulated 3xTg mice. Neural excitability in cingulate cortex pyramidal cells were lower in terms of current-evoked spike frequency, resting membrane potentail and spike half width in slices from the TMS-treated mice. The spike data suggest that large-conductance calcium-activated potassium channels (BK channels) are facilitated by TMS. Thus, TMS alters electrophysi ological properties in the forebrain in plastic manners, some of which might be relevant to TMS-induced cognitive improvement. doi:10.1016/j.neures.2010.07.2403

P1-n03 Dysfunction of autophagy lead to activation of gamma-secretase through GCN2 Kazunori Ohta 1 , Akihito Mizuno 1 , Masashi Ueda 1 , Shimo Li 1 , Yoshihiro Suzuki 1 , Yoko Hida 1 , Yoshika HayakawaYano 1 , Masanori Itoh 1 , Eri Ohta 1 , Masuko Kobori 2 , Toshiyuki Nakagawa 1 1

Department of Neurobiology, Gifu University Graduate School of Medicine National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan 2

␥-Secretase mediates final cleavage of amyloid precursor protein (APP), leading to the production of amyloid-␤ (A␤), which causes synaptic dysfunction and senile plaque in the brain of Alzheimer disease (AD). ␥-Secretase activity is enriched in autophagic vacuoles and it augments A␤ synthesis. However, the modulation of ␥-secretase activity by autophagy-lysosomal dysfunction remains unclear. Here, we examine whether ␥-secretase activity is affected in basal autophagy-disturbed cells. In autophagy-related 5 knockdown (Atg5KD ) human embryonic kidney (HEK293) cells expressing a short hairpin RNA as well as in chloroquine-treated HEK293 cells, Moreover, PS1 induction by chloroquine treatment was prevented in the ␣ subunit of eukaryotic translation initiation factor 2 (eIF2␣) kinase, general control nonderepressible 2 (GCN2) KD and activating transcription factor 4 (ATF4) KD cells. Interestingly, resveratrol, which induces autophagy, markedly reduced PS1 expressions and ␥-secretase activity. These results suggest that the autophagy-lysosomal system regulates ␥-secretase activity through GCN2eIF2␣-ATF4 signal cascade.

sites. We examined here the possibilities. Using GST pull-down assay and a Biacore, we found that Pin1 bound to Tau phosphorylated by Cdk5-p25. Ala mutant of Tau at Thr212 and Thr235 still bound to Pin1 after phosphorylation by Cdk5. Tau-4A, in which Cdk5 phosphorylation sites were mutated to Ala, did not bind to Pin1, suggested that Pin1 binds one of Cdk5 phosphorylation sites. We generated Tau-3A mutants, in which only one of Cdk5-phosphorylation sites remains intact, and examined their binding to Pin1. Pin1 bound to every Tau-3A mutats with the strongest binding at phospho-Ser404. Further, FTDP-17 mutant Tau, P301L or R406W, showed slightly weaker binding to Pin1 than WT Tau. Based on these results, we discuss how P301L and R406W Tau are highly phosphorylated in Tauopathy’s brains. doi:10.1016/j.neures.2010.07.2405

P1-n05 The neuroprotective effect of G-CSF against oA␤142 toxicity Yukiko Doi 1 Suzumura 1

, Tetsuya Mizuno 1 , Hiroyuki Mizoguchi 2 , Akio

1

Department of Neuroimmunology, RIEM, Nagoya University 2 Futuristic Environmental Simulation Center, Research Institute of Environmental Medicine, Nagoya University, Nagoya Introduction Oligomeric amyloid ␤ (oA␤)1-42 shows potent neurotoxicity and possible mediates neuronal damage in Alzheimer’s disease (AD). Granulocyte - colony stimulating factor (G-CSF) is a hematopoietic growth factor, mainly stimulates proliferation, survival and maturation of the neutrophilic granulocyte lineage. In the central nervous system (CNS), G-CSF receptor (G-CSF-R) has been found on the surface of neuronal stem cells, mature neurons, and some glial cells. The precise functions of G-CSF on mature neurons and glial cells are not fully understood. We examined the neuroprotective function of G-CSF against oA␤1-42 toxicity. Method Using neuronal cultures, microglial cultures, and neuron-microglia co-cultures, we examined the role of G-CSF in oA␤ neurotoxicity and microglial A␤ clearance. We assessed the production of A␤ degrading enzyme by G-CSF-treated neurons and microglia using RT-PCR, Western blotting and ELISA. Results G-CSF exhibited neuroprotective effect against oA␤1-42 toxicity through up-regulation of A␤ degrading enzyme, neprilysin. G-CSF also induced A␤ degrading enzyme, angiotensin-converting enzyme-2 in microglia, and enhanced microglial A␤ phagocytosis. Conclusion G-CSF promotes the function of A␤ clearance in neurons and microglia, and may be a possible therapeutic candidate in AD. doi:10.1016/j.neures.2010.07.2406

doi:10.1016/j.neures.2010.07.2404

P1-n06 Effects of presenilin 1 phosphorylation on insulin receptor

P1-n04 Interaction of peptidyl-prolyl isomerase Pin1 with Tau at the Cdk5 phosphorylation sites

Masato Maesako 1 , Kengo Uemura 2 , Masakazu Kubota 1 , Koichi Ando 2 , Akira Kuzuya 2 , Megumi Asada 1,2 , Nao Yamada 1 , Haruhiko Akiyama 3 , Ayae Kinoshita 1

Taeko Kimura 1 , Koji Tsutsumi 1 , Koichi Ishiguro 2 , Taro Sito 1 , Akiko Asada 1 , Takafumi Uchida 3 , Masato Hasegawa 4 , Shin-ichi Hisanaga 1 1

Dept of Biol Sci, Grad Sch.of Sci and Engon, Tokyo Metropolitan Univ Mitsubishi Kagaku Iustitute of Life Science 3 Dept. of Biol., Grad. Sch. of Sci, Tohoku Univ 4 Tokyo Inst. Psych

2

Tau is a microtubule-associated protein. Hyper-phosphorylated Tau is a major component of neurofibrillary tangles (NFTs) in Alzheimer’s brains. Tau is phosphorylated by a variety of protein kinases including Cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3). Cdk5 is a prolinedirected Ser/Thr protein kinase. Deregulation of Cdk5 activity is known to cause neuronal cell death in neurodegenerative diseases and suggested to induce the hyper-phosphorylation of Tau. However, it is not known yet why the hyper-phosphorylation of Tau occurs in disease brains. Pin1 is a peptidyl-prolyl isomerase catalyzing the cis/trans isomerization of phosphoSer/Thr-Pro sequences in a subset of proteins. Hyper-phosphorylated Tau is found in Pin1-deficient mouse brains. Recently we reported that Pin1 enhances dephosphorylation of Tau phosphorylated by Cdk5 (Yotsumoto et al., 2009). Cdk5 phosphorylates Tau mainly at Ser202, Thr205, Ser235 and Ser404. However, it is reported that Pin1 binds only to phosphorylated Thr212 or Thr 231 of Tau. The discrepancy suggests whether Cdk5 can phosphorylate Thr212 or Thr 231, or Pin1 binds to tau at Cdk5-phosphorylation

1

Dept Human Health Sciences, Univ of Kyoto, Kyoto 2 Dept Neurology, Univ of Kyoto, Kyoto 3 Tokyo Institute of Psychiatry

Insulin signaling has recently been highlighted in the pathology of Alzheimer’s disease (AD). Recently, we reported that the impairment of neuronal insulin signaling enhances presenilin 1 (PS1) phosphorylation at serine 353/357. In this study, we examined the function of phosphorylated PS1 at the residues by using the S353/357D PS1 construct; which was substituted with aspartate to mimic phosphorylation state. First, we demonstrated that S353/357D PS1 reduced the expression level of Insulin Receptor (IR), which was caused by the inhibition of IR transcription. Next, we examined the mechanism of this inhibition and demonstrated that the transcriptional inhibition was not caused by PS1/␥-secretase mediated regulated intramembrane proteolysis (RIP). Since phosphorylated PS1 was localized at the cytoplasm, we concluded that PS1 could interact with transcriptional factors via phosphorylation. Since the insulin signaling impairment promotes PS1 phosphorylation, IR reduction by phosphorylated PS1 may constitute a positive feedback mechanism which down-regulates insulin signaling. Several reports suggest that neuronal insulin signaling influences Tau phosphorylation, APP metabolism,