Identification and investigation of gamma-secretase associated proteins from brain

Alzheimer disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. The pathological hallmarks in the AD brain are extracellular deposition of amyloid plaques, mainly composed of the amyloid β-peptide (Aβ), and intracellular neurofibrillary tangles made of hyperphosphorylated tau. Several studies have shown that Aβ aggregation provides the initial insult, and the formation of tangles seems to be a secondary effect. Aβ is generated from the amyloid precursor protein (APP) by cleaving, which is sequentially mediated by β-secretase and γ-secretase. γ-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins such as APP and Notch. In clinical trials, treatment with γ-secretase inhibitors often results in Notch-related side effects and, thus, more specific inhibition of APP processing is necessary. Four proteins, presenilin, nicastrin, Aph-1 and Pen-2, are necessary and sufficient to produce an active γ-secretase complex. It has been suggested that γ-secretase associated proteins (GSAPs) could be of importance for substrate selection. Here, we have established an affinity purification method using a γ-secretase inhibitor derivative to isolate the native complex from brain material. We have identified several novel GSAPs from brain and studied their effect on Aβ production and Notch processing. In Paper I, we designed an efficient and selective method for purification and analysis of γ-secretase and GSAPs. Microsomal membranes were incubated with a γ-secretase inhibitor coupled to biotin via a long linker and an S-S bridge (GCB). After pull-down using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to liquid chromatography directly coupled to tandem mass spectrometry analysis, and proteins were identified by sequence data from an MS/MS spectra. All of the known γ-secretase components were identified, as well as the previously reported GSAP TMP21 and the PS-associated protein, syntaxin1. Hence, we suggest that the present method can be used to further study the composition of the γ-secretase complex. In Paper II, we investigated novel GSAPs from detergent-resistant membranes (DRMs). Recent studies showed that γ-secretase activity is highly enriched in DRMs. Thus, GSAPs localized to DRMs could be of special interest to study. We employed GCB and identified several novel GSAPs in DRMs from brain. From these identified proteins, silencing of voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1) reduced Aβ production. These proteins had a less pronounced effect on Notch processing. We concluded that VDAC1 and CNTNAP1 associate with γ-secretase in DRMs and affect APP processing. In Paper III, we investigated novel GSAPs from synaptic membranes and synaptic vesicles prepared from rat brain. Synaptic degeneration is one of the earliest Indicators of AD and results in loss of cognitive function. We employed GCB and identified several novel GSAPs in synaptic membranes and synaptic vesicles from brain. From these identified proteins, silencing of NADH dehydrogenase [ubiquinone] iron–sulfur protein 7 (NDUFS7) resulted in a decrease in Aβ levels, whereas, silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aβ levels. These proteins had no effect on Notch processing. Association of TPPP and NDUFS7 with γ-secretase was verified using co-immunoprecipitation and Proximity Ligation Assay. In Paper IV, we conduct a large scale affinity purification study of the γ-secretase complex obtained from rat and human brain. Silencing of some of the identified proteins: Probable phospholipid-transporting ATPase IIA (ATP9A), BDNF/NT-3 growth factors receptor precursor (NTRK2), Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2), DnaJ homolog subfamily A member 2 (DNAJA2) and Proton myo-inositol cotransporter (SLC2A13) reduced Aβ42 secretion in a siRNA dose-dependent manner. Two of these proteins, SLC2A13 and HCN2, had a relatively lower effect on Notch processing. Interestingly, overexpression of SLC2A13 increased Aβ40 generation. The interaction between γ-secretase and ATP9A, NTRK2, HCN2 and SLC2A13 was confirmed by using immunoprecipitation. In summary, we have established an affinity purification method to isolate the native γ-secretase complex from brain material, and we have identified several novel GSAPs that affect Aβ processing without affecting Notch cleavage. We suggest that these proteins could be the targets in a strategy to lower Aβ to treat AD

Upgrading of shielding for rare decay search in CANDLES

In the CANDLES experiment aiming to search for the very rare neutrino-less double beta decays (0νββ) using 48Ca, we introduced a new shielding system for high energy γ-rays from neutron captures in massive materials near the detector, in addition to the background reduction for 232Th decays in the 0νββ target of CaF2 crystals. The method of background reduction and the performance of newly installed shielding system are described

無麻酔無拘束の高血圧自然発症ラットの心拍出量

Cardiac output and arterial pressure in the conscious state were measured in spontaneously hypertensive rats and normal control rats with chronically implanted electromagnetic flow probe and arterial indwelling cannula. Cardiac index at rest was greater in hypertensive rats than in controls. However, the mean arterial pressure in probe implanted hypertensive rats was significantly lower than that in those without probe. Even in probe implanted hypertensive rats, those with relatively higher arterial pressures showed elevated total periperal resistance and normal cardiac output. These findings suggest that the hypertensive state is maintained by elevated total peripheral resistance with normal cardiac output and that aortic probe implantation induces a decrease in total peripheral resistance, which, being a decrease in afterload, brings rise to an increase in cardiac output. The increase in cardiac output in transposition response induced by transposing the rat from the home cage to a new cage was greater in hypertensive rats than in controls. The decrease in cardiac output in grasp response induced by grasping the rat by the human hand was insignificant in hypertensive rats

無麻酔 1-クリップ， 2-腎性 腎血管性高血圧ラットの心拍出量

Cardiac output and arterial pressure in the conscious state were observed in one-clip, two-kidney renovascular hypertensive rats with a chronically implanted electromagnetic flow probe and an arterial cannula. Normal Wistar rats were used as controls. At rest, cardiac output per body weight did not show any difference between hypertensive rats and normal controls. This kind of experimental hypertension was maintained entirely by elevation of total peripheral resistance. In transposition response induced by tranposing rats from their home cage to a new cage, after beta adrenoceptor blockade with propranolol, total peripheral resistance remained unchanged in contrast to control rats in which resistance was elevated. This may be due to either a less marked vasoconstriction in the splanchnic area or a greater non-beta adrenergic vasodilatation in the skeletal muscle in transposition response in hypertensive rats than in control rats

V1 Receptor Activation Induced by Hemorrhage and Sympathoinhibition in the Mesentery and Hindquarters of Spontaneously Hypertensive Rats

The aim of this study was to determine the effects of vasopressin V 1 receptor antagonism on regional hemodynamics in spontaneously hypertensive rats (SHR/Izm). Changes in blood flow in the superior mesenteric artery or terminal aorta were measured in rats with a chronically implanted electromagnetic flowmeter. The combination of a non-hypotensive hemorrhage (0.3 ml/100 g weight) and ganglionic blockade with hexamethonium bromide (C6; 25 mg/kg weight) had no effect on mesenteric resistance. On the other hand, subsequent intravenous administration of a peptide vasopressin V1 receptor antagonist (V1A; 10 μg/kg:[d(CH2)5 1-O Methyl-Tyr2-Arg8]-vasopressin) significantly reduced mesenteric resistance in SHR/Izm but had no effect on hindquarter resistance. Furthermore, the infusion of C6 (after pretreatment with hemorrhage plus V 1A) induced a marked reduction of blood pressure and a significant decrease in superior mesenteric resistance only in SHR/Izm. Thus, we showed an altered reactivity to V1A in the superior mesenteric and/or hindquarter vascular regions of SHR/Izm, suggesting that maintenance of elevated resistance in the mesenteric vascular bed mainly relates to a potential vasopressin- mediated vasoconstriction and that a new sympathetic vasoconstrictor tone is generated within the superior mesenteric vascular bed to compensate for hypotensive intervention (minor hemorrhage plus V 1A) in conscious SHR/Izm

Vasoconstricting Effect of Vasopressin on Regional Vascular Beds in Conscious Rats After Ganglionic Blockade

We analyzed the vasoconstrictor effects of arginine vasopressin (AVP) on regional arteries before and after ganglionic blockade with hexamethonium. Simultaneous measurements of mean arterial pressure and regional flows were obtained in conscious rats, using chronically implanted electromagnetic flow probes. Regional vascular resistance was calculated as mean arterial pressure divided by regional flow. AVP was applied intravenously as a bolus, at doses ranging from 5×10-11 to 5×10-8 g/kg. AVP increased mean arterial pressure, decreased superior mesenteric, renal and terminal aortic flows (supplied mainly for the hindquarter vascular area), and increased superior mesenteric, renal and terminal aortic (hindquarter) resistances in a dose-dependent manner. Ganglionic blockade decreased mean arterial pressure and renal resistance significantly, whereas there were no significant differences between changes in resistance before and after ganglionic blockade in superior mesenteric or terminal aortic areas. This suggested the presence of basal sympathetic vasoconstrictor tone in the renal area. After ganglionic blockade, the pressor effect of AVP was enhanced significantly. The increase in renal resistance induced by AVP was augmented after ganglionic blockade, whereas increases in superior mesenteric or terminal aortic resistance remained unchanged following ganglionic blockade. Our data suggest that the vasoconstrictor effect of AVP on renal vascular area is reduced by a mechanism which inhibits renal sympathetic basal tone