Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10-to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na + /K + -ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn 879 and Trp 880 is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.NMR | computational modeling | abnormal protein-protein interaction in synapse | hyperexcitotoxicity | protein-protein interaction inhibitors

Orthovanadate (OVA), a protein tyrosine phosphatase (PTPase) inhibitor, exerts contractile effects on smooth muscle in a Rho-kinase-dependent manner, but the precise mechanisms are not elucidated. The aim of this study was to determine the potential roles of Src and epidermal growth factor receptor (EGFR) in the OVA-induced contraction of rat aortas and the phosphorylation of myosin phosphatase target subunit 1 (MYPT1; an index of Rho-kinase activity) in vascular smooth muscle cells (VSMCs). Aortic contraction by OVA was significantly blocked not only by Rho kinase inhibitors Y-27632 [R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cyclohexanecarboxamide] and hydroxyfasudil [1-(1-hydroxy-5-isoquinolinesulfonyl)homopiperazine] but also by Src inhibitors PP2 [4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine] and Src inhibitor No. 5 [4-(3′-methoxy-6′-chloro-anilino)-6-methoxy-7(morpholino-3-propoxy)-quinazoline], and the EGFR inhibitors AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline] and EGFR inhibitor 1 [cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide]. OVA induced rapid increases in the phosphorylation of MYPT1 (Thr-853), Src (Tyr-416), and EGFR (Tyr-1173) in VSMCs, and Src inhibitors abolished these effects. OVA-induced Src phosphorylation was abrogated by Src inhibitors, but not affected by inhibitors of EGFR and Rho-kinase. Inhibitors of Src and EGFR, but not Rho-kinase, also blocked OVA-induced EGFR phosphorylation. Furthermore, a metalloproteinase inhibitor TAPI-0 [N-(R)-[2-(hydroxyaminocarbonyl) methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine amide] and an inhibitor of heparin-binding EGF (CRM 197) not only abrogated the OVA-induced aortic contraction, but also OVA-induced EGFR and MYPT1 phosphorylation, suggesting the involvement of EGFR transactivation. OVA also induced EGFR phosphorylation at Tyr-845, one of residues phosphorylated by Src. These results suggest that OVA-induced vasocontraction is mediated by the Rho-kinase-dependent inactivation of myosin light-chain phosphatase via signaling downstream of Src-induced transactivation of EGFR.

Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long β-amyloid (Aβ42) in Alzheimer's disease (AD). However, very limited information is available on the structural transition from oligomer to fibril, particularly for pathologically relevant amyloids. To the best of our knowledge, we present here the first site-specific structural characterization of Aβ42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aβ42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aβ42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-μm Aβ42 solution after a ∼10-h incubation at 4 °C, followed by a slow conversion into fibril at ∼180 h. Immunological analysis suggested that the SPA has a surface structure similar to that of amylospheroid (ASPD), a patient-derived toxic Aβ oligomer, which had a diameter of 10–15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a β-loop-β motif, which significantly differed from the triple-β motif observed for the Aβ42 fibril. The comparison of the 13C chemical shifts of SPA with those of the fibril prepared in the above conditions and interstrand distance measurements suggested a large conformational change involving rearrangements of intermolecular β-sheet into in-register parallel β-sheet during the misfolding. A comparison of the SPA and ASPD 13C chemical shifts indicated that SPA is structurally similar to the ASPD relevant to AD. These observations provide insights into the architecture and key structural transitions of amyloid oligomers relevant for AD pathology.

Key words myosin light chain phosphatase; orthovanadate; mesenteric artery Vanadium forms numerous inorganic compounds (vanadyl sulfate, sodium metavanadate, sodium orthovanadate (OVA), vanadium pentoxide) and complexes with organic compounds.1) Because of its structural similarity to phosphate, vanadate possesses various biochemical and pharmacological properties, such as the ability to inhibit ATPases 2) and protein tyrosine phosphatases (PTPases), 3) epidermal growth factor (EGF)-like mitogenic activity, 4) insulin-mimetic properties, 5) anti-apoptotic activities, 6) and antitumor or carcinogenic properties. 7,8) Vanadium compounds exert contractile effects on vascular and non-vascular smooth muscle, such as the guinea pig taenia coli, trachea, and gallbladder smooth muscle.9-11) The contractile effects of vanadates have been considered to be due to inhibition of PTPase, 9,11,12) but not ATPase, 13,14) because genistein, a protein tyrosine kinase inhibitor, attenuates vanadateinduced constriction. Vanadate-induced muscle constriction was regulated by activation of Rho kinase-dependent signaling in ileal longitudinal smooth muscle in guinea pigs, resulting in increased phosphorylation of the myosin light chain (MLC). 15)These reports showed that vanadates inhibited the activity of protein tyrosine phosphatases, leading to Rho kinase-dependent constriction. Recently, we reported that OVA induced Rho kinase-dependent constriction and phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) of myosin light chain phosphatase (MLCP). OVA-induced phosphorylation of MYPT1 was regulated by the EGF receptor (EGFR) and Src, because inhibitors of EGFR and Src abolished phosphorylation of MYPT1.16) Furthermore, metalloproteinase inhibitor TAPI-0 (N-(R)-(2-(hydroxyaminocarbonyl) methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine amide) and an inhibitor of heparin/EGF binding abrogated OVA-induced aortic constriction and phosphorylation of EGFR and MYPT1.16) This finding indicated that OVA transactivated EGFR in vascular smooth muscle cells (VSMCs) derived from the rat thoracic aorta. These findings indicate that OVA can influence smooth muscle constriction through EGFR and Rho kinase-dependent inactivation of MLCP. Furthermore, we showed that EGF induced Ca 2+ sensitization in vascular smooth muscle by Rho kinasedependent inactivation of MLCP through the extracellular signal-regulated kinases 1 and 2 (Erk1/2) and Erk1/2 kinase (MEK) pathway. 17) However, it is not clear whether OVA activates MEK and Erk1/2 signaling through the EGFR. In addition, it is unknown whether OVA induces constriction and phosphorylation of MYPT1 in mesenteric arteries in rats. Therefore, in this study, we aimed to determine whether OVA-induced constriction of rat superior mesenteric arteries is mediated by Src, EGFR, mitogen-activated protein kinase (MAPK) s, and Rho kinase.

Summary We identified ∼30-mer amyloid-β protein (Aβ) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na + ,K + -ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans -Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans -Golgi by increasing Aβ levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.

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Unlike insoluble Ab, ASPD preferentially exist on the endothelium of AD microvessels ASPD inhibit vasorelaxation via binding to ATPase a3 present on endothelial caveolae ASPD-NAKa3 interaction reduces NO release by increasing eNOS-Thr 495 phosphorylation Unlike Ab 1-42 , ASPD induce eNOS inactivation via mitochondrial ROS/ PKC pathway