High-throughput screening for agonists of ROS production in live human vascular endothelial cells.

Reactive oxygen species (ROS) are important physiological molecules, and identifying agonists for ROS production can yield useful tools for future research. Here we present an optimized protocol for high-throughput screening for agonists that induce ROS production. We describe the use of a fluorescent probe in human vascular endothelial cells, which can establish whether ROS production occurs in mitochondria or in the plasma membrane of live cells. For complete details on the use and execution of this profile, please refer to Sasahara et al. (2021).

Alzheimer's Aß assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells.

Amyloid ß-protein (Aß) may contribute to worsening of Alzheimer's disease (AD) through vascular dysfunction, but the molecular mechanism involved is unknown. Using ex vivo blood vessels and primary endothelial cells from human brain microvessels, we show that patient-derived Aß assemblies, termed amylospheroids (ASPD), exist on the microvascular surface in patients' brains and inhibit vasorelaxation through binding to the α3 subunit of sodium, potassium-ATPase (NAKα3) in caveolae on endothelial cells. Interestingly, NAKα3 is also the toxic target of ASPD in neurons. ASPD-NAKα3 interaction elicits neurodegeneration through calcium overload in neurons, while the same interaction suppresses vasorelaxation by increasing the inactive form of endothelial nitric oxide synthase (eNOS) in endothelial cells via mitochondrial ROS and protein kinase C, independently of the physiological relaxation system. Thus, ASPD may contribute to both neuronal and vascular pathologies through binding to NAKα3. Therefore, blocking the ASPD-NAKα3 interaction may be a useful target for AD therapy.

Multi-angle development of therapeutic methods for Alzheimer's disease.

Recent clinical trial results support the idea that treatment based on the so-called amyloid hypothesis is a promising approach in Alzheimer's disease (AD), but actually, developing effective treatments for AD remains highly challenging. The discovery that neuron-specific sodium pump activity is impaired in AD and other neurodegenerative diseases such as Parkinson's disease has suggested a role for the sodium pump in the pathogenesis of these diseases. This opens up new possibilities for intervention, such as inhibiting the aberrant interaction of the sodium pump with the disease-specific ligand(s) or activating the sodium pump itself or its downstream signalling. In this review article, I would like to discuss possible anti-amyloid therapies, focusing especially on our own research. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc.

Principal component analysis of data from NMR titration experiment of uniformly 15N labeled amyloid beta (1-42) peptide with osmolytes and phenolic compounds.

A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.

Region- and neuronal-subtype-specific expression of Na,K-ATPase alpha and beta subunit isoforms in the mouse brain.

Na,K-ATPase is a ubiquitous molecule contributing to the asymmetrical distribution of Na+ and K+ ions across the plasma membrane and maintenance of the membrane potential, a prerequisite of neuronal activity. Na,K-ATPase comprises three subunits (α, ß, and FXYD). The α subunit has four isoforms in mice, with three of them (α1, α2, and α3) expressed in the brain. However, the functional and biological significances of the different brain isoforms remain to be fully elucidated. Recent studies have revealed the association of Atp1a3, a gene encoding α3 subunit, with neurological disorders. To map the cellular distributions of the α subunit isoforms and their coexpression patterns, we evaluated the mRNA expression of Atp1a1, Atp1a2, and Atp1a3 by in situ hybridization in the mouse brain. Atp1a1 and Atp1a3 were expressed in neurons, whereas Atp1a2 was almost exclusively expressed in glial cells. Most neurons coexpressed Atp1a1 and Atp1a3, with highly heterogeneous expression levels across the brain regions and neuronal subtypes. We identified parvalbumin (PV)-expressing GABAergic neurons in the hippocampus, somatosensory cortex, and retrosplenial cortex as an example of a neuronal subtype expressing low Atp1a1 and high Atp1a3. The expression of Atp1b isoforms was also heterogeneous across brain regions and cellular subtypes. The PV-expressing neurons expressed a high level of Atp1b1 and a low level of Atp1b2 and Atp1b3. These findings provide basic information on the region- and neuronal-subtype-dependent expression of Na,K-ATPase α and ß subunit isoforms, as well as a rationale for the selective involvement of neurons expressing high levels of Atp1a3 in neurological disorders.

NMR-based site-resolved profiling of ß-amyloid misfolding reveals structural transitions from pathologically relevant spherical oligomer to fibril.

Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long ß-amyloid (Aß42) 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 Aß42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aß42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aß42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-µm Aß42 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 Aß oligomer, which had a diameter of 10-15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a ß-loop-ß motif, which significantly differed from the triple-ß motif observed for the Aß42 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 ß-sheet into in-register parallel ß-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.

Tracking down a missing trigger for Alzheimer's disease by mass spectrometric imaging based on brain network analysis.

Alzheimer's disease (AD) has been proposed to begin in a cryptic form long before clinical symptoms appear. If this is the case, there might be an initial trigger leading to AD that we have not yet seen. Such a trigger might occur far away from the brain regions that are currently identified as affected sites, and might start long before either functional or histological changes are apparent. How might we detect this putative trigger? In this chapter, I propose an experimental approach using matrix-assisted laser desorption ionization (MALDI)-based imaging mass spectrometry (IMS), combined with pathological and functional connectivity studies, to track down the initial trigger of AD. The deficits in AD brains do not spread randomly, and even though we do not know where to find the initial deficit or which biomolecule is involved, the brain region where the initial change occurs is surely connected functionally to the brain regions that subsequently show detectable damage. Therefore, MALDI IMS taking account of the brain's functional connectivity and the spread of AD symptoms should be a powerful strategy for uncovering molecular signatures related to the AD trigger(s).

Alzheimer Aß Assemblies Accumulate in Excitatory Neurons upon Proteasome Inhibition and Kill Nearby NAKα3 Neurons by Secretion.

We identified ∼30-mer amyloid-ß protein (Aß) 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 Aß 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.

Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aß(1-42) in Amyloid Fibrils.

Intense efforts have been made to understand the molecular structures of misfolded amyloid ß (Aß) in order to gain insight into the pathological mechanism of Alzheimer's disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aß peptide Aß(1-42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22:499-505, 2015). Despite great biological and clinical interest in Aß(1-42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aß(1-42) is described. We illustrate a controlled incubation method to prompt misfolding of Aß(1-42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aß(1-42) fibrils as seeds. Next, we detail analysis of Aß(1-42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aß(1-42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.

Laparoscopic colectomy after self-expanding metallic stent placement through the ileocecal valve for right-sided malignant colonic obstruction: A case report.

A 78-year-old man with a history of open sigmoidectomy for sigmoid cancer presented with abdominal pain and vomiting. Abdominal multi-detector CT revealed an obstructive ileocecal tumor with distended small bowel on the oral side. We performed emergency drainage using a transnasal decompression tube, and 2 days later, we conducted a colonoscopic examination, which lead to a provisional diagnosis of obstruction with a malignant tumor invading the ileocecal valve. We then placed a self-expanding metallic stent (SEMS) through the ileocecal valve. We confirmed patency of the ileocecal valve and removed the transnasal decompression tube 2 days after SEMS placement. We then performed elective laparoscopic colectomy 8 days after SEMS placement. To the best of our knowledge, there has been only one previous report of laparoscopic colectomy after decompression with SEMS placement through the ileocecal valve for right-sided malignant colonic obstruction.

Broad ligament hernia successfully repaired by single-incision laparoscopy: A case report.

A 52-year-old woman with a history of two parturitions presented with lower abdominal pain. Multi-detector CT of the abdomen showed discontinuity of the sigmoid colon near the broad ligament on the left side. We assigned a provisional diagnosis of an internal hernia progressing through a defect in the broad ligament. SILS revealed a total broad ligament defect on the left side but no signs of ischemic, necrotic bowel. We successfully repaired the broad ligament defect with suturing. At the 2-month follow-up, the patient remained well with no signs of recurrence. This case appears to be the first report of a broad ligament hernia successfully diagnosed and repaired by SILS.

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aß) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aß peptides may be complicated. To understand the mechanisms of Aß(1-42) oligomer formation in more detail, we have established a method to produce (15)N-labeled Aß(1-42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aß aggregates must be removed. Here, we succeeded in fractionating a "precipitation-resistant" fraction of Aß(1-42) from an "aggregation-prone" fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aß(1-42). However, both Aß(1-42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the "aggregation seed" was not completely monomerized during HFIP treatment. In addition, Aß(1-42) dissolved in HFIP was found to display a monomer-dimer equilibrium, as shown by two-dimensional (1)H-(15)N NMR. We demonstrated that the initial concentration of Aß during the HFIP pretreatment altered the kinetic profiles of Aß fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aß(1-42) peptide.

[A Case Report - A Case of a Rapidly Growing Anal Canal Origin Malignant Melanoma].

An 85-year-old woman presented to her local physician in early August 2015 with a new-onset "swelling" of the anal region and was referred to our department for evaluation and treatment at the beginning of September. At our initial evaluation, the rectal examination showed a black mass lesion, approximately 3.0×1.5 cm in size, in the anal canal by November, the mass had grown to approximately 7.0×3.5 cm in size. The lesion was diagnosed as malignant melanoma by colonoscopic biopsy. Abdominal cystography computed tomography showed an area of lymphadenopathy around the rectum, but no distant metastasis was detected. The patient underwent abdominoperineal resection of the rectum in December. Her postoperative course was good, and she was discharged on the 30th postoperative day. Arecurrent lesion was detected at a recent follow-up examination(approximately 5 months post-discharge), and the patient has been scheduled for immunotherapy. Malignant melanoma of the anal canal has a poor prognosis, and no standard therapy has been established. This report includes a brief review of current literature about the disease.

[A Case of Angioimmunoblastic T-Cell Lymphoma Merged with Colorectal Cancer That We Were Able to Resect after a Chemotherapy Response].

The patient was 77-year-old man. He visited our hospital with the chief complaint of an abdominal mass in March 2015. We diagnosed the patient with transverse colon cancer and he was suspected of having malignant lymphoma. In March 2016, we attempted to perform right hemicolectomy for the transverse colon cancer, but it was difficult because swollen lymph nodes had formed a large mass with the surrounding tissue, including vessels of the mesentery. We could only complete the surgery after mesenteric lymph node biopsy. On the basis of the results of this biopsy, we diagnosed angioimmunoblastic Tcell lymphoma. At first, we administered THP-COP therapy for malignant lymphoma. However, after 3 courses of this therapy, the patient suddenly developed ileus due to the aforementioned colon cancer. According to enhanced CT performed at the onset of intestinal obstruction, chemotherapy dramatically reduced the size of the intraperitoneal lymph nodes. We therefore concluded that it was able to treat the colon cancer. We performed right hemicolectomy in June 2015. Angioimmunoblastic T-cell lymphoma comprises only 2-3% of all malignant lymphoma cases. We experienced a rare case of complications from angioimmunoblastic T-cell lymphoma and colorectal cancer.

[Successful Treatment of Local Recurrence of Advanced Gastric Cancer Using Curative Gastrectomy via Distal Pancreatectomy after Chemoradiotherapy].

The patient was a 65-year-old woman. She was diagnosed with advanced gastric cancer with liver invasion. After receiving systemic chemotherapy(S-1 plus PTX)for 3 months, she underwent total gastrectomy and partial hepatectomy in May 2008. Because she developed celiac artery circumference lymph node recurrence in November 2010 during postoperative adjuvant chemotherapy consisting of S-1 plus PTX, we changed her chemotherapy regimen to CPT-11 plus CDDP. We observed an increase in the size of the lymph nodes in August 2013 and the response was poor even after switching to DOC. However, the lymph nodes continued to increase in size and we administered radiotherapy of 60.4 Gy/33 Fr that resulted in shrinkage of the nodes. We observed an increase in lymph node size and pancreas invasion in September 2015, including an expansion of the mid pancreatic duct. We performed distal pancreatectomy without identifying the recurrence observed in November 2015 assuming it was an exacerbation. Six months after the surgery, the recurrence was not apparent. We report an example of long-term survival that was achieved for Stage IV gastric cancer. The patient underwent combined modality therapy for 8 years, and local recurrence was controlled via a primary operation.

[Two Cases of Small Intestinal Metastasis of Lung Cancer].

Case 1: A 66-year-old man who had undergone upper right lobe resection for large cell lung carcinoma 2 months earlier presented with abdominal pain and vomiting. Computed tomography showed intussusception of the small intestine. Partial resection of the small intestine was performed. The tumor was pathologically diagnosed as lung cancer metastasis to the small intestine. The patient died 30 days after surgery. Case 2: A 57-year-old man was admitted to hospital due to appetite loss. Computed tomography showed a large intestinal tumor and a small lung tumor, as well as multiple liver metastases and lymph node metastases. We diagnosed perforation of the small intestine tumor, and partial resection of small intestine was performed. Pathological examination and immunohistochemical staining revealed that the tumor was a metastatic adenocarcinoma, and the patient was diagnosed with small intestine metastasis of lung cancer. He died 75 days after surgery. Although small intestine metastasis of lung cancer is rare, the incidence is increasing. The prognosis of lung cancer metastasis of the small intestine is poor, and therefore, we must urgently decide the appropriate treatment.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific 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-Aß-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.

Structural Insight into an Alzheimer's Brain-Derived Spherical Assembly of Amyloid ß by Solid-State NMR.

Accumulating evidence suggests that various neurodegenerative diseases, including Alzheimer's disease (AD), are linked to cytotoxic diffusible aggregates of amyloid proteins, which are metastable intermediate species in protein misfolding. This study presents the first site-specific structural study on an intermediate called amylospheroid (ASPD), an AD-derived neurotoxin composed of oligomeric amyloid-ß (Aß). Electron microscopy and immunological analyses using ASPD-specific "conformational" antibodies established synthetic ASPD for the 42-residue Aß(1-42) as an excellent structural/morphological analogue of native ASPD extracted from AD patients, the level of which correlates with the severity of AD. (13)C solid-state NMR analyses of approximately 20 residues and interstrand distances demonstrated that the synthetic ASPD is made of a homogeneous single conformer containing parallel ß-sheets. These results provide profound insight into the native ASPD, indicating that Aß is likely to self-assemble into the toxic intermediate with ß-sheet structures in AD brains. This approach can be applied to various intermediates relevant to amyloid diseases.

Aß(1-42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease.

Increasing evidence has suggested that formation and propagation of misfolded aggregates of 42-residue human amyloid ß (Aß(1-42)), rather than of the more abundant Aß(1-40), provokes the Alzheimer's disease cascade. However, structural details of misfolded Aß(1-42) have remained elusive. Here we present the atomic model of an Aß(1-42) amyloid fibril, from solid-state NMR (ssNMR) data. It displays triple parallel-ß-sheet segments that differ from reported structures of Aß(1-40) fibrils. Remarkably, Aß(1-40) is incompatible with the triple-ß-motif, because seeding with Aß(1-42) fibrils does not promote conversion of monomeric Aß(1-40) into fibrils via cross-replication. ssNMR experiments suggest that C-terminal Ala42, absent in Aß(1-40), forms a salt bridge with Lys28 to create a self-recognition molecular switch that excludes Aß(1-40). The results provide insight into the Aß(1-42)-selective self-replicating amyloid-propagation machinery in early-stage Alzheimer's disease.