Changes in the amount of nucleotide sugars in aged mouse tissues.

Aging affects tissue glycan profiles, which may alter cellular functions and increase the risk of age-related diseases. Glycans are biosynthesized by glycosyltransferases using the corresponding nucleotide sugar, and the availability of nucleotide sugars affects glycosylation efficiency. However, the effects of aging on nucleotide sugar profiles and contents are yet to be elucidated. Therefore, this study aimed to investigate the effects of aging on nucleotide sugars using a new LC-MS/MS method. Specifically, the new method was used to determine the nucleotide sugar contents of various tissues (brain, liver, heart, skeletal muscle, kidney, lung, and colon) of male C57BL/6NCr mice (7- or 26-month-old). Characteristic age-associated nucleotide sugar changes were observed in each tissue sample. Particularly, there was a significant decrease in UDP-glucuronic acid content in the kidney of aged mice and a decrease in the contents of several nucleotide sugars, including UDP-N-acetylgalactosamine, in the brain of aged mice. Additionally, there were variations in nucleotide sugar profiles among the tissues examined regardless of the age. The kidneys had the highest concentration of UDP-glucuronic acid among the seven tissues. In contrast, the skeletal muscle had the lowest concentration of total nucleotide sugars among the tissues; however, CMP-N-acetylneuraminic acid and CDP-ribitol were relatively enriched. Conclusively, these findings may contribute to the understanding of the roles of glycans in tissue aging.

Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

Endogenous reductase activities for the generation of ribitol-phosphate, a CDP-ribitol precursor, in mammals.

The core M3 O-mannosyl glycan on α-dystroglycan serves as the binding epitope for extracellular matrix molecules. Defects in core M3 glycans cause congenital muscular dystrophies that are collectively known as dystroglycanopathies. The core M3 glycan contains a tandem D-ribitol-5-phosphate (Rbo5P) structure, which is synthesized by the Rbo5P-transferases fukutin and fukutin-related protein using CDP-ribitol (CDP-Rbo) as a donor substrate. CDP-Rbo is synthesized from CTP and Rbo5P by CDP-Rbo pyrophosphorylase A. However, the Rbo5P biosynthesis pathway has yet to be elucidated in mammals. Here, we investigated the reductase activities toward four substrates, including ribose, ribulose, ribose-phosphate and ribulose-phosphate, to identify the intracellular Rbo5P production pathway and elucidated the role of the aldo-keto reductases AKR1A1, AKR1B1 and AKR1C1 in those pathways. It was shown that the ribose reduction pathway is the endogenous pathway that contributes most to Rbo5P production in HEK293T cells and that AKR1B1 is the major reductase in this pathway.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

A characteristic N-glycopeptide signature associated with diabetic cognitive impairment identified in a longitudinal cohort study.

BACKGROUND: Identifying a biomarker for the decline in cognitive function in patients with diabetes is important. Therefore, we aimed to identify the N-glycopeptides on plasma proteins associated with diabetic cognitive impairment in participants in a longitudinal study using N-glycoproteomics. METHODS: We used samples from the 3-year SONIC (Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians) longitudinal cohort study of older Japanese people in the general population. First, we placed the participants with diabetes into two groups: those that did or did not have cognitive decline over a 6-year period. Next, their plasma protein profiles were compared between baseline and the 6-year time point using two-dimensional fluorescence difference gel electrophoresis. Finally, an N-glycoproteomic study of the focused proteins was performed using an enrichment technique and liquid chromatography-tandem mass spectrometry. RESULTS: Approximately 500 N-glycopeptides, derived from 18 proteins, were identified in each sample, from among which we identified the N-glycopeptides that were associated with diabetic cognitive impairment using multivariate analysis. We found that N-glycopeptides with sialylated tri- or tetra-antennary glycans on alpha-2-macroglobulin, clusterin, serum paraoxonase/arylesterase 1, and haptoglobin were less abundant, whereas 3-sialylated tri-antennary N-glycopeptides on serotransferrin were more abundant. CONCLUSION: N-glycopeptides with sialylated multi-antennary glycans comprise a characteristic signature associated with diabetic cognitive impairment. GENERAL SIGNIFICANCE: The characterized N-glycopeptides represent potential biomarker candidates for diabetic cognitive impairment.

Toolbox Accelerating Glycomics (TAG): Improving Large-Scale Serum Glycomics and Refinement to Identify SALSA-Modified and Rare Glycans.

Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N'-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses.

Chemical and Chemo-Enzymatic Syntheses of Glycans Containing Ribitol Phosphate Scaffolding of Matriglycan.

Ribitol phosphate modifications to the core M3 O-mannosyl glycan are important for the functional maturation of α-dystroglycan. Three sequentially extended partial structures of the core M3 O-mannosyl glycan including a tandem ribitol phosphate were regio- and stereo-selectively synthesized: Rbo5P-3GalNAcß, Rbo5P-1Rbo5P-3GalNAcß, and Xylß1-4Rbo5P-1Rbo5P-3GalNAcß (Rbo5P, d-ribitol-5-phosphate; GalNAc, N-acetyl-d-galactosamine; Xyl, d-xylose). Rbo5P-3GalNAcß with p-nitrophenyl at the aglycon part served as a substrate for ribitol phosphate transferase (FKRP, fukutin-related protein), and its product was glycosylated by the actions of a series of glycosyltransferases, namely, ribitol xylosyltransferase 1 (RXYLT1), ß1,4-glucuronyltransferase 1 (B4GAT1), and like-acetyl-glucosaminyltransferase (LARGE). Rbo5P-3GalNAcß equipped with an alkyne-type aglycon was also active for FKRP. The molecular information obtained on FKRP suggests that Rbo5P-3GalNAcß derivatives are the minimal units required as the acceptor glycan for Rbo5P transfer and may serve as a precursor for the elongation of the core M3 O-mannosyl glycan.

CDP-ribitol prodrug treatment ameliorates ISPD-deficient muscular dystrophy mouse model.

Ribitol-phosphate modification is crucial for the functional maturation of α-dystroglycan. Its dysfunction is associated with muscular dystrophy, cardiomyopathy, and central nervous system abnormalities; however, no effective treatments are currently available for diseases caused by ribitol-phosphate defects. In this study, we demonstrate that prodrug treatments can ameliorate muscular dystrophy caused by defects in isoprenoid synthase domain containing (ISPD), which encodes an enzyme that synthesizes CDP-ribitol, a donor substrate for ribitol-phosphate modification. We generated skeletal muscle-selective Ispd conditional knockout mice, leading to a pathogenic reduction in CDP-ribitol levels, abnormal glycosylation of α-dystroglycan, and severe muscular dystrophy. Adeno-associated virus-mediated gene replacement experiments suggested that the recovery of CDP-ribitol levels rescues the ISPD-deficient pathology. As a prodrug treatment strategy, we developed a series of membrane-permeable CDP-ribitol derivatives, among which tetraacetylated CDP-ribitol ameliorated the dystrophic pathology. In addition, the prodrug successfully rescued abnormal α-dystroglycan glycosylation in patient fibroblasts. Consequently, our findings provide proof-of-concept for supplementation therapy with CDP-ribitol and could accelerate the development of therapeutic agents for muscular dystrophy and other diseases caused by glycosylation defects.

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals.

Bacteria contain glycerol phosphate (GroP)-containing glycans, which are important constituents of cell-surface glycopolymers such as the teichoic acids of Gram-positive bacterial cell walls. These glycopolymers comprising GroP play crucial roles in bacterial physiology and virulence. Recently, the first identification of a GroP-containing glycan in mammals was reported as a variant form of O-mannosyl glycan on α-dystroglycan (α-DG). However, the biological significance of such GroP modification remains largely unknown. In this review, we provide an overview of this new discovery of GroP-containing glycan in mammals and then outline the recent progress in elucidating the biosynthetic mechanisms of GroP-containing glycans on α-DG. In addition, we discuss the potential biological role of GroP modification along with the challenges and prospects for further research. The progress in this newly identified glycan modification will provide insights into the phylogenetic implications of glycan.

Toward the development of a vibrant, super-aged society: The future of medicine and society in Japan.

BACKGROUND: As Japan's population continues to age, it is estimated that the number of people aged ≥75 years will exceed 20 million by 2025. Furthermore, over the past 10 years, we have not reduced the difference between life expectancy and healthy life expectancy. Therefore, the extension of healthy life expectancy and the development of a healthy society are the most urgent issues. In terms of medical care, the changing times have inevitably led to changes in disease structures and medical demands; therefore, the medical delivery system has had to be changed to meet these demands. As dementia rapidly increases, it is important to address "frailty," a condition in which people become more vulnerable to environmental factors as they age, and there is a need to provide services to older people, particularly the old-old, that emphasize quality of life in addition to medical care. To realize a super-aged society that will remain vigorous and vibrant for many years, we need to rethink the future of Japanese medicine and healthcare, and the state of society. CURRENT SITUATION AND PROBLEMS: Disparity between healthy life expectancy and average life expectancy in the realization of a healthy society It is a challenge to build a society with a long and healthy life expectancy through comprehensive prevention and management of lifestyle-related diseases, as well as the elucidation of the factors that explain sex differences in healthy life expectancy, based on the recognition that lifestyle-related diseases in midlife are risk factors for frailty and dementia in old age. Challenges in medical care for building a super-aged and healthy society The challenges include promoting clinical guidelines suitable for older people, including lifestyle-related disease management, promoting comprehensive research on aging (basic research, clinical research and community collaboration research), and embodying a paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care." Furthermore, the key to the future of integrated community care is the development of a comprehensive medical care system for older people in each region and the development of the next generation of medical personnel. Dissemination of frailty prevention measures in a super-aged society The concept of frailty encompasses the meaning of multifacetedness and reversibility; therefore, a comprehensive approach is required, including the renewal of conventional prevention activities in each region, such as the nutritional status of older people, physical activity including exercise, and various opportunities for social participation and participation conditions. Challenges of an unstable diet and undernutrition in older people According to the National Health and Nutrition Examination Survey of Japan, energy and protein intakes are low in Japanese people aged ≥75 years; particularly in people aged ≥80 years, low and insufficient intake of nutrients are prominent. Undernutrition in older people is increasing and is more pronounced in women. There are multiple factors behind this, including social factors, such as living alone, eating alone, poverty and other social factors, as well as problems with access to food security. Pharmacotherapy for older people: measures against polypharmacy In addition to the problems of adverse drug events, drug interactions, duplication of effects and the presence of drugs that "require particularly careful administration," it is also necessary to take measures against polypharmacy in older people, as well as medical economic issues, such as high drug costs and large amounts of remaining drugs. Barriers to this measure include multiple medical institution visits for each disease, lack of coordination between professions, and lack of understanding by patients and families. Role of local communities in a healthy society The decline in the working-age population is also a major challenge; however, we need to make a shift to use this declining birthrate and aging population as an opportunity rather than a crisis. As we look ahead to the coming of the 100-year age of life, we rethink the creation of a comprehensive society and community, and aim to create an age-free society where everyone can play an active role and live in peace, regardless of age. CONTENTS OF THE PROPOSAL: In this report, we have put together a vision for the future of an aging Japanese society from a broader perspective of how the environment and local communities should be, rather than simply from the perspective of individual health. We aim to convey this proposal to the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, the Cabinet Office, and various professional organizations. The paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care" should be promoted for the development of a healthy society While further promoting pre-emptive medical care in the medical care for older people, the development of multidisciplinary medical guidelines appropriate for older people should be promoted at the same time. In addition, we should promote basic aging research, clinical research (including the long-term care field) and transitional research that cover regional areas. Furthermore, while promoting the paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care," the development of various comprehensive medical treatment systems for older people and the strengthening of integrated community care systems should be promoted. Development of the next generation of medical personnel to comprehensively deal with geriatric care, including training geriatric specialists, should be promoted As the number of older people with multimorbidities and frailty rapidly increases in the future, we should promote the development of the next generation of medical personnel who can comprehensively handle medical care for older people, including training leading geriatricians in cooperation with multiple professions in the integrated community care system to provide sufficient medical care. Countermeasures for frailty in older people should be promoted from medical and community planning perspectives To address frailty, which requires comprehensive evaluation and intervention, the three pillars of frailty prevention (nutrition, exercise and social participation) should be incorporated and addressed as part of community development within each municipality, taking into account local characteristics. In particular, it is necessary to revise the way of thinking about nutrition management in older people and the guidelines of the societies in the field. In addition, it is important to strengthen industry-academia-government-private partnerships in each region, taking into account not only medical issues, but also social factors, and encourage the development of momentum in the entire region regarding measures against undernutrition in older people. Polypharmacy measures should be promoted in pharmacotherapy for older people It is necessary to promote cooperation between physicians and pharmacists, establish other multiprofessional cooperation systems, and develop medical and long-term care insurance systems to support this. It is also essential to change the public's mindset, and awareness-raising activities at all levels are required, including the enhancement of educational materials for medical caregivers and the general public. In addition, the economic impact of healthcare using big data should be timely clarified. Innovation in medical and urban planning perspectives should be promoted In the future, it will be necessary to modify and update multidisciplinary approaches such as social participation (e.g. participation in a salon) with a view to innovation in both medical care and community development, especially on the idea of a symbiotic community. In addition, industry-academia-government-private partnership is necessary, including all aforementioned, such as places where people can play an active role in the rest of their lives (such as employment), promotion of human connections, promotion of technology to support older people and support for daily life. Geriatr Gerontol Int 2021; 21: 601-613.

PCYT2 synthesizes CDP-glycerol in mammals and reduced PCYT2 enhances the expression of functionally glycosylated α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface protein. Defective O-mannosyl glycan on α-DG is associated with muscular dystrophies and cancer. In the biosynthetic pathway of the O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP) transfer ribitol phosphate (RboP). Previously, we reported that FKTN and FKRP can also transfer glycerol phosphate (GroP) from CDP-glycerol (CDP-Gro) and showed the inhibitory effects of CDP-Gro on functional glycan synthesis by preventing glycan elongation in vitro. However, whether mammalian cells have CDP-Gro or associated synthetic machinery has not been elucidated. Therefore, the function of CDP-Gro in mammals is largely unknown. Here, we reveal that cultured human cells and mouse tissues contain CDP-Gro using liquid chromatography tandem-mass spectrometry (LC-MS/MS). By performing the enzyme activity assay of candidate recombinant proteins, we found that ethanolamine-phosphate cytidylyltransferase (PCYT2), the key enzyme in de novo phosphatidylethanolamine biosynthesis, has CDP-Gro synthetic activity from glycerol-3-phosphate (Gro3P) and CTP. In addition, knockdown of PCYT2 dramatically reduced cellular CDP-Gro. These results indicate that PCYT2 is a CDP-Gro synthase in mammals. Furthermore, we found that the expression of functionally glycosylated α-DG is increased by reducing PCYT2 expression. Our results suggest an important role for CDP-Gro in the regulation of α-DG function in mammals.

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O-mannosylation site on α-dystroglycan.

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds ß1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure.

Lactone-Driven Ester-to-Amide Derivatization for Sialic Acid Linkage-Specific Alkylamidation.

Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.

FAM3B/PANDER-Like Carbohydrate-Binding Domain in a Glycosyltransferase, POMGNT1.

Protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is one of the gene products responsible for α-dystroglycanopathy, which is a type of congenital muscular dystrophy caused by O-mannosyl glycan defects. The originally identified function of POMGNT1 was as a glycosyltransferase that catalyzes the formation of the GlcNAcß1-2Man linkage of O-mannosyl glycan, but the enzyme function is not essential for α-dystroglycanopathy pathogenesis. Our recent study revealed that the stem domain of POMGNT1 has a carbohydrate-binding ability, which recognizes the GalNAcß1-3GlcNAc structure. This carbohydrate-binding activity is required for the formation of the ribitol phosphate (RboP)-3GalNAcß1-3GlcNAc structure by fukutin. This protocol describes methods to assess the carbohydrate-binding activity of the POMGNT1 stem domain.

Crystal structures of fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.

Decreased ADAM17 expression in the lungs of α-Klotho reduced mouse.

The deficiency of α-Klotho in mice causes phenotypes resembling human age-associated disorders at 3-4 weeks after birth and shows short lifespans of ∼2 months. One of the crucial symptoms is pulmonary emphysema, although α-Klotho is not expressed in the lungs. α-Klotho secreted from the kidneys is probably involved in the pathology of emphysema because kidney-specific knockout mice exhibit emphysematous structural changes. We examined whether any glycan changes in α-Klotho mouse lungs were observed, because α-Klotho is reported to have glycosidase activity. Here, we found the accumulation of heparan sulphate in the microsomal fraction of α-Klotho mouse lungs. Meanwhile, a disintegrin and metalloproteinase 17 (ADAM17) expression was decreased in α-Klotho mice. From these results, it is thought that the increase in heparan sulphate is due to insufficient cleavage of the core protein by ADAM17. Additionally, a reduction in α-Klotho and a decline of ADAM17 were also observed both in normal aged mice and in senescence marker protein-30 (SMP30) knockout mice, a mouse model of premature ageing. Thus, the decrease in ADAM17 is caused by the reduction in α-Klotho. These may be involved in the deterioration of lung function during ageing and may be associated with the pathology of pulmonary emphysema.

Fumarate accumulation involved in renal diabetic fibrosis in Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is a spontaneous animal model of type 2 diabetes and early stage of diabetic nephropathy. However, the pathophysiological mechanisms contributing to the progression of diabetic nephropathy in GK rats remain unclear. Kidneys from 15-week old male diabetic GK/Jcl rats and age-matched Wistar rats, which have the same genetic background as GK rats, were used. Proteomic analyses of GK and Wistar kidneys were performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Differentially expressed proteins in GK rats were subjected to pathway analysis, and expression levels of hypoxia inducible factor 1α (HIF-1α) and transforming growth factor-ß1 (TGF-ß1), and fumarate accumulation in GK kidneys were examined. Azan staining and immunohistochemical staining of α-smooth muscle actin were performed in relation to fibrosis in GK kidneys. Proteomic analysis using 2D-DIGE, analysis of fumarate content, and expression analysis of HIF-1α, TGF-ß1, and α-smooth muscle actin of GK rat's kidney, suggested the mechanism of fibrosis characterized as two stages in diabetic nephropathy of GK rats. Abnormalities of glucose metabolism such as elevated levels of 2-oxoglutarate dehydrogenase and reduction of fumarate hydratase caused the accumulation of fumarate followed by the upregulation of HIF-1α and TGF-ß1 leading to fibrosis in diabetic nephropathy. Alterations in proteins involved in the tricarboxylic acid cycle are associated with fibrosis through fumarate accumulation in diabetic nephropathy of GK rats.

O-GlcNAcylation and phosphorylation of ß-actin Ser199 in diabetic nephropathy.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of ß-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199 of ß-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199 (gS199)-actin and in the phosphorylation of Ser199 (pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199 occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

Age-associated proteomic alterations in human aortic media.

AIM: Human vascular senescence, which mainly occurs in media, is not completely understood. Here, we used proteomic approaches to investigate age-associated changes in human aortic media with the goal of understanding the molecular mechanisms underlying vascular senescence. METHOD: Cryopreserved autopsy samples of aortic media from older-aged (aged 70-100 years, n = 25), middle-aged (aged 49-68 years, n = 24), and young (aged 21-39 years, n = 12) subjects were collected. We used two proteomic techniques, two-dimensional differential gel electrophoresis and isobaric tags for relative and absolute quantitation, and we subjected differentially-expressed proteins among age groups to immunohistochemical analyses. RESULTS: Proteomic analyses showed that the expression of lactadherin, which produces medin, was elevated in aortic media of older-aged individuals. Immunohistochemical and Congo red staining showed that lactadherin and apolipoprotein E were deposited, and that amyloidosis was enhanced in older-aged aortic media. Furthermore, the markers of oxidative damage (8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal) were significantly elevated in aortic media of middle-aged or older-aged individuals. The immunohistochemical expression of anti-oxidant proteins (thioredoxin and extracellular superoxide dismutase) was also high in middle-aged and older-aged groups. Oxidative damage might induce the disruption of smooth muscle cells, resulting in the decrement of α-actin, a highly-expressed protein in smooth muscle cells, and matrix remodeling, in which several proteins associated with extracellular matrix were altered with aging. CONCLUSIONS: Proteomic approaches showed that the elevated expression of lactadherin might contribute to amyloid deposition, enhancement of oxidative stress, induction of antioxidant proteins and matrix remodeling in older-aged aortic media. Geriatr Gerontol Int 2019; 19: 1054-1062.

Establishment of Novel Murine Model showing Vascular Inflammation-derived Cognitive Dysfunction.

Inflammation is a critical feature of aging and its related diseases, including cardiovascular diseases. Recent epidemiological studies demonstrated that abdominal aortic aneurysm (AAA), an aging-related vascular pathological condition, is associated with cognitive decline. However, the underlying mechanism, especially the role of vascular inflammation, is largely unknown because of lack of an available animal model. In this study, we examined whether vascular inflammation affects synaptic and cognitive dysfunction, using an AAA mouse model. In young (3 months) and middle-aged (12 months) C57BL/6J mice, AAA was induced by angiotensin II infusion with calcium chloride application. After 4 weeks of induction, aortic diameter was significantly increased and excessive Mac3-positive inflammatory cells infiltrated the destroyed aorta in middle-aged mice. AAA-induced middle-aged mice further exhibited cognitive impairment. Neuronal loss was observed in the CA3 region of the hippocampus. IBA1/MHCII-double-positive microglia activation was also seen in the hippocampus, suggesting that vascular inflammation drives neuroinflammation and subsequent cognitive dysfunction. Furthermore, we found that senescence-accelerated mice prone 8 exhibited robust AAA formation and a marked decrease of cognitive and synaptic function in the hippocampus mediated by inflammation. In conclusion, this novel murine model convincingly suggested the occurrence of vascular inflammation-derived cognitive dysfunction.