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Amyloid precursor protein processing. APP can be processed either through the non-amyloidogenic (left) or the amyloidogenic pathway (right). In the non-amyloidogenic processing pathway, APP is initially cleaved by α-secretase generating sAPPα and C83. C83 is further processed by γ-secretase producing AICD and the non-toxic p3 fragment. In contrary, APP is first processed by β-secretase in the amyloidogenic pathway, leading to formation of sAPPβ and C99, where C99 is further cleaved by γ-secretase generating AICD and Aβ peptides. These peptides are prone to aggregate into the toxic Aβ plaques, characteristic for AD.
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Alzheimer’s disease (AD) is the most common cause of dementia, affecting millions of people worldwide, and no cure is currently available. The major pathological hallmarks of AD are considered to be amyloid beta plaques and neurofibrillary tangles, generated by respectively APP processing and Tau phosphorylation. Recent evidence imply that glycosyl...
Citations
... Glycosylation abnormalities are common in AD, and a variety of abnormalities have been described [52,53]. Tau undergoes glycosylation in AD brain tissue and not in control brain tissue, and this has been shown to be important for maintenance of paired helical filament structure [54]. ...
INTRODUCTION: Normal pressure hydrocephalus (NPH) patients undergoing cortical shunting frequently show early AD pathology on cortical biopsy, which is predictive of progression to clinical AD. The objective of this study was to use samples from this cohort to identify CSF biomarkers for AD-related CNS pathophysiologic changes using tissue and fluids with early pathology, free of post-mortem artifact.
METHODS: We analyzed Simoa, proteomic, and metabolomic CSF data from 81 patients with previously documented pathologic and transcriptomic changes.
RESULTS: AD pathology on biopsy correlates with CSF β-amyloid-40/42, neurofilament light chain (NfL), and phospho-tau-181(p-tau181)/β-amyloid-42, while several gene expression modules correlate with NfL. Proteomic analysis highlights 7 core proteins that correlate with pathology and gene expression changes on biopsy, and metabolomic analysis of CSF identifies disease-relevant groups that correlate with biopsy data..
DISCUSSION: As additional biomarkers are added to AD diagnostic panels, our work provides insight into the CNS pathophysiology these markers are tracking.
... In fact, it has been reported that AD-causing PSs mutants increase cellular cholesterol level [28,33,34]. However, although altered protein glycosylation in the brains of AD patients has been indicated by several studies [72,101,102], it remains unknown whether AD-causing PS mutations, including those of PS2, affect protein glycosylation events. Thus, the effect of the mutants on protein glycosylation and cholesterol metabolism, as well as the causal relationship between the two cellular events, should be evaluated in the future. ...
Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.
... In contrast, O-glycosylation modifications have diverse core structures. These glycans are covalently bound to hydroxyl oxygen groups on serine or threonine amino acids with additional modification of SA by glycosyltransferase enzymes and can be further modified with other PTMs such as acylation and sulfonation [46]. Prior to the present study, visualization of these glycan modifications within the brain has been limited. ...
Glycosylation is the most common form of post‐translational modification in the brain. Aberrant glycosylation has been observed in cerebrospinal fluid and brain tissue of Alzheimer's disease (AD) cases, including dysregulation of terminal sialic acid (SA) modifications. While alterations in sialylation have been identified in AD, the localization of SA modifications on cellular or aggregate‐associated glycans is largely unknown because of limited spatial resolution of commonly utilized methods. The present study aims to overcome these limitations with novel combinations of histologic techniques to characterize the sialylation landscape of O‐ and N‐linked glycans in autopsy‐confirmed AD post‐mortem brain tissue. Sialylated glycans facilitate important cellular functions including cell‐to‐cell interaction, cell migration, cell adhesion, immune regulation, and membrane excitability. Previous studies have not investigated both N‐ and O‐linked sialylated glycans in neurodegeneration. In this study, the location and distribution of sialylated glycans were evaluated in three brain regions (frontal cortex, hippocampus, and cerebellum) from 10 AD cases using quantitative digital pathology techniques. Notably, we found significantly greater N‐sialylation of the Aβ plaque microenvironment compared with O‐sialylation. Plaque‐associated microglia displayed the most intense N‐sialylation proximal to plaque pathology. Further analyses revealed distinct differences in the levels of N‐ and O‐sialylation between cored and diffuse Aβ plaque morphologies. Interestingly, phosphorylated tau pathology led to a slight increase in N‐sialylation and no influence of O‐sialylation in these AD brains. Confirming our previous observations in mice with novel histologic approach, these findings support microglia sialylation appears to have a relationship with AD protein aggregates while providing potential targets for therapeutic strategies.
... Alterations in the glycomic profiles of glycoproteins, e.g., overexpression of sialylated or core fucosylated glycans, or increased levels of complex-type branched glycans, may promote the acquisition of cellular features required for the malignant transformation of cells 1,[9][10][11][12] . Recent studies also reported altered glycosylation patterns in patients with Alzheimer's disease 13,14 . ...
The development of reliable single-cell dispensers and substantial sensitivity improvement in mass spectrometry made proteomic profiling of individual cells achievable. Yet, there are no established methods for single-cell glycome analysis due to the inability to amplify glycans and sample losses associated with sample processing and glycan labeling. In this work, we present an integrated platform coupling online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling of single mammalian cells. Direct and unbiased quantitative characterization of single-cell surface N-glycomes are demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations are unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow is also applied to the profiling of ng-level amounts (5–500 ng) of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively.
... The importance of N-glycosylation in the study of neurodegenerative disease has been further demonstrated by the promising use of glycoproteomics to uncover diagnostic biomarkers in circulating biofluids for brain-related diseases [13,18,[23][24][25][26][27][28][29][30]. N-glycosylation influences protein folding, protein activity, and interaction with other biomolecules, and also regulates ligand-receptor interactions, cell-tocell communication, immunological response, membrane trafficking, and signal transduction [31]. ...
Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities with the aging of individuals, such as language or visual/spatial comprehension. MCI is considered a prodromal phase of more complicated neurodegenerative diseases such as Alzheimer's. Therefore, accurate diagnosis and better understanding of the disease prognosis will facilitate prevention of neurodegeneration. However, the existing diagnostic methods fail to provide precise and well‐timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of the serum N‐ glycoproteome expression could represent an essential contributor to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using less invasive procedures. In this approach, we identified N‐ glycopeptides with different expressions between healthy and MCI patients from serum glycoproteins. Seven of the N‐ glycopeptides showed outstanding AUC values, among them the antithrombin‐III Asn224 + 4‐5‐0‐2 with an AUC value of 1.00 and a p value of 0.0004. According to proteomics and ingenuity pathway analysis (IPA), our data is in line with recent publications, and the glycoproteins carrying the identified N‐ sites play an important role in neurodegeneration.
... It thus comes as no surprise that defects in protein glycosylation are associated with a variety of diseases. We direct the reader to excellent reviews on glycosylation in cancer [43][44][45][46][47][48], neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, autism spectrum disorder, and schizophrenia [49][50][51], congenital disorders [52,53], infection, and inflammation [54][55][56][57][58]. To understand how the nature and conformation of the glycan can drastically change the interaction of a protein with another in the context of health and disease, we direct the reader to several review articles [47,[59][60][61]. ...
Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to macromolecules. Dysregulated glycosylation is linked to a spectrum of diseases, encompassing cancer, neurodegenerative disorders, congenital disorders, infections, and inflammation. This review delves into the intricate interplay between glycosylation and protein conformation, with a specific focus on the profound impact of N-glycans on the selection of distinct protein conformations characterized by distinct interactomes—namely, protein assemblies—under normal and pathological conditions across various diseases. We begin by examining the spike protein of the SARS virus, illustrating how N-glycans regulate the infectivity of pathogenic agents. Subsequently, we utilize the prion protein and the chaperone glucose-regulated protein 94 as examples, exploring instances where N-glycosylation transforms physiological protein structures into disease-associated forms. Unraveling these connections provides valuable insights into potential therapeutic avenues and a deeper comprehension of the molecular intricacies that underlie disease conditions. This exploration of glycosylation’s influence on protein conformation effectively bridges the gap between the glycome and disease, offering a comprehensive perspective on the therapeutic implications of targeting conformational mutants and their pathologic assemblies in various diseases. The goal is to unravel the nuances of these post-translational modifications, shedding light on how they contribute to the intricate interplay between protein conformation, assembly, and disease.
... Glycoproteins are essential macromolecules, which perform many important functions such as lubrication and protection, intracellular migration, sorting and secretion, antigen-antibody interaction, signal transduction, virus-cell interaction, and many others [2]. It has been proved that the alteration in glycosylation can be used as specific biomarkers for cancer [3], Alzheimer's diseases [4], or diabetes [5]. Moreover, in the last decade, biotherapeutic products based on glycoproteins have been introduced to the pharmaceutical industry. ...
The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.
... It thus comes as no surprise that defects in protein glycosylation are associated with a variety of diseases. We direct the reader to excellent reviews on glycosylation in cancer [42][43][44][45][46][47], neurodegenerative disorders, including Alzheimerʹs disease, Parkinsonʹs disease, autism spectrum disorder, and schizophrenia [48][49][50], congenital disorders [51,52], infection and inflammation [53][54][55][56][57]. To understand how the nature and conformation of the glycan can drastically change the interaction of a protein with another in the context of health and disease, we direct the reader to several review articles [46,[58][59][60]. ...
Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to macromolecules. Dysregulated glycosylation is linked to a spectrum of diseases, encompassing cancer, neurodegenerative disorders, congenital disorders, infections, and inflammation. This review delves into the intricate interplay between glycosylation and protein conformation, with a specific focus on the profound impact of N-glycans on the selection of distinct protein conformations, characterized by distinct interactomes – namely protein assemblies - under normal and pathological conditions across various diseases. We begin by examining the spike protein of the SARS virus, illustrating how N-glycans regulate the infectivity of pathogenic agents. Subsequently, we utilize the prion protein and the chaperone glucose-regulated protein 94 as examples, exploring instances where N-glycosylation transforms physiological protein structures into disease-associated forms. Unraveling these connections provides valuable insights into potential therapeutic avenues and a deeper comprehension of the molecular intricacies that underlie disease conditions. This exploration of glycosylation's influence on protein conformation effectively bridges the gap between the glycome and disease, offering a comprehensive perspective on the therapeutic implications of targeting conformational mutants and their pathologic assemblies in various diseases. The goal is to unravel the nuances of these post-translational modifications, shedding light on how they contribute to the intricate interplay between protein conformation, assembly and disease.
... Metabolic shifts in diseased cardiac fibroblasts are not well characterised but changes to rates of glycolysis have been reported (Lombardi et al., 2019;. As alterations to glycosylation (Haukedal & Freude, 2021;Peixoto et al., 2019) and the pentose phosphate pathway (Jin & Zhou, 2019;Tu et al., 2019) are implicated in other tissue remodelling associated diseases (e.g., cancer, neurodegeneration), investigation into cardiac fibrosis-related changes may provide further insight into pathogenesis. ...
Pathological reprogramming of cardiomyocyte and fibroblast proteome landscapes drive the initiation and progression of cardiac fibrosis. Although the secretome of dysfunctional cardiomyocytes is emerging as an important driver of pathological fibroblast reprogramming, our understanding of the downstream molecular players remains limited. Here, we show that cardiac fibroblast activation (αSMA ⁺ ) and oxidative stress mediated by the secretome of TGFβ‐stimulated cardiomyocytes is associated with a profound reprogramming of their proteome and phosphoproteome landscape. Within the fibroblast global proteome there was a striking dysregulation of proteins implicated in extracellular matrix, protein localisation/metabolism, KEAP1‐NFE2L2 pathway, lysosomes, carbohydrate metabolism, and transcriptional regulation. Kinase substrate enrichment analysis of phosphopeptides revealed potential role of kinases (CK2, CDK2, PKC, GSK3B) during this remodelling. We verified upregulated activity of casein kinase 2 (CK2) in secretome‐treated fibroblasts, and pharmacological CK2 inhibitor TBB (4,5,6,7‐Tetrabromobenzotriazole) significantly abrogated fibroblast activation and oxidative stress. Our data provides molecular insights into cardiomyocyte to cardiac fibroblast crosstalk, and the potential role of CK2 in regulating cardiac fibroblast activation and oxidative stress.
... Nglycans on glycoproteins are increasingly recognized as functional effectors of genetic and epigenetic disease risk (16). Accumulating evidence points to a link between aberrant protein Nglycosylation and AD pathogenesis (17)(18)(19). Glycomic analyses of N-glycans detached from glycoproteins have revealed changes in global levels of N-glycans in AD brains (20)(21)(22), but the information of glycoproteins and glycosites bearing the N-glycans was lost. We recently performed an integrated proteomic and glycoproteomic study using 18 O-tagging to label in vivo Nglycosylation sites and identified disease-associated N-glycoproteins and glycosites with altered N-glycosylation site occupancy in AD brains (23). ...
... We and others previously reported aberrant tau N-glycosylation in AD (17)(18)(19)23), but the sitespecific N-glycans and N-glycoforms of tau in AD remain unknown. Our analysis revealed that tau was modified at the N410 residue of tau441 isoform (corresponding to N727 of tau758 isoform) by a high-mannose-type glycan exclusively in AD but not controls ( Fig. 2 and table S2), consistent with our prior finding of tau N410 as the N-glycosite in AD (23). ...
Despite the importance of protein glycosylation to brain health, current knowledge of glycosylated proteoforms or glycoforms in human brain and their alterations in Alzheimer's disease (AD) is limited. Here, we present a new paradigm of proteome-wide glycoform profiling study of human AD and control brains using intact glycopeptide-based quantitative glycoproteomics coupled with systems biology. Our study identified over 10,000 human brain N-glycoforms from nearly 1200 glycoproteins and uncovered disease signatures of altered glycoforms and glycan modifications, including reduced sialylation and N-glycan branching as well as elevated mannosylation and N-glycan truncation in AD. Network analyses revealed a higher-order organization of brain glycoproteome into networks of co-regulated glycoforms and glycans and discovered glycoform and glycan modules associated with AD clinical phenotype, amyloid-β accumulation, and tau pathology. Our findings provide novel insights and a rich resource of glycoform and glycan changes in AD and pave the way forward for developing glycosylation-based therapies and biomarkers for AD.
