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Early detection of Alzheimer's disease is vital for timely treatment. Existing biomarkers for Alzheimer's disease reflect amyloid- and tau-related pathology, but it is unknown whether the disease can be detected before cerebral amyloidosis is observed. N-glycosylation has been suggested as an upstream regulator of both amyloid and tau pathology, and levels of the N-glycan structure bisecting N-acetylglucosamine (GlcNAc) correlate with tau in blood and CSF already at pre-clinical stages of the disease. Therefore, we aimed to evaluate whether bisecting GlcNAc could predict future cognitive decline in patients from a memory clinic cohort, stratified by amyloid/tau status. We included 251 patients (mean age: 65.6 ± 10.6 years, 60.6% female) in the GEDOC cohort, from the Memory Clinic at Karolinska University Hospital, Stockholm, Sweden. Patients were classified as amyloid/tau positive or negative based on CSF biomarkers. Cognitive decline, measured by longitudinal Mini-Mental State Examination scores, was followed for an average of 10.7 ± 4.1 years and modelled using non-linear mixed effects models. Additionally, bisecting GlcNAc levels were measured in hippocampus and cortex with lectin-based immunohistochemistry in 10 Alzheimer's disease and control brains. We found that CSF bisecting GlcNAc levels were elevated in tau-positive individuals compared with tau-negative individuals, but not in amyloid-positive individuals compared with amyloid-negative individuals. In the whole sample, high levels of CSF bisecting GlcNAc predicted earlier cognitive decline. Strikingly, amyloid/tau stratification showed that high CSF bisecting GlcNAc levels predicted earlier cognitive decline in amyloid-negative patients ( β = 2.53 ± 0.85 years, P = 0.003) and tau-negative patients ( β = 2.43 ± 1.01 years, P = 0.017), but not in amyloid- or tau-positive patients. Finally, histochemical analysis of bisecting GlcNAc showed increased levels in neurons in hippocampus and cortex of Alzheimer's disease compared with control brain (fold change = 1.44-1.49, P < 0.001). In conclusion, high CSF levels of bisecting GlcNAc reflected neuronal pathology and predicted cognitive decline in amyloid- and tau-negative individuals, suggesting that abnormal glycosylation precedes cerebral amyloidosis and tau hyper-phosphorylation in Alzheimer's disease. Bisecting GlcNAc is a promising novel early biomarker for Alzheimer's disease., Competing Interests: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

At the time of diagnosis, Alzheimer's disease (AD) patients already suffer from significant neuronal loss. The identification of proteins that influence disease progression before the onset of symptoms is thus an essential part of the development of new effective drugs and biomarkers. Here, we used an unbiased 18 O labelling proteomics approach to identify proteins showing altered levels in the AD brain. We studied the relationship between the protein with the highest increase in hippocampus, DEAD box Helicase 24 (DDX24), and AD pathology. We visualised DDX24 in the human brain and in a mouse model for Aβ42-induced AD pathology- App NL-F -and studied the interaction between Aβ and DDX24 in primary neurons. Immunohistochemistry in the AD brain confirmed the increased levels and indicated an altered subcellular distribution of DDX24. Immunohistochemical studies in App NL-F mice showed that the increase of DDX24 starts before amyloid pathology or memory impairment is observed. Immunocytochemistry in App NL-F primary hippocampal neurons showed increased DDX24 intensity in the soma, nucleus and nucleolus. Furthermore, siRNA targeting of DDX24 in neurons decreased APP and Aβ42 levels, and the addition of Aβ42 to the medium reduced DDX24. In conclusion, we have identified DDX24 as a protein with a potential role in Aβ-induced AD pathology.

Background: The COVID-19 pandemic prompted a refocus of health care resources to acute care which has impacted on the capacity of healthcare systems to conduct neurological surgeries. The elderly population has been shown to be particularly vulnerable to the consequences of the pandemic. Less neurosurgery can result in great impact on public health by increasing morbidity and mortality in patients with malignancies and traumatic injuries. The aim of this study was to investigate the effects of the COVID-19 pandemic on neurosurgical procedures in the elderly population in Sweden., Methods: In this retrospective observational study, the reported incidence of all neurosurgical procedures registered in the 21 Regions of Sweden during 2015-2021 in people aged 65 year or older was collected. Surgical procedures were classified according to the NOMESCO system of classification. Neurosurgery incidence was defined as the number of NOMESCO associated interventions per 100.000 inhabitants. ICD-10 codes associated with neurosurgery-related diagnoses and deaths were also collected. Expected incidence of neurosurgery, neurosurgery-associated deaths and brain cancer diagnoses was estimated and compared to actual outcomes. Decrease in the incidence of neurosurgery was compared to regional COVID-19 incidence, other types of surgery and surgery waiting times., Results: The incidence of several categories of neurosurgery decreased in Sweden during 2020 and 2021, although not as much as other surgical categories. Women were more affected than men by the decrease in neurosurgery which could be partly explained by a decrease in brain cancer diagnoses amongst women. There was an association between regional decrease in neurosurgery incidence and longer surgery waiting time. COVID-19 incidence in the region did not have an effect on regional decreases in neurosurgery incidence., Conclusions: The COVID-19 pandemic resulted in a reduction in the number of neurosurgical procedures performed in Sweden during 2020-2021, although not as much as in other European countries. There was regional difference in Sweden with respect to number of surgeries, and waiting time for elective surgeries although there was no increase in mortality., (© 2024. The Author(s).)

Background: The N-glycan structure bisecting N-acetylglucosamine (bisecting GlcNAc) is present on several N-glycans that are elevated in Alzheimer's disease (AD), and previous studies have shown that bisecting GlcNAc levels correlate with total tau and phospho-tau181 in cerebrospinal fluid at early stages of AD. A recent population-based study showed that bisecting GlcNAc correlates with total tau also in blood and that this correlation could predict conversion to dementia., Objective: In this study, we have further investigated how bisecting GlcNAc relates to total tau and phospho-tau 181 in cerebrospinal fluid samples from controls and cases with early cognitive deficits, stratified by amyloid/tau status and gender., Methods: Relative levels of bisecting GlcNAc in cerebrospinal fluid were measured by an enzyme-linked lectin assay in individuals with subjective cognitive decline, mild cognitive impairment and controls from the Norwegian Dementia Disease Initiation cohort., Results: As in our previous study, the correlation between bisecting GlcNAc and total tau or phospho-tau181 was particularly strong in the subjective cognitive decline group. The correlation was observed in amyloid negative and tau negative as well as amyloid positive and tau positive individuals, both in females and in males. Interestingly, among the amyloid negative and tau negative individuals, the correlation was observed in individuals with subjective cognitive decline but not in the controls., Conclusions: Thus, bisecting GlcNAc could be a biomarker for early cognitive decline.

Introduction: There is an urgent need for novel blood biomarkers for the detection of Alzheimer's disease (AD). We previously showed that levels of the bisecting N-acetylglucosamine glycan epitope was elevated in cerebrospinal fluid in AD. However, its diagnostic value in blood is unknown., Methods: We analyzed blood levels of bisecting N-acetylglucosamine and total tau in a retrospective cohort of 233 individuals. Progression to AD was compared between the groups using Cox regression. The predictive value of the biomarkers was determined by logistic regression., Results: Bisecting N-acetylglucosamine correlated with tau levels (p < 0.0001). Individuals with an intermediate tau/bisecting N-acetylglucosamine ratio had elevated AD risk (hazard ratio = 2.06, 95% confidence interval [CI]: 1.18-3.6). Moreover, a combined model including tau/bisecting N-acetylglucosamine ratio, apolipoprotein E (APOE) ε4 status, and Mini-Mental State Examination score predicted future AD (area under the curve = 0.81, 95% CI: 0.68-0.93)., Discussion: Bisecting N-acetylglucosamine in combination with tau is a valuable blood biomarker for predicting AD., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Objective: It has been found that COVID-19 increases deaths within common diseases in countries that have implemented strict lockdowns. In order to elucidate the proper national response to a pandemic, the mortality rates within COVID-19 and various diseases need to be studied in countries whose pandemic response differ. Sweden represents a country with lax pandemic restrictions, and we aimed to study the effects of COVID-19 on historical mortality rates within common diseases during 2020., Methods: Regression models and moving averages were used to predict expected premature mortality per the ICD-10 during 2020 using historical data sets. Predicted values were then compared to recorded premature mortality to identify changes in mortality trends., Results: Seasonal increased mortality was found within neurological diseases. Infectious diseases, tumours and cardiac disease mortality rates decreased compared to expected outcome., Conclusions: Changes in mortality trends were observed for several common diseases during the COVID-19 pandemic. Neurological and cardiac conditions, infections and tumours are examples of diseases that were heavily affected by the pandemic. The indirect effects of COVID-19 on certain patient populations should be considered when determining pandemic impact.

The amyloid precursor protein (APP) is a ubiquitously expressed type 1 transmembrane protein mostly known for serving as a precursor to the amyloid-β peptide (Aβ), a culprit in Alzheimer disease (AD). However, APP also has important physiological functions by being implicated in, for instance, adhesion, signaling, neuronal development, and synaptic function. Human APP contains 2 N-glycosylation sites, at asparagine (N) 467 (N467) and N496. Here, we studied the role of N-glycosylation on APP trafficking and processing by constructing APP-SNAP plasmid vectors for wildtype APP and N-glycosylation site mutants in which N467 or N496 was replaced by glutamine (Q) and expressed these in HEK293T cells. Lack of either of the 2 N-glycans resulted in a reduction in the size of intracellular APP-SNAP-positive vesicles and a reduction of APP-SNAP in the plasma membrane and lysosomes. Importantly, loss of either of the 2 N-glycans resulted in elevated levels of intracellular as well as secreted Aβ42. These data suggest that N-glycans have a major impact on trafficking and processing of APP and could play an important role in the development of AD., (© The Author(s) 2022. Published by Oxford University Press.)

Introduction: The COVID-19 pandemic has caused large disruptions to healthcare systems. Refocus on COVID-19 related care might have contributed to indirect effects on other healthcare areas. Care focused on acute conditions have been negatively affected although research into the effects on chronic and care intensive patient groups such as patients with dementia diseases is lacking. In this study we evaluated dementia diagnosis trends in Sweden during 2015-2020 according to International Classification of Disease version 10 coding of common dementia diseases., Methods: Regional and national statistics in the form of International Classification of Disease version 10 coding, COVID-19 incidence, mortality data, and population census data were collected from the National Institute of Health and Welfare. Logistic regression analysis was performed to identify trends of dementia diagnosis during 2015-2020. Correlation test was performed between COVID-19 incidence, mortality rates, and dementia coding., Results: Dementia diagnosis incidence has been declining since 2015 and further decline was noted in many regions in Sweden during 2020. As COVID-19 incidence increased, fewer cases of dementia were diagnosed, a decrease that differentially impacted women and those who were advanced in age., Conclusions: Dementia diagnosis incidence in Sweden has been on a decline since 2015. The COVID-19 pandemic caused a further larger decline in dementia diagnosis incidence during 2020. COVID-19 incidence, but not mortality, was associated with decrease in dementia diagnosis incidence. There might be a large number of undiagnosed patients with dementia and healthcare reforms should be enacted to address this. Women and elderly are particularly vulnerable groups., (© 2022. The Author(s).)

Mass spectrometry (MS)-based proteomics is a powerful tool to explore pathogenic changes of a disease in an unbiased manner and has been used extensively in Alzheimer disease (AD) research. Here, by performing a meta-analysis of high-quality proteomic studies, we address which pathological changes are observed consistently and therefore most likely are of great importance for AD pathogenesis. We retrieved datasets, comprising a total of 21,588 distinct proteins identified across 857 postmortem human samples, from ten studies using labeled or label-free MS approaches. Our meta-analysis findings showed significant alterations of 757 and 1,195 proteins in AD in the labeled and label-free datasets, respectively. Only 33 proteins, some of which were associated with synaptic signaling, had the same directional change across the individual studies. However, despite alterations in individual proteins being different between the labeled and the label-free datasets, several pathways related to synaptic signaling, oxidative phosphorylation, immune response and extracellular matrix were commonly dysregulated in AD. These pathways represent robust changes in the human AD brain and warrant further investigation., (© 2021. The Author(s).)

Novel insights on proteins involved in Alzheimer's disease (AD) are needed. Since multiple cell types and matrix components are altered in AD, bulk analysis of brain tissue maybe difficult to interpret. In the current study, we isolated pyramidal cells from the cornu ammonis 1 (CA1) region of the hippocampus from five AD and five neurologically healthy donors using laser capture microdissection (LCM). The samples were analyzed by proteomics using 18 O-labeled internal standard and nano-high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for relative quantification. Fold change between AD and control was calculated for the proteins that were identified in at least two individual proteomes from each group. From the 10 cases analyzed, 62 proteins were identified in at least two AD cases and two control cases. Creatine kinase B-type (CKB), 14-3-3-γ, and heat shock cognate 71 (Hsc71), which have not been extensively studied in the context of the human AD brain previously, were selected for further studies by immunohistochemistry (IHC). In hippocampus, semi-quantitative measures of IHC staining of the three proteins confirmed the findings from our proteomic analysis. Studies of the same proteins in the frontal cortex revealed that the alterations remained for CKB and 14-3-3-γ but not for Hsc71. Protein upregulation in CA1 neurons of final stage AD is either a result of detrimental, pathological effects, or from cell-specific protective response mechanisms in surviving neurons. Based on previous findings from experimental studies, CKB and Hsc71 likely exhibit protective effects, whereas 14-3-3-γ may represent a detrimental pathway. These new players could reflect pathways of importance for the development of new therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sandebring-Matton, Axenhus, Bogdanovic, Winblad, Schedin-Weiss, Nilsson and Tjernberg.)

Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac-mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Protein glycosylation is crucial for the central nervous system and brain functions, including processes that are defective in Alzheimer disease (AD) such as neurogenesis, synaptic function, and memory formation. Still, the roles of glycans in the development of AD are relatively unexplored. Glycomics studies of cerebrospinal fluid (CSF) have previously shown altered glycosylation pattern in patients with different stages of cognitive impairment, including AD, compared to healthy controls. As a consequence, we hypothesized that the glycan profile is altered in the brain of patients with AD and analyzed the asparagine-linked (N-linked) glycan profile in hippocampus and cortex in AD and control brain. Glycans were enzymatically liberated from brain glycoproteins and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eleven glycans showed significantly different levels in hippocampus compared to cortex in both control and AD brain. Two glycans in cortex and four in hippocampus showed different levels in AD compared to control brain. All glycans that differed between controls and AD brain had similar structures with one sialic acid, at least one fucose and a confirmed or potential bisecting N-acetylglucosamine (GlcNAc). The glycans that were altered in AD brain differed from those that were altered in AD CSF. One glycan found to be present in significantly lower levels in both hippocampus and cortex in AD compared to control contained a structurally and functionally interesting epitope that we assign as a terminal galactose decorated with fucose and sialic acid. Altogether, these studies suggest that protein glycosylation is an important component in the development of AD and warrants further studies., (© 2020 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Alzheimer's disease (AD) is the most common type of dementia, contributing to 60-80% of cases. It is a neurodegenerative disease that usually starts symptomless in the first two to three decades and then propagates into a long-term, irreversible disease, resulting in the progressive loss of memory, reasoning, abstraction and language capabilities. It is a complex disease, involving a large number of entangled players, and there is no effective treatment to cure it or alter its progressive course. Therefore, a thorough understanding of the disease pathology and an early diagnosis are both necessary. AD has two significant pathological hallmarks: extracellular senile plaques composed of amyloid β-peptide (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein, and the aggregation of Aβ, which starts in earlier stages, is usually claimed to be the primary cause of AD. Secretases that cleave Aβ precursor protein (APP) and produce neurotoxic Aβ reside in distinct organelles of the cell, and current concepts suggest that APP moves between distinct intracellular compartments. Obviously, APP transport and processing are intimately related processes that cannot be dissociated from each other, and, thus, how and where APP is transported determines its processing fate. In this review, we summarize critical mechanisms underlying neuronal APP transport, which we divide into separate parts: (1) secretory pathways and (2) endocytic and autophagic pathways. We also include two lipoprotein receptors that play essential roles in APP transport: sorting-related receptor with A-type repeats and sortilin. Moreover, we consider here some major disruptions in the neuronal transport of APP that contribute to AD physiology and pathology. Lastly, we discuss current methods and technical difficulties in the studies of APP transport.

Proteomics-large-scale studies of proteins-has over the last decade gained an enormous interest for studies aimed at revealing proteins and pathways involved in disease. To fully understand biological and pathological processes it is crucial to also include post-translational modifications in the "omics". To this end, glycomics (identification and quantification of glycans enzymatically or chemically released from proteins) and glycoproteomics (identification and quantification of peptides/proteins with the glycans still attached) is gaining interest. The study of protein glycosylation requires a workflow that involves an array of sample preparation and analysis steps that needs to be carefully considered. Herein, we briefly touch upon important steps such as sample preparation and preconcentration, glycan release, glycan derivatization and quantification and advances in mass spectrometry that today are the work-horse for glycomics and glycoproteomics studies. Several proteins related to Alzheimer disease pathogenesis have altered protein glycosylation, and recent glycomics studies have shown differences in cerebrospinal fluid as well as in brain tissue in Alzheimer disease as compared to controls. In this review, we discuss these techniques and how they have been used to shed light on Alzheimer disease and to find glycan biomarkers in cerebrospinal fluid.

Amyloid β-peptide (Aβ) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aβ oligomerization in neurons still need to be revealed. Förster resonance energy transfer (FRET) is a simple but effective way to study molecular interactions. Here, we used a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detected Aβ42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled Aβ42 in the cell culture medium for 24 h. Aβ42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aβ42 were significantly higher than for Aβ40. These findings provide a better understanding of Aβ42 oligomerization in neurons.

Background: Processing of the amyloid-β protein precursor (AβPP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid β-peptide (Aβ42), which is a key player in Alzheimer's disease., Objective: Our aim was to clarify the subcellular locations of the fragments involved in the amyloidogenic pathway in primary neurons with a focus on Aβ42 and its immediate substrate AβPP C-terminal fragment (APP-CTF). To overcome the difficulties of resolving these compartments due to their small size, we used super-resolution microscopy., Methods: Mouse primary hippocampal neurons were immunolabelled and imaged by stimulated emission depletion (STED) microscopy, including three-dimensional three-channel imaging, and quantitative image analyses., Results: The first (β-secretase) and second (γ-secretase) cleavages of AβPP were localized to functionally and distally distinct compartments. The β-secretase cleavage was observed in early endosomes in soma, where we were able to show that the liberated N- and C-terminal fragments were sorted into distinct vesicles budding from the early endosomes. Lack of colocalization of Aβ42 and APP-CTF in soma suggested that γ-secretase cleavage occurs in neurites. Indeed, APP-CTF was, in line with Aβ42 in our previous study, enriched in the presynapse but absent from the postsynapse. In contrast, full-length AβPP was not detected in either the pre- or the postsynaptic side of the synapse. Furthermore, we observed that endogenously produced and endocytosed Aβ42 were localized in different compartments., Conclusion: These findings provide critical super-resolved insight into amyloidogenic AβPP processing in primary neurons.

Throughout development, neuronal progenitors undergo complex transformation into polarized nerve cells, warranting the directional flow of information in the neural grid. The majority of neuronal polarization studies have been carried out on rodent-derived precursor cells, programmed to develop into neurons. Unlike rodent neuronal cells, SH-SY5Y cells derived from human bone marrow present a sub-clone of neuroblastoma line, with their transformation into neuron-like cells showing a range of highly instructive neurobiological characteristics. We applied two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) protocol to monitor the conversion of undifferentiated SH-SY5Y into neuron-like cells with distinctly polarized axon-dendritic morphology and formation of bona fide synaptic connections. We show that BDNF is a key driver and regulator of the expression of axonal marker tau and dendritic microtubule-associated protein-2 (MAP2), with their sorting to distinct cellular compartments. Using selective kinase inhibitors downregulating BDNF-TrkB signaling, we demonstrate that constitutive activation of TrkB receptor is essential for the maintenance of established polarization morphology. Importantly, the proximity ligation assay applied in our preparation demonstrates that differentiating neuron-like cells develop elaborate synaptic connections enriched with hallmark pre- and postsynaptic proteins. Described herein findings highlight several fundamental processes related to neuronal polarization and synaptogenesis in human-derived cells, which are of major relevance to neurobiology and translational neuroscience., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Alzheimer disease (AD) is a devastating disease and a global health problem, and current treatments are only symptomatic. A wealth of clinical studies support that the disease starts to develop decades before the first symptoms appear, emphasizing the importance of studying early changes for improving early diagnosis and guiding toward novel treatment strategies. Protein glycosylation is altered in AD but it remains to be clarified why these alterations occur and how they affect the disease development. Here, we used a glycomics approach to search for alterations in protein glycosylation in cerebrospinal fluid (CSF) in AD compared with nondemented controls. Using both matrix-assisted laser desorption ionization-time of flight and liquid chromatography-electrospray mass spectrometry, we observed an increase in N-glycans carrying bisecting N-acetylglucosamine in AD. Based on those findings, we designed an enzyme-linked multiwell plate assay to quantify N-glycans binding to the lectin Phaseolus vulgaris Erythroagglutinin (PHA-E), which is specific for N-glycans containing bisecting N-acetylglucosamine. Using this assay, we found a similar increase in CSF in AD compared with controls. Further analysis of CSF from 242 patients with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), or AD dementia revealed significantly increased binding to PHA-E in MCI and AD compared to SCI. Interestingly, PHA-E binding correlated with CSF levels of phosphorylated tau and total tau and this correlation was most prominent in the SCI group (R = 0.53-0.54). This study supports a link between N-glycosylation, neurodegeneration, and tau pathology in AD and suggests that glycan biomarkers have potential to identify SCI cases at risk of developing AD., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: The 42 amino acids long amyloid-β peptide, Aβ42, may initiate a cascade of events leading to the severe neurodegeneration observed in Alzheimer's disease (AD) brain. However, the underlying molecular mechanisms remain to be established., Objective: To find early Aβ42-induced AD related mechanisms, we performed a brain proteomics time-course study on a novel App knock-in AD mouse model, AppNL-F, expressing high levels of Aβ42 without AβPP overexpression artifacts., Methods: Hippocampus and cortex were analyzed separately by using 18O-labelling mass spectrometry to reveal alterations in protein levels. Pathway analysis of proteomics data was used to identify altered biological functions. Immunohistochemistry was used to further investigate a significant key regulatory protein., Results: Around 100 proteins were differently expressed in AppNL-F mice at each time point (3, 6, 9, and 18 months of age) as compared to wild type mice. Strikingly, already at 3 months of age-long before Aβ plaque development and memory impairment-several pathways, including long-term potentiation and synaptic plasticity, were downregulated, and neuritogenesis was increased. Huntingtin (HTT) was identified as an upstream regulator, i.e., a key protein affecting the levels of several proteins. Increased levels of HTT in hippocampus of AppNL-F mice was supported by immunofluorescence microscopy., Conclusion: Notably, the proteome was significantly altered already at 3 months of age, 6 months before the development of plaques. Differentially expressed proteins varied over time, indicating that increased Aβ42 levels initiate a cascade of events that eventually manifests in amyloid depositions, inflammation, and decline in memory.

Background: We have recently identified Huntingtin (Htt), the pathogenic protein in Huntington's disease, as a mediator of Alzheimer's disease (AD) pathology in an amyloid precursor protein (APP) knock-in mouse model of AD. That finding prompted us to examine if Htt is accumulated in the brains of AD patients and in which cell type Htt is present in the AD brain., Objective: To investigate whether location and levels of Htt are affected in hippocampus and frontal cortex in AD., Methods: Brains from AD patients (n=11) and controls (n=11) were stained for Htt using immunohistochemistry and signal intensity of Htt was quantified and localized in subregions and neurons. Confocal microscopy was used to characterize neuronal Htt localisation and its relationship with tau tangles and astrocytes., Results: Htt levels were increased in neuronal cells in the granular layer of the dentate gyrus, in CA1 and CA3 in hippocampus and in layer III of the frontal cortex. Htt was found in the soma, perinuclear space, thin neurites and nucleus of pyramidal neurons. Htt was present in neurons containing tau tangles but did not colocalize with astrocytes., Conclusion: Htt accumulates in pyramidal neuron-rich areas including hippocampal subregions associated with memory and frontal cortex layer III. The accumulation of Htt in AD shows distinct cellular and morphological patterns and is not present in astrocytes. Clearly, further research is warranted to elucidate the role of Htt as a mediator of AD pathology and the potential use of Htt as a target in future therapeutic strategies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The amyloid-β protein precursor (AβPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AβPP generates the neurotoxic amyloid-β peptide (Aβ). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AβPP. The C-terminal tail of AβPP was fluorescently labeled using a SNAP-tag, while the Aβ region of AβPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AβPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AβPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AβPP variants was validated with ELISA and immunoblotting, and seven AβPP mutants that showed similar cleavage pattern as wild-type AβPP were identified. The AβPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AβPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aβ and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aβ with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AβPP.

The amyloid β-peptide (Aβ) is a physiological ubiquitously expressed peptide suggested to be involved in synaptic function, long-term potentiation, and memory function. The 42 amino acid-long variant (Aβ42) forms neurotoxic oligomers and amyloid plaques and plays a key role in the loss of synapses and other pathogenic events of Alzheimer disease. Still, the exact localization of Aβ42 in neurons and at synapses has not been reported. Here, we used super-resolution microscopy and show that Aβ42 was present in small vesicles in presynaptic compartments, but not in postsynaptic compartments, in the neurites of hippocampal neurons. Some of these vesicles appeared to lack synaptophysin, indicating that they differ from the synaptic vesicles responsible for neurotransmitter release. The Aβ42-containing vesicles existed in presynapses connected to stubby spines and mushroom spines, and were also present in immature presynapses. These vesicles were scarce in other parts of the neurites, where Aβ42 was instead present in large, around 200-600 nm, vesicular structures. Three-dimensional super-resolution microscopy confirmed that Aβ42 was present in the presynapse and absent in the postsynapse., Competing Interests: The authors declare that they have no conflict of interest.

How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Increased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons., Methods: MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis., Results: Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of MAO-B enhanced Aβ production., Conclusions: This study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aβ production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aβ levels using protein-protein interaction breakers.

The toxic amyloid β-peptide (Aβ) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase (to yield APP-C-terminal fragment β (APP-CTFβ) and soluble APPβ (sAPPβ)) and γ-secretase (to yield Aβ). We reasoned that proteins that associate with γ-secretase are likely to regulate Aβ production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aβ levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aβ, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aβ levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by β-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The transmembrane protein assembly γ-secretase is a key protease in regulated intramembrane processing (RIP) of around 100 type-1 transmembrane proteins. Importantly, it has a pathological role in Alzheimer disease (AD) as it generates the neurotoxic amyloid β-peptide from the amyloid precursor protein (APP). Studies on γ-secretase location are therefore crucial both from a biological and a therapeutic perspective. Despite several years of efforts in many laboratories, it is not clear where in the neuron γ-secretase exerts it's activities. Technical challenges include the fact that the active enzyme contains four protein components and that most subcellular compartments cannot be spatially resolved by traditional light microscopy. Here, we have used a powerful combination of the two nanoscopy techniques STORM and STED microscopy to visualize the location of γ-secretase in neurons using an active-site specific probe, with a focus on the synapse. We show that γ-secretase is present in both the pre-and postsynaptic compartments. We further show that the enzyme is enriched very close to the synaptic cleft in the postsynaptic membrane, as well as to NMDA receptors, demonstrating that γ-secretase is present in the postsynaptic plasma membrane. Importantly, the expression of γ-secretase increased in the pre- and postsynaptic compartments with the size of the synapse, suggesting a correlation between γ-secretase activity and synapse maturation. Thus, our data shows the synaptic location with high precision in three dimensions and settles the long-lasting debate on the synaptic location of γ-secretase.

Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.

Synaptic degeneration and accumulation of the neurotoxic amyloid β-peptide (Aβ) in the brain are hallmarks of Alzheimer disease. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP), by the β-secretase β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. However, Aβ generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aβ can be produced locally at the synapse. To study the synaptic localization of the APP processing enzymes we used western blotting to demonstrate that, compared to total brain homogenate, ADAM10 and BACE1 were greatly enriched in synaptic vesicles isolated from rat brain using controlled-pore glass chromatography, whereas Presenilin1 was the only enriched component of the γ-secretase complex. Moreover, we detected ADAM10 activity in synaptic vesicles and enrichment of the intermediate APP-C-terminal fragments (APP-CTFs). We confirmed the western blotting findings using in situ proximity ligation assay to demonstrate close proximity of ADAM10 and BACE1 with the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. In contrast, only sparse co-localization of active γ-secretase and synaptophysin was detected. These results indicate that the first step of APP processing occurs in synaptic vesicles whereas the final step is more likely to take place elsewhere., (© 2015 International Society for Neurochemistry.)

γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid β-peptide (Aβ), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aβ production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aβ and these proteins were selected for further studies on Aβ production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aβ secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aβ40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aβ production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aβ lowering therapy aimed at treating Alzheimer's disease., (© 2015 FEBS.)

Glycosylation is one of the most common, and the most complex, forms of post-translational modification of proteins. This review serves to highlight the role of protein glycosylation in Alzheimer disease (AD), a topic that has not been thoroughly investigated, although glycosylation defects have been observed in AD patients. The major pathological hallmarks in AD are neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles are composed of phosphorylated tau, and the plaques are composed of amyloid β-peptide (Aβ), which is generated from amyloid precursor protein (APP). Defects in glycosylation of APP, tau and other proteins have been reported in AD. Another interesting observation is that the two proteases required for the generation of amyloid β-peptide (Aβ), i.e. γ-secretase and β-secretase, also have roles in protein glycosylation. For instance, γ-secretase and β-secretase affect the extent of complex N-glycosylation and sialylation of APP, respectively. These processes may be important in AD pathogenesis, as proper intracellular sorting, processing and export of APP are affected by how it is glycosylated. Furthermore, lack of one of the key components of γ-secretase, presenilin, leads to defective glycosylation of many additional proteins that are related to AD pathogenesis and/or neuronal function, including nicastrin, reelin, butyrylcholinesterase, cholinesterase, neural cell adhesion molecule, v-ATPase, and tyrosine-related kinase B. Improved understanding of the effects of AD on protein glycosylation, and vice versa, may therefore be important for improving the diagnosis and treatment of AD patients., (© 2013 FEBS.)

Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies.

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease (AD) and results in loss of cognitive function. One of the causative agents for the synaptic degeneration is the amyloid β-peptide (Aβ), which is formed from its precursor protein by two sequential cleavages mediated by β- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aβ is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aβ can therefore be potential therapeutic targets. We used a recently developed affinity purification approach to identify γ-secretase associated proteins (GSAPs) in synaptic membranes and synaptic vesicles prepared from rat brain. Liquid chromatography-tandem mass spectrometry analysis of the affinity purified samples revealed the known γ-secretase components presenilin-1, nicastrin and Aph-1b along with a number of novel potential GSAPs. To investigate the effect of these GSAPs on APP processing, we performed siRNA experiments to knock down the expression of the GSAPs and measured the Aβ levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aβ levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aβ levels. Treatment with γ-secretase inhibitors often results in Notch-related side effects and therefore we also studied the effect of the siRNAs on Notch processing. Interestingly, silencing of TPPP or NDUFS7 did not affect cleavage of Notch. We also studied the expression of TPPP and NDUFS7 in control and AD brain and found NDUFS7 to be highly expressed in vulnerable neurons such as pyramidal neurons in the hippocampus, whereas TPPP was found to accumulate in intraneuronal granules and fibrous structures in hippocampus from AD cases. In summary, we here report on two proteins, TPPP and NDUFS7, which interact with γ-secretase and alter the Aβ levels without affecting Notch cleavage., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-α signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA-deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages.

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH₂-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc₃Hex₅HexNAc₄, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x). A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH₂-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⁻¹ s⁻¹ for the natural full-length PCI and a form lacking six amino acids at the NH₂-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⁻¹ s⁻¹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH₂-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation, fertilization, prevention of tumors and pathogen defence. It has been reported that PCI isolated from human blood plasma is highly heterogeneous, and that this heterogeneity is caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of two forms that differ by the presence or absence of 6 amino acids at the amino-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single individuals, and that individuals of two different ethnicities possess a similar PCI pattern, verifying that the micro-heterogeneity is conserved among humans. Furthermore, we have provided experimental evidence that PCI in both individuals is O-glycosylated on Thr20 with a core type 1 O-glycan, which is mostly NeuAcGalGalNAc. Modeling suggested that the O-glycan attachment site is located in proximity to several ligand-binding sites of the inhibitor., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible approximately 40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K D was 7.4 +/- 1.5 microM and thus correlated well with the apparent K I. In the presence of physiological Ca2+ concentration the K I and K D values were three to four fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn2+ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.

Protein C inhibitor (PCI) is a versatile serine protease inhibitor with both pro- and anticoagulant and other properties. Interactions of certain ligands with PCI, including heparin, affect its specificity for proteases. In this study, heparin was found to enhance PCI inhibition of factor Xa up to 42-fold in the presence of a physiological Ca(2+) concentration, whereas no heparin-induced activation was observed in the absence of Ca(2+). These results thus show that factor Xa adds to the group of proteases whose inhibition by PCI is enhanced by heparin and that such inhibition contributes to the anticoagulant properties of PCI by a Ca(2+)-dependent mechanism.

A specific pentasaccharide sequence of heparin binds with high affinity to native antithrombin and induces a conformational change in the inhibitor by a previously described two-step interaction mechanism. In this work, the interactions of heparin with the antiangiogenic latent and cleaved antithrombin forms were studied. Binding of heparin to these antithrombin forms was specific for the same pentasaccharide sequence as native antithrombin. Rapid kinetic studies demonstrated that this pentasaccharide induced a conformational change also in latent and cleaved antithrombin. The binding affinities of these antithrombin forms for the pentasaccharide, as compared to native antithrombin, were approximately 30-fold lower due to two to three fewer ionic interactions, resulting in less stable conformationally altered states. Affinities of latent and cleaved antithrombin for longer heparin chains, containing the pentasaccharide sequence, were 2-fold lower than for the pentasaccharide itself. This contrasts the interaction with native antithrombin and demonstrates that residues flanking the pentasaccharide sequence of heparin are repelled by the latent and cleaved forms. These findings contribute to delineating the mechanism by which heparin or heparan sulfate mediates antiangiogenic activity of antithrombin.

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., and Björk, I. (2001) J. Biol. Chem. 276, 11996-12002). Here, we show that the previously characterized prelatent antithrombin is a mixture of native antithrombin and a modified, true prelatent antithrombin that are resolvable by heparin-agarose chromatography. Kinetic analyses revealed that prelatent antithrombin is an intermediate in the conversion of native to latent antithrombin whose formation is favored by stabilizing anions of the Hofmeister series. Purified prelatent antithrombin had reduced anticoagulant function compared with native antithrombin, due to a reduced heparin affinity and consequent impaired ability of heparin to either bridge prelatent antithrombin and coagulation proteases in a ternary complex or to induce full conformational activation of the serpin. Significantly, prelatent antithrombin possessed an antiangiogenic activity more potent than that of latent antithrombin, based on the relative abilities of the two forms to inhibit endothelial cell growth. The prelatent form was conformationally altered from native antithrombin as judged from an attenuation of tryptophan fluorescence changes following heparin activation and a reduced thermal stability. The alterations are consistent with the limited structural changes involving strand 1C observed in a prelatent form of plasminogen activator inhibitor-1 (Dupont, D. M., Blouse, G. E., Hansen, M., Mathiasen, L., Kjelgaard, S., Jensen, J. K., Christensen, A., Gils, A., Declerck, P. J., Andreasen, P. A., and Wind, T. (2006) J. Biol. Chem. 281, 36071-36081), since the (1)H NMR spectrum, electrophoretic mobility, and proteolytic susceptibility of prelatent antithrombin most resemble those of native rather than those of latent antithrombin. Together, these results demonstrate that limited conformational alterations of antithrombin that modestly reduce anticoagulant activity are sufficient to generate antiangiogenic activity.

Fatty acid esterification of dolichol and cholesterol in Niemann-Pick type C1 mouse (Balb/c NIH npc1(-/-)) livers was investigated in response to treatment with peroxisomal proliferators. These inducers have hypolipidemic properties and influence the mevalonate pathway and the intracellular transport of the final products of this biosynthetic route. Such inducers are consequently interesting to use in a disease model with defective intracellular transport of lipids. In wild-type mice, the levels of dolichol and cholesterol found as free alcohols were not changed to any great extent upon treatment with the peroxisomal inducers dehydroepiandrosterone, clofibrate and diethylhexylphtalate. In contrast, the amounts of dolichyl esters increased whereas cholesteryl esters decreased by the same treatments. The rate of enzymatic esterification of dolichol in isolated microsomes was accordingly elevated after 5- to 7-day treatments with the efficient peroxisomal proliferators DEHP and PFOA, while the corresponding esterification of cholesterol was decreased. Upon peroxisomal induction in npc1(-/-) mice, the enzymatic dolichol esterification in vitro increased whereas the low concentration of dolichyl esters remained unchanged. The results thus demonstrate that the induction of fatty acid esterification of dolichol in vivo is impaired in npc1(-/-) mouse liver. It is therefore proposed that the intracellular lipid transport defect in npc1(-/-) mouse liver disables either dolichol and/or the fatty acid from reaching the site of esterification in vivo. This proposal was strengthened by the finding that the amount of dolichol was decreased in an isolated Golgi fraction from npc1(-/-) mice.

Idraparinux is a synthetic O-sulfated, O-methylated pentasaccharide that binds tightly to antithrombin (AT) and thereby specifically and efficiently induces the inactivation of the procoagulant protease, factor Xa. In this study, the affinity and kinetics of the interaction of this high-affinity pentasaccharide with alpha- and beta-AT were compared with those of a synthetic pentasaccharide comprising the natural AT-binding sequence of heparin. Dissociation equilibrium constants, Kd, for the interactions of Idraparinux with alpha- and beta-AT were approximately 0.4 and 0.1 nM, respectively, corresponding to an over 100-fold enhancement in affinity compared with that of the normal pentasaccharide. This large enhancement was due to a approximately 400-fold tighter conformationally activated complex formed in the second binding step, whereas the encounter complex established in the first step was approximately 4-fold weaker. The high-affinity and normal pentasaccharides both made a total of four ionic interactions with AT, although the high-affinity saccharide only established one ionic interaction in the first binding step and was compensated by three in the second step, whereas the normal pentasaccharide established two ionic interactions in each step. In contrast, the affinities of the nonionic interactions (Kd approximately 450 and 90 nM for the binding to alpha- and beta-AT, respectively) were considerably higher than those for the normal pentasaccharide and the highest of all AT-saccharide interactions reported so far. The nonionic contribution to the total free energy of the high-affinity pentasaccharide binding to AT thus amounted to approximately 70%. These findings show that nonionic interactions can play a predominant role in the binding of highly charged saccharide ligands to proteins and can be successfully exploited in the design of such biologically active ligands.

The N-terminal region residues, Lys11, Arg13, and Arg24, of the plasma coagulation inhibitor, antithrombin, have been implicated in binding of the anticoagulant polysaccharide, heparin, from the identification of natural mutants with impaired heparin binding or by the X-ray structure of a complex of the inhibitor with a high-affinity heparin pentasaccharide. Mutations of Lys11 or Arg24 to Ala in this work each reduced the affinity for the pentasaccharide approximately 40-fold, whereas mutation of Arg13 to Ala led to a decrease of only approximately 7-fold. All three substitutions resulted in the loss of one ionic interaction with the pentasaccharide and those of Lys11 or Arg24 also in 3-5-fold losses in affinity of nonionic interactions. Only the mutation of Lys11 affected the initial, weak interaction step of pentasaccharide binding, decreasing the affinity of this step approximately 2-fold. The mutations of Lys11 and Arg13 moderately, 2-7-fold, altered both rate constants of the second, conformational change step, whereas the substitution of Arg24 appreciably, approximately 25-fold, reduced the reverse rate constant of this step. The N-terminal region of antithrombin is thus critical for high-affinity heparin binding, Lys11 and Arg24 being responsible for maintaining appreciable and comparable binding energy, whereas Arg13 is less important. Lys11 is the only one of the three residues that is involved in the initial recognition step, whereas all three residues participate in the conformational change step. Lys11 and Arg13 presumably bind directly to the heparin pentasaccharide by ionic, and in the case of Lys11, also nonionic interactions. However, the role of Arg24 most likely is indirect, to stabilize the heparin-induced P-helix by interacting intramolecularly with Glu113 and Asp117, thereby positioning the crucial Lys114 residue for optimal ionic and nonionic interactions with the pentasaccharide. Together, these findings show that N-terminal residues of antithrombin make markedly different contributions to the energetics and dynamics of binding of the pentasaccharide ligand to the native and activated conformational states of the inhibitor that could not have been predicted from the X-ray structure.