The impact of the COVID-19 pandemic on neurosurgery in the elderly population in Sweden.

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.

DEAD Box Helicase 24 Is Increased in the Brain in Alzheimer's Disease and AppN-LF Mice and Influences Presymptomatic Pathology.

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 18O 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 Aß42-induced AD pathology-AppNL-F-and studied the interaction between Aß 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 AppNL-F mice showed that the increase of DDX24 starts before amyloid pathology or memory impairment is observed. Immunocytochemistry in AppNL-F primary hippocampal neurons showed increased DDX24 intensity in the soma, nucleus and nucleolus. Furthermore, siRNA targeting of DDX24 in neurons decreased APP and Aß42 levels, and the addition of Aß42 to the medium reduced DDX24. In conclusion, we have identified DDX24 as a protein with a potential role in Aß-induced AD pathology.

A glycan epitope correlates with tau in serum and predicts progression to Alzheimer's disease in combination with APOE4 allele status.

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.

Lack of N-glycosylation increases amyloidogenic processing of the amyloid precursor protein.

The amyloid precursor protein (APP) is a ubiquitously expressed type 1 transmembrane protein mostly known for serving as a precursor to the amyloid-ß peptide (Aß), 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 Aß42. 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 Proteome of the Dentate Terminal Zone of the Perforant Path Indicates Presynaptic Impairment in Alzheimer Disease.

Synaptic dysfunction is an early pathogenic event in Alzheimer disease (AD) that contributes to network disturbances and cognitive decline. Some synapses are more vulnerable than others, including the synapses of the perforant path, which provides the main excitatory input to the hippocampus. To elucidate the molecular mechanisms underlying the dysfunction of these synapses, we performed an explorative proteomic study of the dentate terminal zone of the perforant path. The outer two-thirds of the molecular layer of the dentate gyrus, where the perforant path synapses are located, was microdissected from five subjects with AD and five controls. The microdissected tissues were dissolved and digested by trypsin. Peptides from each sample were labeled with different isobaric tags, pooled together and pre-fractionated into 72 fractions by high-resolution isoelectric focusing. Each fraction was then analyzed by liquid chromatography-mass spectrometry. We quantified the relative expression levels of 7322 proteins, whereof 724 showed significantly altered levels in AD. Our comprehensive data analysis using enrichment and pathway analyses strongly indicated that presynaptic signaling, such as exocytosis and synaptic vesicle cycle processes, is severely disturbed in this area in AD, whereas postsynaptic proteins remained unchanged. Among the significantly altered proteins, we selected three of the most downregulated synaptic proteins; complexin-1, complexin-2 and synaptogyrin-1, for further validation, using a new cohort consisting of six AD and eight control cases. Semi-quantitative analysis of immunohistochemical staining confirmed decreased levels of complexin-1, complexin-2 and synaptogyrin-1 in the outer two-thirds of the molecular layer of the dentate gyrus in AD. Our in-depth proteomic analysis provides extensive knowledge on the potential molecular mechanism underlying synaptic dysfunction related to AD and supports that presynaptic alterations are more important than postsynaptic changes in early stages of the disease. The specific synaptic proteins identified could potentially be targeted to halt synaptic dysfunction in AD.

Changes in dementia diagnoses in Sweden during the COVID-19 pandemic.

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.

The N-glycan profile in cortex and hippocampus is altered in Alzheimer disease.

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.

Engineered Human Induced Pluripotent Cells Enable Genetic Code Expansion in Brain Organoids.

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.

Live Cell FRET Imaging Reveals Amyloid ß-Peptide Oligomerization in Hippocampal Neurons.

Amyloid ß-peptide (Aß) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aß 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 Aß42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled Aß42 in the cell culture medium for 24 h. Aß42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aß42 were significantly higher than for Aß40. These findings provide a better understanding of Aß42 oligomerization in neurons.

Proximity ligation assay reveals both pre- and postsynaptic localization of the APP-processing enzymes ADAM10 and BACE1 in rat and human adult brain.

BACKGROUND: Synaptic degeneration and accumulation of amyloid ß-peptides (Aß) are hallmarks of the Alzheimer diseased brain. Aß is synaptotoxic and produced by sequential cleavage of the amyloid precursor protein (APP) by the ß-secretase BACE1 and by γ-secretase. If APP is instead cleaved by the α-secretase ADAM10, Aß will not be generated. Although BACE1 is considered to be a presynaptic protein and ADAM10 has been reported to mainly localize to the postsynaptic density, we have previously shown that both ADAM10 and BACE1 are highly enriched in synaptic vesicles of rat brain and mouse primary hippocampal neurons. RESULTS: Here, using brightfield proximity ligation assay, we expanded our previous result in primary neurons and investigated the in situ synaptic localization of ADAM10 and BACE1 in rat and human adult brain using both pre- and postsynaptic markers. We found that ADAM10 and BACE1 were in close proximity with both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. The substrate APP was also detected both pre- and postsynaptically. Subcellular fractionation confirmed that ADAM10 and BACE1 are enriched to a similar degree in synaptic vesicles and as well as in the postsynaptic density. CONCLUSIONS: We show that the α-secretase ADAM10 and the ß-secretase BACE1 are located in both the pre- and postsynaptic compartments in intact brain sections. These findings increase our understanding of the regulation of APP processing, thereby facilitating development of more specific treatment strategies.

APP depletion alters selective pre- and post-synaptic proteins.

The normal role of Alzheimer's disease (AD)-linked amyloid precursor protein (APP) in the brain remains incompletely understood. Previous studies have reported that lack of APP has detrimental effects on spines and electrophysiological parameters. APP has been described to be important in synaptic pruning during development. The effect of APP knockout on mature synapses is complicated by this role in development. We previously reported on differential changes in synaptic proteins and receptors in APP mutant AD transgenic compared to wild-type neurons, which revealed selective decreases in levels of pre- and post-synaptic proteins, including of surface glutamate receptors. In the present study, we undertook a similar analysis of synaptic composition but now in APP knockout compared to wild-type mouse neurons. Here we demonstrate alterations in levels of selective pre- and post-synaptic proteins and receptors in APP knockout compared to wild-type mouse primary neurons in culture and brains of mice in youth and adulthood. Remarkably, we demonstrate selective increases in levels of synaptic proteins, such as GluA1, in neurons with APP knockout and with RNAi knockdown, which tended to be opposite to the reductions seen in AD transgenic APP mutant compared to wild-type neurons. These data reinforce that APP is important for the normal composition of synapses.

Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen.

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 (Aß42) protofibrils, but not with Aß40 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 Aß42, 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.

Cerebrospinal fluid from Alzheimer patients affects cell-mediated nerve growth factor production and cell survival in vitro.

Alzheimer's disease (AD) is characterized by early degeneration of cholinergic neurons and decreased levels of nerve growth factor (NGF). Thus, increasing the NGF levels by for instance encapsulated cell bio-delivery (ECB) is a potential treatment strategy. The results from our previous first-in-human studies on ECB of NGF to the basal forebrain cholinergic neurons were promising, but indicated some variability of long-term viability of the encapsulated cells and associated reduced NGF-release. Here we studied the effect of amyloid beta-peptides (Aß), interleukin 1-beta (IL-1ß), and CSF from AD, Lewy body dementia (LBD) or subjective cognitive impairment (SCI) patients on the NGF overproducing cell line NGC-0295. At physiological concentrations, neither Aß40 nor Aß42 had any major impact on cell viability or NGF-production. In contrast, IL-1ß dose-dependently affected NGF-production over time. Exposure of NGF-producing cells to CSF from AD patients showed significantly reduced NGF-release as compared to CSF from LBD or SCI patients. By mass spectrometry we found 3 proteins involved in inflammatory pathways to have an altered expression in AD CSF compared to LBD and SCI. Cell survival and NGF-release were not affected by Aß. NGF-release was affected by IL-1ß, suggesting that inflammation has a negative effect on ECB cells.

Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2).

The toxic amyloid ß-peptide (Aß) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aß is produced by the sequential cleavage of the Aß precursor protein (APP) by ß-secretase (to yield APP-C-terminal fragment ß (APP-CTFß) and soluble APPß (sAPPß)) and γ-secretase (to yield Aß). We reasoned that proteins that associate with γ-secretase are likely to regulate Aß 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 Aß 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 Aß, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aß levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by ß-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.

Loss of neprilysin alters protein expression in the brain of Alzheimer's disease model mice.

Alzheimer's disease (AD) is a neurodegenerative disease displaying extracellular plaques formed by the neurotoxic amyloid ß-peptide (Aß), and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relate to the massive neuronal death that occurs in AD brains remain elusive. Neprilysin is the major Aß-degrading enzyme and a lack thereof increases Aß levels in the brain twofold. To identify altered protein expression levels induced by increased Aß levels, we performed a proteomic analysis of the brain of the AD mouse model APPsw and compared it to that of APPsw mice lacking neprilysin. To this end we established an LC-MS/MS method to analyze brain homogenate, using an (18) O-labeled internal standard to accurately quantify the protein levels. To distinguish between alterations in protein levels caused by increased Aß levels and those induced by neprilysin deficiency independently of Aß, the brain proteome of neprilysin deficient APPsw mice was also compared to that of neprilysin deficient mice. By this approach we identified approximately 600 proteins and the levels of 300 of these were quantified. Pathway analysis showed that many of the proteins with altered expression were involved in neurological disorders, and that tau, presenilin and APP were key regulators in the identified networks. The data have been deposited to the ProteomeXchange Consortium with identifiers PXD000968 and PXD001786 (http://proteomecentral.proteomexchange.org/dataset/PXD000968 and (http://proteomecentral.proteomexchange.org/dataset/PXD001786). Interestingly, the levels of several proteins, including some not previously reported to be linked to AD, were associated with increased Aß levels.

ADAM10 and BACE1 are localized to synaptic vesicles.

Synaptic degeneration and accumulation of the neurotoxic amyloid ß-peptide (Aß) in the brain are hallmarks of Alzheimer disease. Aß is produced by sequential cleavage of the amyloid precursor protein (APP), by the ß-secretase ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. However, Aß generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aß 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.

Highly Sensitive FRET-FCS Detects Amyloid ß-Peptide Oligomers in Solution at Physiological Concentrations.

Oligomers formed by the amyloid ß-peptide (Aß) are pathogens in Alzheimer's disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aß oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Förster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aß, FRET-FCS detected oligomers consisting of less than 10 Aß molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aß oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain.

In Alzheimer disease, oligomeric amyloid ß-peptide (Aß) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aß from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aß production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aß production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aß production (Aß40 and Aß42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aß production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

Analysis of microdissected neurons by 18O mass spectrometry reveals altered protein expression in Alzheimer's disease.

It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post-mortem brains by laser capture microdissection. By using (18)O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat-shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron-specific analysis. Hence, we conclude that cell-specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis.

Mutations in nicastrin protein differentially affect amyloid beta-peptide production and Notch protein processing.

The γ-secretase complex is responsible for intramembrane processing of over 60 substrates and is involved in Notch signaling as well as in the generation of the amyloid ß-peptide (Aß). Aggregated forms of Aß have a pathogenic role in Alzheimer disease and, thus, reducing the Aß levels by inhibiting γ-secretase is a possible treatment strategy for Alzheimer disease. Regrettably, clinical trials have shown that inhibition of γ-secretase results in Notch-related side effects. Therefore, it is of great importance to find ways to inhibit amyloid precursor protein (APP) processing without disturbing vital signaling pathways such as Notch. Nicastrin (Nct) is part of the γ-secretase complex and has been proposed to be involved in substrate recognition and selection. We have investigated how the four evenly spaced and conserved cysteine residues in the Nct ectodomain affect APP and Notch processing. We mutated these cysteines to serines and analyzed them in cells lacking endogenous Nct. We found that two mutants, C213S (C2) and C230S (C3), differentially affected APP and Notch processing. Both the formation of Aß and the intracellular domain of amyloid precursor protein (AICD) were reduced, whereas the production of Notch intracellular domain (NICD) was maintained on a high level, although C230S (C3) showed impaired complex assembly. Our data demonstrate that single residues in a γ-secretase component besides presenilin are able to differentially affect APP and Notch processing.