Long-term health sequelae and quality of life at least 6 months after infection with SARS-CoV-2: design and rationale of the COVIDOM-study as part of the NAPKON population-based cohort platform (POP).

PURPOSE: Over the course of COVID-19 pandemic, evidence has accumulated that SARS-CoV-2 infections may affect multiple organs and have serious clinical sequelae, but on-site clinical examinations with non-hospitalized samples are rare. We, therefore, aimed to systematically assess the long-term health status of samples of hospitalized and non-hospitalized SARS-CoV-2 infected individuals from three regions in Germany. METHODS: The present paper describes the COVIDOM-study within the population-based cohort platform (POP) which has been established under the auspices of the NAPKON infrastructure (German National Pandemic Cohort Network) of the national Network University Medicine (NUM). Comprehensive health assessments among SARS-CoV-2 infected individuals are conducted at least 6 months after the acute infection at the study sites Kiel, Würzburg and Berlin. Potential participants were identified and contacted via the local public health authorities, irrespective of the severity of the initial infection. A harmonized examination protocol has been implemented, consisting of detailed assessments of medical history, physical examinations, and the collection of multiple biosamples (e.g., serum, plasma, saliva, urine) for future analyses. In addition, patient-reported perception of the impact of local pandemic-related measures and infection on quality-of-life are obtained. RESULTS: As of July 2021, in total 6813 individuals infected in 2020 have been invited into the COVIDOM-study. Of these, about 36% wished to participate and 1295 have already been examined at least once. CONCLUSION: NAPKON-POP COVIDOM-study complements other Long COVID studies assessing the long-term consequences of an infection with SARS-CoV-2 by providing detailed health data of population-based samples, including individuals with various degrees of disease severity. TRIAL REGISTRATION: Registered at the German registry for clinical studies (DRKS00023742).

N-cadherin specifies first asymmetry in developing neurons.

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.

Down-regulation of the ATP-binding cassette transporter 2 (Abca2) reduces amyloid-ß production by altering Nicastrin maturation and intracellular localization.

Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-ß (Aß) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aß generation, without affecting Notch cleavage.

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy.

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein alphaA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.

Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.

Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions (both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology, a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing and fluorescence in situ hybridization on mechanically stretched chromosomes. Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis. Besides the production of truncated PGRN proteins due to premature stop codons, we identified a mutation within the splice donor site of intron 0 (IVS0 + 5G > C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family. Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation (c.3G > A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.

The ascent of the blessed: regulatory issues on health effects and health claims for probiotics in Europe and the rest of the world.

The outcome of the first series of health claim applications for probiotics in Europe as evaluated by the European Food Safety Authority (EFSA) has, up to 2013 almost completely yielded negative results. All recent applications also have been rejected, including the latest on prevention of mastitis in breastfeeding mothers. In other developed countries, such as Switzerland, Japan and Canada, the health effects of probiotics, for which scientific evidence has been provided, can be communicated to potential consumers. The number of clinical trials with probiotics over recent years shows a trend to level off or even decline. At the same time, clinical research into the role of (gut) microbiota in a wide variety of diseases and conditions is booming. Ultimately, this may offer new indications for gut microbiota management by probiotics, prebiotics or other food supplements.

Mean age-of-onset of familial alzheimer disease caused by presenilin mutations correlates with both increased Abeta42 and decreased Abeta40.

The varied ways in which mutations in presenilins (PSEN1 and PSEN2) affect amyloid b precursor protein (APP) processing in causing early-onset familial Alzheimer disease (FAD) are complex and not yet properly understood. Nonetheless, one useful diagnostic marker is an increased ratio of Ab42 to Ab40 (Ab42/Ab40) in patients' brain and biological fluids as well as in transgenic mice and cells. We studied Ab and APP processing for a set of nine clinical PSEN mutations on a novel and highly reproducible enzyme-linked immunosorbent assay (ELISA)-based in vitro method and also sought correlation with brain Ab analyzed by image densitometry and mass spectrometry. All mutations significantly increased Ab42/Ab40 in vitro by significantly decreasing Ab40 with accumulation of APP C-terminal fragments, a sign of decreased PSEN activity. A significant increase in absolute levels of Ab42 was observed for only half of the mutations tested. We also showed that age-of-onset of PSEN1-linked FAD correlated inversely with Ab42/Ab40 (r = -0.89; P = 0.001) and absolute levels of Ab42 (r = -0.83; P = 0.006), but directly with Ab40 levels (r = 0.69; P = 0.035). These changes also partly correlated with brain Ab42 and Ab40 levels. Together, our data suggested that Ab40 might be protective by perhaps sequestering the more toxic Ab42 and facilitating its clearance. Also, the in vitro method we describe here is a valid tool for assaying the pathogenic potential of clinical PSEN mutations in a molecular diagnostic setting.

Characterization of ubiquitinated intraneuronal inclusions in a novel Belgian frontotemporal lobar degeneration family.

The most common histologic feature in patients with frontotemporal lobar degeneration (FTLD) is intracellular brain inclusions of yet uncharacterized proteins that react with antiubiquitin (Ub) antibodies, but not with tau or synuclein (FTLD-U). We identified a four-generation Belgian FTLD family in which 8 patients had dominantly inherited FTLD. In one patient, we showed frontotemporal atrophy with filamentous Ub-positive intracellular inclusions in absence of tau pathology or any alterations in the levels of soluble tau. We characterized the cellular and subcellular localization and morphology of the inclusions. Ub-positive inclusions predominantly occurred within neurons (>97%), but were also observed within oligodendroglia (approximately 2%) and microglia (<1%), but not within astroglia. Regarding the subcellular localization, the intranuclear inclusions (INI) were up to approximately four-fold more frequent than the cytoplasmic inclusions, although the latter were more specific to neurons. The INIs frequently appeared spindle-shaped and 3-dimensional confocal reconstructions identified flattened, leaf-like structures. Ultrastructurally, straight 10- to 18-nm-diameter filaments constituted the spindle-shaped inclusions that occurred in close proximity to the nuclear membrane. Staining for HSP40, p62, and valosin/p97 was observed in only a minority of the inclusions. Whereas the precise nature of the protein remains elusive, characterization of such familial FTLD-U patients would be helpful in identifying a common denominator in the pathogenesis of familial and the more prevalent sporadic FTLD-U.

Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum.

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered. Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grünwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1. Furthermore, macrophage subtypes were characterized by ultrastructural analysis. Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96%. The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions. The contaminating cells were mainly mesothelial. A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10. Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

An ultrastructural study of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum.

An ultrastructural study was performed to examine the presence and distribution of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum. Non-myelinated nerve fibres were located perivascularly as well as throughout the milky spots. Dopamine immunoreactivity was demonstrated in the nerve fibres and in a portion of the macrophage population. These results demonstrate a discrepancy between human and non-human milky spots.

Do the research goal and databases match? A checklist for a systematic approach.

To test the appropriateness of a given database for specific research questions, we designed a checklist starting with the definition of an ideal database. This ideal database contains all relevant data on patients, providers and services. It is safe and accessible, input is always accurate, continuity is guaranteed and linkage with other information is easy. Of course no such database exists. Still these features are often taken for granted, but highly influenced by organizational processes in healthcare and prioritization. Starting with the characteristics of an ideal database, one can systematically list the required aspects for research goals and compare these with the available systems. This checklist is used to address important aspects of administrative database research and ethical issues. The increasing possibility to misuse sensitive data needs to be discussed by researchers, administrators, individuals and society. This checklist can also be valuable to others to design or interpret studies based on claims databases.

Alterations in phosphatidylethanolamine levels affect the generation of Aß.

Several studies suggest that the generation of Aß is highly dependent on the levels of cholesterol within membranes' detergent-resistant microdomains (DRM). Indeed, the ß-amyloid precursor protein (APP) cleaving machinery, namely ß- and γ-secretases, has been shown to be present in DRM and its activity depends on membrane cholesterol levels. Counterintuitive to the localization of the cleavage machinery, the substrate, APP, localizes to membranes' detergent-soluble microdomains enriched in phospholipids (PL), indicating that Aß generation is highly dependent on the capacity of enzyme and substrate to diffuse along the lateral plane of the membrane and therefore on the internal equilibrium of the different lipids of DRM and non-DRM domains. Here, we studied to which extent changes in the content of a main non-DRM lipid might affect the proteolytic processing of APP. As phosphatidylethanolamine (PE) accounts for the majority of PL, we focused on its impact on the regulation of APP proteolysis. In mammalian cells, siRNA-mediated knock-down of PE synthesis resulted in decreased Aß owing to a dual effect: promoted α-secretase cleavage and decreased γ-secretase processing of APP. In vivo, in Drosophila melanogaster, genetic reduction in PL synthesis results in decreased γ-secretase-dependent cleavage of APP. These results suggest that modulation of the membrane-soluble domains could be a valuable alternative to reduce excessive Aß generation.

Intraneuronal amyloid beta and reduced brain volume in a novel APP T714I mouse model for Alzheimer's disease.

Transgenic mouse models of Alzheimer's disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.

Macrophage depletion in the rat after intraperitoneal administration of liposome-encapsulated clodronate: depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund's adjuvant.

The purpose of this study was to develop a method for the depletion of macrophages from the peritoneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with liposome-encapsulated clodronate (dichloromethylene bisphosphonate: Cl2MBP-liposomes). This treatment resulted in complete elimination of mature tissue macrophages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were completely depleted, few ED1-positive cells remained and repopulation of ED1-positive cells was faster. The treatment further depleted macrophages from the spleen, especially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of Cl2MBP-liposomes considerably accelerated repopulation in the omentum. The protocol described might be used to investigate the contribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimination of intestinal tumours.

Milky spots in the greater omentum are predominant sites of local tumour cell proliferation and accumulation in the peritoneal cavity.

The role that milky spots in the greater omentum play in tumour cell spread in the peritoneal cavity is presently not fully understood. To study whether intraperitoneally injected tumour cells appear preferentially in milky spots of the greater omentum and to study the changes in the greater omentum, and especially in the cell population of milky spots after tumour cell infiltration, the following study was performed. A detailed temporal sequences of changes in morphology and cellular composition in milky spots of the greater omentum of Wag/Rij rats 5, 15, 30, 60 min, 2, 4, 8, 16, 24 h, 2, 4, 8 days and 2 and 4 weeks after intraperitoneal administration of 2.0 x 10(6) CC 531 tumour cells was investigated by light microscopy and electron microscopy (pre-embedding labelling). Our data showed that the milky spots in the greater omentum were the sites to which tumour cells migrated preferentially from the peritoneal cavity. The tumour cells infiltrated the milky spots and formed clusters within. The cellular population in milky spots reacted by a very rapid influx of young macrophages during the first hour and an increase of the total number of cells (P < 0.01). After 4 h tumour cells were also located on the greater omentum outside the area of the milky spots. Around these tumour cell deposits, new milky spots are formed, which increased the total number of milky spots. The cells present in milky spots are not capable of reversing the growth of tumours and finally a solid omental cake of tumour cells is formed.

Ontogeny of milky spots in the human greater omentum: an immunochemical study.

BACKGROUND: Milky spots in the human greater omentum are preformed specific accumulations of primarily macrophages within the stroma of the greater omentum. To obtain a better understanding of milky spots in the human greater omentum, the development and the earliest forms of milky spots in the human greater omentum were studied, with special attention to the macrophage population. METHODS: Specimens of human greater omentum were obtained from fetuses of 20 to 40 weeks gestation and one newborn three days old (n = 6). Using mature macrophages (RFD 7), activated macrophages (RFD 1), B-lymphocytes (CD 22), and T-lymphocytes (CD 2), and immunoperoxydase labeling, the percentage of these cells in developing milky spots and the development of milky spots were studied by light microscopy. A time-dependent increase in the percentage of positive staining cells and the size of clusters was analyzed using the non-parametric Spearman rank correlation test. RESULTS: Small accumulations of cells with about 50% monocytes/macrophages were present at 20 weeks of gestation. With increasing gestational age the number of clusters of cells increased significantly (P < 0.01) as well as their size (P < 0.01). Starting at 29 weeks, vascularized clusters of cells were seen; true milky spots were present at 35 weeks. A significant (P < 0.05) increase in the percentage of mature macrophages was found in developing milky spots, whereas no activated macrophages were seen. The percentage of B-lymphocytes and T-lymphocytes found in the clusters of cells and milky spots increased significantly (P < 0.05) but did not exceed 10% of the total number of cells. CONCLUSIONS: From our data it can be concluded that milky spots are specific structures in the greater omentum formed between the 20th and 35th week of gestation. Further, we concluded that immature cells (promonocytes) mature locally in developing milky spots.

Cellular composition of milky spots in the human greater omentum: an immunochemical and ultrastructural study.

BACKGROUND: Milky spots in the greater omentum of some animals are well organized perivascular infiltrates of leucocytes, and are considered to have characteristics of secondary lymphoid tissue. To determine whether milky spots in the human greater omentum can also be regarded as secondary lymphoid tissue, we studied milky spots in an unstimulated state. METHODS: Patients were selected on the basis of absence of disease in the peritoneal cavity that might influence the state of the milky spots. Using monoclonal antibodies against macrophages, B-lymphocytes and T-lymphocytes, and immunoperoxidase labeling, the number of these cells and their location in milky spots were studied by light microscopy. However, the stromal components of the greater omentum, especially those within the milky spots, were studied by electron microscopy. RESULTS: Milky spots in the human greater omentum are relatively uniform vascularized accumulations of mononuclear cells comprising macrophages (67.9% +/- 9.4, mean +/- standard deviation), B-cells (10.1% +/- 3.4), T-cells (10.2% +/- 3.7), and mast cells. However, no special B-cell and T-cell areas could be distinguished. On the ultrastructural level it was demonstrated that macrophages are present in different stages of maturation and can enter or leave the milky spots. Furthermore, no cells characteristic of secondary lymphoid organs, such as interdigitating cells or follicular dendritic cells, were seen. CONCLUSIONS: These data indicate that unstimulated milky spots in the human greater omentum are to a great extent just a preformed specific accumulation of primarily macrophages within the stroma of the greater omentum, and therefore, cannot be regarded as true secondary lymphoid tissue. Milky spots could serve as a gateway for, as well as a provider of peritoneal macrophages when the intra-abdominal status so requires. Finally, the data from this study are compared with the data of other studies of human milky spots and those in animals.

Alzheimer-associated C allele of the promoter polymorphism -22C>T causes a critical neuron-specific decrease of presenilin 1 expression.

We, amongst others, have shown that CC homozygosity at the -22C>T promoter polymorphism in presenilin 1 (PSEN1) is associated with increased risk for Alzheimer's disease (AD). Also, studies in AD brains suggested that CC homozygosity increased the risk for AD by increasing the Abeta load. We characterized the PSEN1 promoter by deletion mapping, and analysed the effect of the -22C and -22T alleles on the transcriptional activity of PSEN1 in a transient transfection system. We showed a neuron-specific 2-fold decrease in promoter activity for the -22C risk allele, which in homozygous individuals would lead to a critical decrease in PSEN1 expression. The deletion mapping suggested that the 13 bp region (-33/-20) spanning the -22C>T polymorphism harbours a binding site for a negative regulatory factor. This factor has a higher affinity for the -22C risk allele and is strongly dependent on downstream sequences for cell-type-specific expression differences. Together, these studies provide evidence that the increased risk for AD associated with PSEN1 may result from genetic variations in the regulatory region, leading to altered expression levels of PSEN1 in neurons.

The gene encoding nicastrin, a major gamma-secretase component, modifies risk for familial early-onset Alzheimer disease in a Dutch population-based sample.

Nicastrin regulates gamma-secretase cleavage of the amyloid precursor protein by forming complexes with presenilins, in which most mutations causing familial early-onset Alzheimer disease (EOAD) have been found. The gene encoding nicastrin (NCSTN) maps to 1q23, a region that has been linked and associated with late-onset Alzheimer disease (LOAD) in various genome screens. In 78 familial EOAD cases, we found 14 NCSTN single-nucleotide polymorphisms (SNPs): 10 intronic SNPs, 3 silent mutations, and 1 missense mutation (N417Y). N417Y is unlikely to be pathogenic, since it did not alter amyloid beta secretion in an in vitro assay and its frequency was similar in case and control subjects. However, SNP haplotype estimation in two population-based series of Dutch patients with EOAD (n=116) and LOAD (n=240) indicated that the frequency of one SNP haplotype (HapB) was higher in the group with familial EOAD (7%), compared with the LOAD group (3%) and control group (3%). In patients with familial EOAD without the APOE epsilon4 allele, the HapB frequency further increased, to 14%, resulting in a fourfold increased risk (odds ratio = 4.1; 95% confidence interval 1.2-13.3; P=.01). These results are compatible with an important role of gamma-secretase dysfunction in the etiology of familial EOAD.

In vitro studies of Flemish, Dutch, and wild-type beta-amyloid provide evidence for two-staged neurotoxicity.

Mutations in the beta-amyloid (Abeta) sequence of the amyloid precursor protein gene (APP) present with variable disease phenotypes. While patients with the Dutch APP mutation (E693Q) have predominantly hemorrhagic strokes, Flemish APP (A692G) patients develop both strokes and Alzheimer's disease (AD). To determine whether these diverse clinical and pathological presentations are due to mutant Abeta or APP, we studied the effect of Flemish, Dutch, and wild-type Abeta/APP on phosphorylation of specific tau epitopes observed in AD. No effect was observed in differentiated SH-SY5Y cells either stably expressing APP or treated with synthetic Abeta(12-42). However, we did observe a paradoxical temporal difference in the neurotoxic potential of mutant and wild-type Abeta. While long 24-h incubation at physiological levels of Abeta (2 microM) showed a higher amount of apoptosis for Dutch Abeta, a short 2-h incubation showed elevated apoptosis for Flemish and wild-type Abeta. The altered aggregating properties of Abeta, with Dutch Abeta aggregating faster and Flemish Abeta slower than wild type, elucidated a discrete two-phase Abeta neurotoxicity. We propose here that, at least in vitro, Abeta might be neurotoxic in an initial phase due to its soluble oligomeric or other early toxic Abeta intermediate(s), which is perhaps distinct from the late neurotoxicity incurred by aggregated larger assemblies of Abeta.