The pathogenesis of Alzheimer's disease (AD) is believed to be closely dependent on deposits of neurotoxic amyloid-? peptides (A?), which become abundantly present throughout the central nervous system in advanced stages of the disease. The different A? peptides existing are generated by subsequent cleavage of the amyloid-? protein precursor (A?PP) and may vary in length and differ at their C-terminus. Despite extensive studies on the most prevalent species A?40 and A?42, A? peptides with other C-termini such as A?38 have not received much attention. In the present study, we used a highly specific and sensitive antibody against A?38 to analyze the distribution of this A? species in cases of sporadic and familial AD, as well as in the brains of a series of established transgenic AD mouse models. We found A?38 to be present as vascular deposits in the brains of the majority of sporadic AD cases, whereas it is largely absent in non-demented control cases. A?38-positive extracellular plaques were virtually limited to familial cases. Interestingly we observed A?38-positive plaques not only among familial cases due to A?PP mutations, but also in cases of familial AD caused by presenilin (PSEN) mutations. Furthermore we demonstrate that A?38 deposits in the form of extracellular plaques are common in several AD transgenic mouse models carrying either only A?PP, or combinations of A?PP, PSEN1, and tau transgenes.
Amyloid-? (A?) immunotherapy for Alzheimer's disease (AD) has good preclinical support from transgenic mouse models and clinical data suggesting that a long-term treatment effect is possible. Soluble A? protofibrils have been shown to exhibit neurotoxicity in vitro and in vivo, and constitute an attractive target for immunotherapy. Here, we demonstrate that the humanized antibody BAN2401 and its murine version mAb158 exhibit a strong binding preference for A? protofibrils over A? monomers. Further, we confirm the presence of target by showing that both antibodies efficiently immunoprecipitate soluble A? aggregates in human AD brain extracts. mAb158 reached the brain and reduced the brain protofibril levels by 42% in an exposure-dependent manner both after long-term and short-term treatment in tg-ArcSwe mice. Notably, a 53% reduction of protofibrils/oligomers in cerebrospinal fluid (CSF) that correlated with reduced brain protofibril levels was observed after long-term treatment, suggesting that CSF protofibrils/oligomers could be used as a potential biomarker. No change in native monomeric A?42 could be observed in brain TBS extracts after mAb158-treatment in tg-ArcSwe mice. By confirming the specific ability of mAb158 to selectively bind and reduce soluble A? protofibrils, with minimal binding to A? monomers, we provide further support in favor of its position as an attractive new candidate for AD immunotherapy. BAN2401 has undergone full Phase 1 development, and available data indicate a favorable safety profile in AD patients.
An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-? (A?) peptide become self-propagating in Alzheimer's disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of A? prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of A? isoforms and the morphology of cerebrovascular A? deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent A?38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that A? strains are serially transmissible. We conclude that at least two distinct strains of A? prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.
Amyloid beta (A?) is the main component of plaques, the central neuropathological hallmark in Alzheimer's disease (AD). A? is derived from the amyloid precursor protein (APP) by ?- and ?-secretase-mediated cleavages. A large number of A? peptides are found in cerebrospinal fluid and these peptides are produced in specific metabolic pathways, which are important for diagnosis, in drug development and to explore disease pathogenesis. To investigate whether a similar pattern could be found also in blood samples, an immunoprecipitation (IP) based method for enrichment of A? peptides from human plasma was developed. The peptides were analyzed using matrix-assisted-laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry for A? profiling and selected reaction monitoring (SRM) for MS quantification of A?1-38, A?1-40 and A?1-42 using tripe quadrupole MS. Sixteen N- or C-terminally truncated A? peptides were reproducibly detected in human plasma, of which 11 were verified by tandem MS. In a pilot study including 9 AD patients and 10 controls, where A?1-38, A?1-40 and A?1-42 were quantified using SRM, no AD-associated change in plasma levels of the peptides were observed. Using MS-based measurement techniques, we show that several A? peptides can be monitored in a single analysis and the developed methods have the potential to be used as a read out in clinical trials of drugs affecting APP processing or A? homeostasis.
Incidence and mortality for sex-unspecific cancers are higher among men, a fact that is largely unexplained. Furthermore, age-related loss of chromosome Y (LOY) is frequent in normal hematopoietic cells, but the phenotypic consequences of LOY have been elusive. From analysis of 1,153 elderly men, we report that LOY in peripheral blood was associated with risks of all-cause mortality (hazards ratio (HR) = 1.91, 95% confidence interval (CI) = 1.17-3.13; 637 events) and non-hematological cancer mortality (HR = 3.62, 95% CI = 1.56-8.41; 132 events). LOY affected at least 8.2% of the subjects in this cohort, and median survival times among men with LOY were 5.5 years shorter. Association of LOY with risk of all-cause mortality was validated in an independent cohort (HR = 3.66) in which 20.5% of subjects showed LOY. These results illustrate the impact of post-zygotic mosaicism on disease risk, could explain why males are more frequently affected by cancer and suggest that chromosome Y is important in processes beyond sex determination. LOY in blood could become a predictive biomarker of male carcinogenesis.
Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (?-synuclein) in the central nervous system (CNS) is an early pathogenic event in Parkinson's disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against ?-synuclein as a promising novel treatment strategy. Since large ?-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. In support of this, (Thy-1)-h[A30P] ?-synuclein transgenic mice with motor dysfunction symptoms were found to display increased levels of ?-synuclein protofibrils in the CNS. An ?-synuclein protofibril-selective monoclonal antibody (mAb47) was evaluated in this ?-synuclein transgenic mouse model. As measured by ELISA, 14month old mice treated for 14weeks with weekly intraperitoneal injections of mAb47 displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. Besides the lower levels of pathogenic ?-synuclein demonstrated, a reduction of motor dysfunction in transgenic mice upon peripheral administration of mAb47 was indicated. Thus, immunotherapy with antibodies targeting toxic ?-synuclein species holds promise as a future disease-modifying treatment in Parkinson's disease and related disorders.
We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.
According to the modified amyloid hypothesis the main event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid ?-peptide (A?) within neurons. Additionally to full-length peptides, a great diversity of N-truncated A? variants is derived from the larger amyloid precursor protein (APP). Vast evidence suggests that A?x??? isoforms play an important role triggering neurodegeneration due to its high abundance, amyloidogenic propensity and toxicity. Although N-truncated and A?x??? species have been pointed as crucial players in AD etiology, the A??-x isoforms have not received much attention.
Immunotherapy targeting ?-synuclein has evolved as a potential therapeutic strategy for neurodegenerative diseases, such as Parkinson's disease, and initial studies on cellular and animal models have shown promising results. ?-synuclein vaccination of transgenic mice reduced the number of brain inclusions, whereas passive immunization studies demonstrated that antibodies against the C-terminus of ?-synuclein can pass the blood-brain barrier and affect the pathology. In addition, preliminary evidence suggests that transgenic mice treated with an antibody directed against ?-synuclein oligomers/protofibrils resulted in reduced levels of such species in the CNS. The underlying mechanisms of immunotherapy are not yet fully understood, but may include antibody-mediated clearance of pre-existing aggregates, prevention of protein propagation between cells and microglia-dependent protein clearance. Thus, immunotherapy targeting ?-synuclein holds promise, but needs to be further developed as a future disease-modifying treatment in Parkinson's disease and other ?-synucleinopathies.
The increased risk of Alzheimer's disease (AD) associated with specific apolipoprotein E (ApoE) isoforms appears to relate to altered amyloid-? (A?) homeostasis. Clearance of A? from the brain is reduced in the presence of the AD-associated ApoE4 isoform, which may contribute to the accumulation of A? deposits in the parenchyma and vasculature. The low-density lipoprotein receptor-related protein 1 (LRP1) and heparan sulfate proteoglycans (HSPGs), both established ApoE receptors, are involved in A? uptake, with LRP1 additionally implicated in A? transcytosis across the blood-brain barrier. In this study, we detected the co-distribution of heparan sulfate (HS), ApoE and LRP1 in A?(1-40)-positive brain microvessels from individuals with Down's syndrome diagnosed with AD. In addition, ApoE was pulled-down from AD cerebrospinal fluid with anti-A? antibodies. Using Chinese hamster ovary cells deficient in HS or LRP1, we found that ApoE increases cell association of A? in a HSPG- and LRP1-dependent manner; and further, ApoE processing is altered in the absence of cellular HS. These interactions may facilitate A? clearance from the brain, but if overwhelmed could contribute to A? accumulation and the pathogenesis of AD.
To investigate whether total sleep deprivation (TSD) affects circulating concentrations of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in humans. These factors are usually found in the cytoplasm of neurons and glia cells. Increasing concentrations of these factors in blood may be therefore indicative for either neuronal damage, impaired blood brain barrier function, or both. In addition, amyloid ? (A?) peptides 1-42 and 1-40 were measured in plasma to calculate their ratio. A reduced plasma ratio of A? peptides 1-42 to 1-40 is considered an indirect measure of increased deposition of A? 1-42 peptide in the brain.
The symptomatic drugs currently on the market for Alzheimer's disease (AD) have no effect on disease progression, and this creates a large unmet medical need. The type of drug that has developed most rapidly in the last decade is immunotherapy: vaccines and, especially, passive vaccination with monoclonal antibodies. Antibodies are attractive drugs as they can be made highly specific for their target and often with few side effects. Data from recent clinical AD trials indicate that a treatment effect by immunotherapy is possible, providing hope for a new generation of drugs. The first anti-amyloid-beta (anti-A?) vaccine developed by Elan, AN1792, was halted in phase 2 because of aseptic meningoencephalitis. However, in a follow-up study, patients with antibody response to the vaccine demonstrated reduced cognitive decline, supporting the hypothesis that A? immunotherapy may have clinically relevant effects. Bapineuzumab (Elan/Pfizer Inc./Johnson & Johnson), a monoclonal antibody targeting fibrillar A?, was stopped because the desired clinical effect was not seen. Solanezumab (Eli Lilly and Company) was developed to target soluble, monomeric A?. In two phase 3 studies, Solanezumab did not meet primary endpoints. When data from the two studies were pooled, a positive pattern emerged, revealing a significant slowing of cognitive decline in the subgroup of mild AD. The Arctic mutation has been shown to specifically increase the formation of soluble A? protofibrils, an A? species shown to be toxic to neurons and likely to be present in all cases of AD. A monoclonal antibody, mAb158, was developed to target A? protofibrils with high selectivity. It has at least a 1,000-fold higher selectivity for protofibrils as compared with monomers of A?, thus targeting the toxic species of the peptide. A humanized version of mAb158, BAN2401, has now entered a clinical phase 2b trial in a collaboration between BioArctic Neuroscience and Eisai without the safety concerns seen in previous phase 1 and 2a trials. Experiences from the field indicate the importance of initiating treatment early in the course of the disease and of enriching the trial population by improving the diagnostic accuracy. BAN2401 is a promising candidate for A? immunotherapy in early AD. Other encouraging efforts in immunotherapy as well as in the small-molecule field offer hope for new innovative therapies for AD in the future.
Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls.
Alzheimer's disease (AD) has been associated with increased local inflammation in the affected brain regions, and in some studies also with elevated levels of proinflammatory cytokines in peripheral blood. Cytomegalovirus (CMV) is known to promote a more effector-oriented phenotype in the T-cell compartment, increasing with age. The aim of this study was to investigate the inflammatory response of peripheral blood mononuclear cells (PBMCs) from AD patients and non-demented (ND) controls. Using a multiplex Luminex xMAP assay targeting GM-CSF, IFN-?, IL-1?, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-?, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid ? (A?) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-? levels after anti-CD3/CD28 and CMV pp65 but not after A? stimulation, compared to CMV seropositive ND controls. When analysing IFN-? response to anti-CD3/CD28 stimulation on a subgroup level, CMV seropositive AD subjects presented with higher levels compared to both CMV seronegative AD and CMV seropositive ND subjects. Taken together, our data from patients with clinically manifest AD suggest a possible role of CMV as an inflammatory promoter in AD immunology. Further studies of AD patients at earlier stages of disease, could provide better insight into the pathophysiology.
Aggregated ?-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinsons disease and dementia with Lewy bodies. Experimental evidence indicates that ?-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of ?-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when ?-synuclein oligomers were added to monomeric ?-synuclein. In contrast, exogenously added ?-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing ?-synuclein that were treated with the oligomers displayed reduced ?-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected ?-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of ?-synuclein oligomers into the neocortex of ?-synuclein transgenic mice did not induce formation of proteinase K resistant ?-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced ?-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.
Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease.
The amyloid hypothesis in Alzheimer disease (AD) considers amyloid ? peptide (A?) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated A? peptides especially N-truncated pyroglutamate A?pE3-42 have been extensively studied. Together with full-length A?1-42 and A?1-40, N-truncated A?pE3-42 and A?4-42 are major variants in AD brain. Although A?4-42 has been known for a much longer time, there is a lack of studies addressing the question whether A?pE3-42 or A?4-42 may precede the other in Alzheimers disease pathology.
The Arctic mutation (p.E693G/p.E22G)fs within the ?-amyloid (A?) region of the ?-amyloid precursor protein gene causes an autosomal dominant disease with clinical picture of typical Alzheimers disease. Here we report the special character of Arctic AD neuropathology in four deceased patients.
Inclusions of intraneuronal alpha-synuclein (?-synuclein) can be detected in brains of patients with Parkinsons disease and dementia with Lewy bodies. The aggregation of ?-synuclein is a central feature of the disease pathogenesis. Among the different ?-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large ?-synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinsons disease and dementia with Lewy bodies and detect pathology earlier in ?-synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of ?-synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of ?-synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective ?-synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.
Cytomegalovirus (CMV) has been suggested as a contributing force behind the impaired immune responsiveness in the elderly, with decreased numbers of naïve T-cells and an increased proportion of effector T-cells. Immunological impairment is also implicated as a part of the pathogenesis in Alzheimers disease (AD). The aim of this study was to investigate whether AD patients present with a different CMV-specific CD8 immune profile compared to non-demented controls. Blood samples from 50 AD patients and 50 age-matched controls were analysed for HLA-type, CMV serostatus and systemic inflammatory biomarkers. Using multi-colour flow cytometry, lymphocytes from peripheral blood mononuclear cells were analysed for CMV-specific CD8 immunity with MHC-I tetramers A01, A02, A24, B07, B08 and B35 and further classified using CD27, CD28, CD45RA and CCR7 antibodies. Among CMV seropositive subjects, patients with AD had significantly lower proportions of CMV-specific CD8 T-cells compared to controls, 1.16 % vs. 4.13 % (p=0.0057). Regardless of dementia status, CMV seropositive subjects presented with a lower proportion of naïve CD8 cells and a higher proportion of effector CD8 cells compared to seronegative subjects. Interestingly, patients with AD showed a decreased proportion of CMV-specific CD8 cells but no difference in general CD8 differentiation.
Calcium signaling in the brain is fundamental to the learning and memory process and there is evidence to suggest that its dysfunction is involved in the pathological pathways underlying Alzheimers disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca(2+)-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca(2+) permeability and is associated with an earlier age-at-onset of AD. To investigate the role of CALHM1 variants in early-onset AD (EOAD), we sequenced all CALHM1 coding regions in three independent series comprising 284 EOAD patients and 326 controls. Two missense mutations in patients (p.G330D and p.R154H) and one (p.A213T) in a control individual were identified. Calcium imaging analyses revealed that while the mutation found in a control (p.A213T) behaved as wild-type CALHM1 (CALHM1-WT), a complete abolishment of the Ca(2+) influx was associated with the mutations found in EOAD patients (p.G330D and p.R154H). Notably, the previously reported p. P86L mutation was associated with an intermediate Ca(2+) influx between the CALHM1-WT and the p.G330D and p.R154H mutations. Since neither expression of wild-type nor mutant CALHM1 affected amyloid ß-peptide (Aß) production or Aß-mediated cellular toxicity, we conclude that rare genetic variants in CALHM1 lead to Ca(2+) dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.
Frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions, and linkage to chromosome 17 was recently found to be caused by mutations in the progranulin (PGRN) gene. In this study, we screened a group of 51 FTLD patients for PGRN mutations and identified a novel exon 6 splice donor site deletion (IVS6+5_8delGTGA) in 2 unrelated patients. This mutation displayed an altered splicing pattern generating 2 aberrant transcripts and causing frameshifts of the coding sequence, premature termination codons, and a near absence of PGRN mRNA from the mutated alleles most likely through nonsense-mediated decay. The subsequent PGRN haploinsufficiency is consistent with previously described PGRN mutations. We present a molecular characterization of the IVS6+5_8delGTGA mutation and also describe clinical and neuropathologic features from the 2 patients carrying this PGRN mutation. From the screening of these 51 FTLD patients, we could also identify the earlier reported mutation Gln130fs, and several coding sequence variants that are most likely nonpathogenic.
The Arctic (p. E693G) mutation in the amyloid-? precursor protein (A?PP) facilitates amyloid-? (A?) protofibril formation and generates clinical symptoms of Alzheimers disease (AD). Here, molecular details of A? in post mortem brain were investigated with biochemical and morphological techniques. The basic structure of Arctic plaques resembled cotton wool plaques. However, they appeared ring-formed with A?42-specific antibodies, but were actually targetoid, since the periphery and center of many parenchymal A? deposits stained differently with mid-domain, N- and C-terminal A? antibodies. A? fibrils were similar in shape, albeit shorter than in sporadic AD brain, when examined by electron microscopy. A?wild-type and A?arctic codeposited and parenchymal deposits were highly enriched in both N- and C-terminally truncated A?. In contrast, cerebral amyloid angiopathy (CAA) contained a substantial amount of A?1-40. The absence of plaques with cores of fibrillary A? might be due to the scarcity of full-length A?, although other mechanisms could be involved. Our findings are discussed in relation to mechanisms and relevance of amyloid formation and to the clinical features of AD.
Deposition of fibrillar ?-synuclein as Lewy bodies is the neuropathological hallmark of Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Apart from ?-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in ?-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and ?-synuclein in brain tissue from PD and DLB patients as well as in a cell model for ?-synuclein aggregation. Moreover, the potential effect of gelsolin on ?-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with ?-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in ?-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of ?-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the ?-synuclein aggregation process.
Recent research implicates soluble aggregated forms of ?-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinsons disease and related disorders. The pathway by which ?-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing ?-synuclein fused to hemi:GFP constructs to study the effects of ?-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective ?-synuclein antibodies reduced the extent of ?-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower ?-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of ?-synuclein, thus providing further support for passive immunization against diseases with ?-synuclein pathology.
Humans carrying the prevalent rs9939609 A allele of the fat mass and obesity-associated (FTO) gene are more susceptible to developing obesity than noncarries. Recently, polymorphisms in the FTO gene of elderly subjects have also been linked to a reduced volume in the frontal lobe as well as increased risk for incident Alzheimer disease. However, so far there is no evidence directly linking the FTO gene to functional cognitive processes. Here we examined whether the FTO rs9939609 A allele is associated with verbal fluency performance in 355 elderly men at the age of 82 years who have no clinically apparent cognitive impairment. Retrieval of verbal memory is a good surrogate measure reflecting frontal lobe functioning. Here we found that obese and overweight but not normal weight FTO A allele carriers showed a lower performance on verbal fluency than non-carriers (homozygous for rs9939609 T allele). This effect was not observed for a measure of general cognitive performance (i.e., Mini-Mental State Examination score), thereby indicating that the FTO gene primarily affects frontal lobe-dependent cognitive processes in elderly men.
The identification of disease-causing mutations in Alzheimers disease has contributed greatly to the understanding of the pathogenesis of this disease. The amyloid-? (A?) peptide has come into focus and is believed to be central to the pathogenesis of Alzheimers disease. With only symptomatic treatment available, efforts to develop new therapeutics aimed at lowering the amount of A? peptides in the affected brain have intensified. In particular, immunotherapy against A? peptides has attracted considerable interest, as it offers the possibility to generate highly specific molecules targeting highly specific moieties. Due to intense research efforts and massive investments at universities and in the pharmaceutical industry, the outlook for patients and their relatives has never been brighter.
N-terminally truncated A? peptides starting with pyroglutamate (A?pE3) represent a major fraction of all A? peptides in the brain of Alzheimer disease (AD) patients. A?pE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length A?. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of A?pE3 and studied the potential involvement of oligomeric A?pE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall A? plaque load and A?pE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight A?pE3 oligomers.
Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimers disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.
Oxidative stress has been implicated in the etiology of neurodegenerative disorders with ?-synuclein pathology. Lipid peroxidation products such as 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE) can covalently modify and structurally alter proteins. Herein, we have characterized ONE- or HNE-induced ?-synuclein oligomers. Our results demonstrate that both oligomers are rich in ?-sheet structure and have a molecular weight of about 2000 kDa. Atomic force microscopy analysis revealed that ONE-induced ?-synuclein oligomers were relatively amorphous, with a diameter of 40-80 nm and a height of 4-8 nm. In contrast, the HNE-induced ?-synuclein oligomers had a protofibril-like morphology with a width of 100-200 nm and a height of 2-4 nm. Furthermore, neither oligomer type polymerized into amyloid-like fibrils despite prolonged incubation. Although more SDS and urea stable, because of a higher degree of cross-linking, ONE-induced ?-synuclein oligomers were less compact and more sensitive to proteinase K treatment. Finally, both ONE- and HNE-induced ?-synuclein oligomers were cytotoxic when added exogenously to a neuroblastoma cell line, but HNE-induced ?-synuclein oligomers were taken up by the cells to a significantly higher degree. Despite nearly identical chemical structures, ONE and HNE induce the formation of off-pathway ?-synuclein oligomers with distinct biochemical, morphological, and functional properties.
Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A? peptides are cornerstones in the pathology of Alzheimers disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A? protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.
We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Abeta) peptides from their aggregates. From solutions of Abeta(1-40) or Abeta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of Abeta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for Abeta(1-40) and Abeta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the Abeta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the Abeta-selective antibody mAb1C3, where a monomeric Abeta(1-16) peptide was added to the protofibril preparation. Abeta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.
Traumatic brain injury (TBI) in the mouse results in the rapid appearance of scattered clusters of cells expressing the chemokine Cxcl10 in cortical and subcortical areas. To extend the observation of this unique pattern, we used neuropathological mouse models using quantitative reverse transcriptase-polymerase chain reaction, gene array analysis, in-situ hybridization and flow cytometry. As for TBI, cell clusters of 150-200 mum expressing Cxcl10 characterize the cerebral cortex of mice carrying a transgene encoding the Swedish mutation of amyloid precursor protein, a model of amyloid Alzheimer pathology. The same pattern was found in experimental autoimmune encephalomyelitis in mice modelling multiple sclerosis. In contrast, mice carrying a SOD1(G93A) mutant mimicking amyotrophic lateral sclerosis pathology lacked such cell clusters in the cerebral cortex, whereas clusters appeared in the brainstem and spinal cord. Mice homozygous for a null mutation of the Cxcl10 gene did not show detectable levels of Cxcl10 transcript after TBI, confirming the quantitative reverse transcriptase-polymerase chain reaction and in-situ hybridization signals. Moreover, unbiased microarray expression analysis showed that Cxcl10 was among 112 transcripts in the neocortex upregulated at least threefold in both TBI and ageing TgSwe mice, many of them involved in inflammation. The identity of the Cxcl10(+) cells remains unclear but flow cytometry showed increased numbers of activated microglia/macrophages as well as myeloid dendritic cells in the TBI and experimental autoimmune encephalomyelitis models. It is concluded that the Cxcl10(+) cells appear in the inflamed central nervous system and may represent a novel population of cells that it may be possible to target pharmacologically in a broad range of neurodegenerative conditions.
Transgenic animals expressing mutant human amyloid precursor protein (APP) are used as models for Alzheimer disease (AD). Ideally, behavioral tests improve the predictive validity of studies on animals by mirroring the functional impact of AD-like neuropathology. Learning and memory studies in APP transgenic models have been difficult to replicate. Standardization of procedures, automatization or improved protocol design can improve reproducibility. Here the IntelliCage, an automated system, was used for behavioral testing of APP female transgenic mice with both the Arctic and Swedish mutations, the tg-ArcSwe model. Protocols covering exploration, operant learning, place learning and extinction of place preference as well as passive avoidance tests were used for longitudinal characterization of behavior. Differences in exploratory activity were significant at four months of age, when plaque-free tg-ArcSwe mice visited less frequently the IntelliCage corners and initially performed fewer visits with licks compared to non-tg animals, inside the new environment. Fourteen months old tg-ArcSwe mice required a longer time to re-habituate to the IntelliCages than non-tg mice. At both ages tg-ArcSwe mice perseverated in place preference extinction test. Fourteen months old tg-ArcSwe mice were impaired in hippocampus-dependent spatial passive avoidance learning. This deficit was found to inversely correlate to calbindin-D28k immunoreactivity in the polymorphic layer of the dentate gyrus. Reduced water intake and body weight were observed in 4 months old tg-ArcSwe animals. The body weight difference increased with age. Thus behavioral and metabolic changes in the tg-ArcSwe APP model were detected using the IntelliCage, a system which provides the opportunity for standardized automated longitudinal behavioral phenotyping.
In the early 1990s, breakthrough discoveries on the genetics of Alzheimers disease led to the identification of missense mutations in the amyloid-beta precursor protein gene. Research findings quickly followed, giving insights into molecular pathogenesis and possibilities for the development of new types of animal models. The complete toolbox of transgenic techniques, including pronuclear oocyte injection and homologous recombination, has been applied in the Alzheimers disease field, to produce overexpressors, knockouts, knockins and regulatable transgenics. Transgenic models have dramatically advanced our understanding of pathogenic mechanisms and allowed therapeutic approaches to be tested. Following a brief introduction to Alzheimers disease, various nontransgenic and transgenic animal models are described in terms of their values and limitations with respect to pathogenic, therapeutic and functional understandings of the human disease.
Amyloid-? (A?) protofibrils are neurotoxic soluble intermediates in the A? aggregation process eventually forming senile plaques in Alzheimers disease. This A? species is a potential biomarker for Alzheimers disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent A? antibodies specific for A? protofibrils.
Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimers disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.
Recently, the P86L alteration in CALHM1 (calcium homeostasis modulator-1) was reported to be associated with Alzheimers disease (AD). Moreover, the risk allele increased amyloid-beta (A beta) levels in conditioned media from cultured cells. Therefore, we hypothesized that CALHM1 P86L may modulate A beta or tau levels in cerebrospinal fluid (CSF). Nearly 200 individuals with AD or other cognitive disorders were included for CSF analysis and CALHM1 genotyping. No significant differences in CSF levels of A beta 42, tau or phospho-tau were found across the various CALHM1 genotypes. In conclusion, we found no evidence that CALHM1 P86L is associated with altered CSF levels of the investigated AD biomarkers.
Amyloid-beta (Abeta) is a major drug target in Alzheimers disease. Here, we demonstrate that deposited Abeta is SDS insoluble in tgAPP-ArcSwe, a transgenic mouse model harboring the Arctic (E693G) and Swedish (KM670/671NL) APP mutations. Formic acid was needed to extract the majority of deposited Abeta in both tgAPP-ArcSwe and Alzheimers disease brain, but not in a commonly used type of mouse model with the Swedish mutation alone. Interestingly, the insoluble state of Arctic Abeta was determined early on and did not gradually evolve with time. In tgAPP-ArcSwe, Abeta plaques displayed a patchy morphology with bundles of Abeta fibrils, whereas amyloid cores in tgAPP-Swe were circular with radiating fibrils. Amyloid was more densely stacked in tgAPP-ArcSwe, as demonstrated with a conformation sensitive probe. A reduced increase in plasma Abeta was observed following acute administration of an Abeta antibody in tgAPP-ArcSwe, results that might imply reduced brain to plasma Abeta efflux. TgAPP-ArcSwe, with its insoluble state of deposited Abeta, could serve as a complementary model to better predict the outcome of clinical trials.
Alterations in gene dosage have recently been associated with neurodegenerative disorders, such as Alzheimers disease and Parkinsons disease, and deletions of the progranulin (PGRN) locus were recently described in patients with frontotemporal lobar degeneration (FTLD). FTLD is a genetically complex neurodegenerative disorder with mutations in the PGRN and the microtubule-associated protein tau (MAPT) genes being the most common known causes of familial FTLD. In this study, we investigated 39 patients with FTLD, previously found negative for mutations in PGRN and MAPT, for copy number alterations of these 2 genes.
The presence of Abeta(pE3) (N-terminal truncated Abeta starting with pyroglutamate) in Alzheimers disease (AD) has received considerable attention since the discovery that this peptide represents a dominant fraction of Abeta peptides in senile plaques of AD brains. This was later confirmed by other reports investigating AD and Downs syndrome postmortem brain tissue. Importantly, Abeta(pE3) has a higher aggregation propensity, and stability, and shows an increased toxicity compared to full-length Abeta. We have recently shown that intraneuronal accumulation of Abeta(pE3) peptides induces a severe neuron loss and an associated neurological phenotype in the TBA2 mouse model for AD. Given the increasing interest in Abeta(pE3), we have generated two novel monoclonal antibodies which were characterized as highly specific for Abeta(pE3) peptides and herein used to analyze plaque deposition in APP/PS1KI mice, an AD model with severe neuron loss and learning deficits. This was compared with the plaque pattern present in brain tissue from sporadic and familial AD cases. Abundant plaques positive for Abeta(pE3) were present in patients with sporadic AD and familial AD including those carrying mutations in APP (arctic and Swedish) and PS1. Interestingly, in APP/PS1KI mice we observed a continuous increase in Abeta(pE3) plaque load with increasing age, while the density for Abeta(1-x ) plaques declined with aging. We therefore assume that, in particular, the peptides starting with position 1 of Abeta are N-truncated as disease progresses, and that, Abeta(pE3) positive plaques are resistant to age-dependent degradation likely due to their high stability and propensity to aggregate.
Mutations in the progranulin (PGRN) gene have recently been identified in families with frontotemporal lobar degeneration and ubiquitin-positive brain inclusions linked to chromosome 17q21. We have previously described a Swedish family displaying frontotemporal dementia with rapid progression and linkage to chromosome 17q21. In this study, we performed an extended clinical and neuropathological investigation of affected members of the family and a genetic analysis of the PGRN gene. There was a large variation of the initial presenting symptoms in this family, but common clinical features were non-fluent aphasia and loss of spontaneous speech as well as personality and behavioural changes. Mean age at onset was 54 years with disease duration of close to 4 years. Neuropathological examination revealed frontotemporal neurodegeneration with ubiquitin and TAR DNA binding protein-43 immunoreactive intraneuronal inclusions. Mutation screening of the PGRN gene identified a 1 bp deletion in exon 1 causing a frameshift of the coding sequence and introducing a premature termination codon in exon 2 (Gly35GlufsX19). Analysis of PGRN messenger RNA (mRNA) levels revealed a considerable decrease in lymphoblasts from mutation carriers and fragment size separation, and sequence analysis confirmed that the mutated mRNA allele was almost absent in these samples. In conclusion, the PGRN Gly35fs mutation causes frontotemporal dementia with variable clinical presentation in a large Swedish family, most likely through nonsense-mediated decay of mutant PGRN mRNA and resulting haploinsufficiency.
Intraneuronal punctate immunostaining in Alzheimers disease brain and amyloid-beta precursor protein (APP) transgenic mice has been suggested to represent Abeta, but this is somewhat controversial. Here we show that both biochemical Abeta levels and intraneuronal immunostaining are reduced in APP transgenic mice when gamma-secretase is inhibited. Moreover, BACE-1 deficient APP transgenic mice show neither Abeta production nor intraneuronal immunostaining. Our findings suggest that the punctate immunostaining with APP antibodies is due to Abeta that has accumulated inside neurons. Similar type of intraneuronal Abeta accumulation, which precedes senile plaque formation, may link Abeta to tauopathy and neurodegeneration in Alzheimers disease pathogenesis.
We describe a case of late onset neurodegeneration with brain iron accumulation (NBIA) presenting as frontotemporal dementia (FTD) with amyotrophic lateral sclerosis (ALS). A male patient presented at age 66 with change of personality: disinhibition, emotional blunting, and socially inappropriate behavior, coupled with dysarthria, dystonia, and corticospinal tract involvement. Magnetic resonance imaging showed general cortical atrophy, iron deposits in the globus pallidus, and the "eye of the tiger" sign. Neuropsychologic performance was globally reduced, especially executive functions. Fluorodeoxyglucose positron emission tomography showed hypometabolism predominantly in frontal and temporal areas. Repeated neurophysiologic examinations showed signs of chronic denervation. The patient was diagnosed with NBIA but fulfilled consensus criteria for FTD and had a clinical picture of ALS, without neurophysiologic confirmation. Our finding introduces NBIA as a possible cause of FTD and as a differential diagnosis of the FTD-ALS complex.
Common FTO (fat mass and obesity associated) gene variants have recently been strongly associated with body mass index and obesity in several large studies. Here we set out to examine the association of the FTO variant rs9939609 with BMI in a 32 year follow up study of men born 1920-1924. Moreover, we analyzed the effect of physical activity on the different genotypes.
Human genetics link Alzheimers disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimers disease brain.
In the present work, we investigated the level of IgM autoantibodies directed against different A? epitopes as potential diagnostic biomarker for Alzheimers disease (AD). Anti-A? autoantibody levels were measured in 75 plasma samples from patients with AD, individuals with mild cognitive impairment (MCI), and healthy age- and sex-matched controls (HC). To validate the presence of anti-A? IgMs, pooled plasma samples were subjected to gel-filtration analysis. The mean level of pGluA?-IgM (N-terminal truncated starting at position three with pyroglutamate) was significantly decreased in AD patients as compared to HC. In the group of MCI patients there was a significant positive correlation between pGluA?-IgM and cognitive decline analyzed by MMSE (rho = 0.58, d.f. = 13, p = 0.022). These observations indicate that the level of IgM autoantibodies against pGluA? is a promising plasma biomarker for AD and correlates with the cognitive status of individuals at risk to develop AD.
Six young related pre-symptomatic carriers of a His163Tyr mutation in the presenilin 1 gene who will develop early onset familial Alzheimers disease (eoFAD), and a control group of 23 non-carriers underwent (18)F-fluorodeoxyglucose positron emission tomography (FDG PET). The mutation carriers were followed-up after 2 years. Multivariate analysis showed clear separation of carriers from non-carriers on both occasions, with the right thalamus being the region contributing most to group differentiation. Statistical parametric mapping (SPM) revealed in the carriers non-significantly lower thalamic cerebral glucose metabolism (CMRglc) at baseline and significantly decreased CMRglc in the right thalamus at follow-up. One mutation carrier was followed-up with FDG PET 10 years after baseline and showed reductions in cognition and CMRglc in the posterior cingulate and the frontal cortex. This subject was diagnosed with AD 1 year later and assessed with an additional FDG as well as an (11)C-PIB PET scan 12 years after baseline. Global cortical CMRglc and cognition were distinctly decreased. PIB binding was comparable with sporadic AD patterns but showing slightly higher striatal levels.
Neurofilament proteins (Nf) are highly specific biomarkers for neuronal death and axonal degeneration. As these markers become more widely used, an inter-laboratory validation study is required to identify assay criteria for high quality performance.
The lowering of natively analyzed Abeta42 in cerebrospinal fluid (CSF) is used as a diagnostic tool in Alzheimers disease (AD). The presence of Abeta oligomers can interfere with such analyses causing underestimation of Abeta levels due to epitope masking. The aim was to investigate if the lowering of CSF Abeta42 seen in AD is caused by oligomerization.
Oligomeric assemblies of amyloid-beta (Abeta) are suggested to be central in the pathogenesis of Alzheimers disease because levels of soluble Abeta correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Abeta species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long-term potentiation. The tg-ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Abeta protofibrils in the brain, making tg-ArcSwe a highly suitable model for investigating the pathogenic role of these Abeta assemblies. In the present study, we estimated Abeta protofibril levels in the brain and cerebrospinal fluid of tg-ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg-ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg-ArcSwe mice with considerable plaque deposition, Abeta protofibrils were approximately 50% higher than in younger mice, whereas levels of total Abeta were exponentially increased. Young tg-ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Abeta protofibrils, but not with total Abeta levels. We conclude that Abeta protofibrils accumulate in an age-dependent manner in tg-ArcSwe mice, although to a far lesser extent than total Abeta. Our findings suggest that increased levels of Abeta protofibrils could result in spatial learning impairment.
Increased cerebrospinal fluid (CSF) tau, decreased CSF amyloid-beta42 (Abeta42) and the apolipoprotein E gene (APOE) epsilon4 allele predict progression from mild cognitive impairment (MCI) to Alzheimers disease (AD). Here, we investigated these markers to assess their predictive value and influence on the rate of disease progression.
It has previously been shown that immune complexes (IC) of a given biomarker with class M immunoglobulins (IgM) provide better performances compared to the unbound biomarker in a number of cancer entities. In the present work, we investigated IC of IgM-Abeta as a potential biomarker for Alzheimers disease (AD). Abeta-IgM concentration has been measured in 75 plasma samples from patients with AD, individuals with mild cognitive impairment (MCI), and healthy age- and sex-matched controls (HC). To characterize the fractions associated with Abeta, pooled plasma samples were subjected to gel-filtration analysis. Size-separated fractions were analyzed for the presence of Abeta using a sandwich ELISA assay. A strong reactivity was observed in the high molecular weight IgM (>500 kDa) and 150 kDa (IgG) fractions indicating that blood Abeta is strongly associated with antibodies. Using an ELISA assay detecting Abeta-IgM complexes, we observed that high levels of Abeta-IgMs were detectable in HC and MCI patients; however, there was no significant difference to the AD group.
Recently, the aldehyde 4-oxo-2-nonenal (ONE) was identified as a product of lipid peroxidation and found to be an effective protein modifier. In this in vitro study we investigated structural implications of the interaction between ONE and alpha-synuclein, a protein which forms intraneuronal inclusions in neurodegenerative disorders such as Parkinsons disease and dementia with Lewy bodies. Our results demonstrate that ONE induced an almost complete conversion of monomeric alpha-synuclein into 40-80 nm wide and 6-8 nm high soluble beta-sheet-rich oligomers with a molecular weight of approximately 2000 kDa. Furthermore, the ONE-induced alpha-synuclein oligomers displayed a high stability and were not sensitive to treatment with sodium dodecyl sulfate, indicating that ONE stabilized the oligomers by cross-linking individual alpha-synuclein molecules. Despite prolonged incubation the oligomers did not continue to aggregate into a fibrillar state, thus suggesting that these alpha-synuclein species were not on a fibrillogenic pathway.
Genetic factors influencing common complex conditions have proven difficult to identify, and data from numerous investigations have provided incomplete conclusions as to the identity of these genes. Here we aimed to identify susceptibility genes for late-onset Alzheimers disease (AD).
Parkinsons disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of ?-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.
Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimers disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-? peptide, implicated in Alzheimers disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-? elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimers disease.
Recent genome-wide association studies have described many loci implicated in type 2 diabetes (T2D) pathophysiology and ?-cell dysfunction but have contributed little to the understanding of the genetic basis of insulin resistance. We hypothesized that genes implicated in insulin resistance pathways might be uncovered by accounting for differences in body mass index (BMI) and potential interactions between BMI and genetic variants. We applied a joint meta-analysis approach to test associations with fasting insulin and glucose on a genome-wide scale. We present six previously unknown loci associated with fasting insulin at P < 5 × 10(-8) in combined discovery and follow-up analyses of 52 studies comprising up to 96,496 non-diabetic individuals. Risk variants were associated with higher triglyceride and lower high-density lipoprotein (HDL) cholesterol levels, suggesting a role for these loci in insulin resistance pathways. The discovery of these loci will aid further characterization of the role of insulin resistance in T2D pathophysiology.
Soluble amyloid-? (A?) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimers Disease (AD). To investigate the properties of biologically relevant A? species, brain extracts from amyloid ? protein precursor (A?PP) transgenic mice and AD patients as well as synthetic A? preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic A?42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic A? aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In A?PP transgenic mouse brain, the most abundant soluble A? species were found in fraction 2 and consisted mainly of A?40. Also in AD brains, A? was mainly found in fraction 2 but primarily as A?42. All biologically derived A? from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble A?, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80-500 kDa synthetic A? protofibrils and were equally detected with mAb158.
Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ?60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ?55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7-19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.