Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights.
Understanding how FI and FG levels are regulated is important because their derangement is a feature of T2D. Despite recent success from GWAS in identifying regions of the genome influencing glycemic traits, collectively these loci explain only a small proportion of trait variance. Unlocking the biological mechanisms driving these associations has been challenging because the vast majority of variants map to non-coding sequence, and the genes through which they exert their impact are largely unknown. In the current study, we sought to increase our understanding of the physiological pathways influencing both traits using exome-array genotyping in up to 33,231 non-diabetic individuals to identify coding variants and consequently genes associated with either FG or FI levels. We identified novel association signals for both traits including the receptor for GLP-1 agonists which are a widely used therapy for T2D. Furthermore, we identified coding variants at several GWAS loci which point to the genes underlying these association signals. Importantly, we found that multiple coding variants in G6PC2 result in a loss of protein function and lower fasting glucose levels.
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.
In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10−6) and 14 (IGHV1-67 p = 7.9×10−8) which indexed novel susceptibility loci.
The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.
Eleven susceptibility loci for late-onset Alzheimer’s disease (LOAD) were identified by previous studies; however, a large portion of the genetic risk for this disease remains unexplained. We conducted a large, two-stage meta-analysis of genome-wide association studies (GWAS) in individuals of European ancestry. In stage 1, we used genotyped and imputed data (7,055,881 SNPs) to perform meta-analysis on 4 previously published GWAS data sets consisting of 17,008 Alzheimer’s disease cases and 37,154 controls. In stage 2,11,632 SNPs were genotyped and tested for association in an independent set of 8,572 Alzheimer’s disease cases and 11,312 controls. In addition to the APOE locus (encoding apolipoprotein E), 19 loci reached genome-wide significance (P < 5 × 10−8) in the combined stage 1 and stage 2 analysis, of which 11 are newly associated with Alzheimer’s disease.
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-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid β (Aβ) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-γ levels after anti-CD3/CD28 and CMV pp65 but not after Aβ stimulation, compared to CMV seropositive ND controls. When analysing IFN-γ 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.
According to the modified amyloid hypothesis the main event in the pathogenesis of Alzheimer’s disease (AD) is the deposition of neurotoxic amyloid β-peptide (Aβ) within neurons. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid precursor protein (APP). Vast evidence suggests that Aβx-42 isoforms play an important role triggering neurodegeneration due to its high abundance, amyloidogenic propensity and toxicity. Although N-truncated and Aβx-42 species have been pointed as crucial players in AD etiology, the Aβ5-x isoforms have not received much attention.
The present study is the first to show immunohistochemical evidence of Aβ5-x in familial cases of AD (FAD) and its distribution in APP/PS1KI, 5XFAD and 3xTG transgenic mouse models. In order to probe Aβ5-x peptides we generated the AB5-3 antibody. Positive plaques and congophilic amyloid angiopathy (CAA) were observed among all the FAD cases tested carrying either APP or presenilin 1 (PS1) mutations and most of the sporadic cases of AD (SAD). Different patterns of Aβ5-x distribution were found in the mouse models carrying different combinations of autosomal mutations in the APP, PS1 and Tau genes. All of them showed extracellular Aβ deposits but none CAA. Additionally, they were all affected by a severe amyloid pathology in the hippocampus among other areas. Interestingly, neither 5XFAD nor APP/PS1KI showed any evidence for intraneuronal Aβ5-x.
Different degrees of Aβ5-x accumulations can be found in the transgenic AD mouse models and human cases expressing the sporadic or the familial form of the disease. Due to the lack of intracellular Aβ5-x, these isoforms might not be contributing to early mechanisms in the cascade of events triggering AD pathology. Brain sections obtained from SAD cases showed higher Aβ5-x–immunoreactivity in vascular deposits than in extracellular plaques, while both are equally important in the FAD cases. The difference may rely on alternative mechanisms involving Aβ5-x peptides and operating in a divergent way in the late and early onset forms of the disease.
Aβ5-x; Amyloid plaques; 5XFAD; 3xTG; APP/PS1KI; Vascular deposits; N-truncated Aβ
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.
Gene-targeting was used to delete exon 10 in murine tau on both alleles (E10−/−) to study its functional role. Moreover, mice devoid of exon 10 (E10+/−) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10−/− mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/− and wild-type mice, and their muscular grip strength was less than that of E10+/− mice. The performance of E10+/− mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.
Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions.
Alzheimer’s disease; Tau; Knockout mice; Alternative splicing; Microtubule
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 Alzheimer’s 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 Alzheimer’s disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca2+-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.
The Arctic mutation (p.E693G/p.E22G)fs within the β-amyloid (Aβ) region of the β-amyloid precursor protein gene causes an autosomal dominant disease with clinical picture of typical Alzheimer’s disease. Here we report the special character of Arctic AD neuropathology in four deceased patients.
Aβ deposition in the brains was wide-spread (Thal phase 5) and profuse. Virtually all parenchymal deposits were composed of non-fibrillar, Congo red negative Aβ aggregates. Congo red only stained angiopathic vessels. Mass spectrometric analyses showed that Aβ deposits contained variably truncated and modified wild type and mutated Aβ species. In three of four Arctic AD brains, most cerebral cortical plaques appeared targetoid with centres containing C-terminally (beyond aa 40) and variably N-terminally truncated Aβ surrounded by coronas immunopositive for Aβx-42. In the fourth patient plaque centres contained almost no Aβ making the plaques ring-shaped. The architectural pattern of plaques also varied between different anatomic regions. Tau pathology corresponded to Braak stage VI, and appeared mainly as delicate neuropil threads (NT) enriched within Aβ plaques. Dystrophic neurites were scarce, while neurofibrillary tangles were relatively common. Neuronal perikarya within the Aβ plaques appeared relatively intact.
In Arctic AD brain differentially truncated abundant Aβ is deposited in plaques of variable numbers and shapes in different regions of the brain (including exceptional targetoid plaques in neocortex). The extracellular non-fibrillar Aβ does not seem to cause overt damage to adjacent neurons or to induce formation of neurofibrillary tangles, supporting the view that intracellular Aβ oligomers are more neurotoxic than extracellular Aβ deposits. However, the enrichment of NTs within plaques suggests some degree of intra-plaque axonal damage including accumulation of hp-tau, which may impair axoplasmic transport, and thereby contribute to synaptic loss. Finally, similarly as the cotton wool plaques in AD resulting from exon 9 deletion in the presenilin-1 gene, the Arctic plaques induced only modest glial and inflammatory tissue reaction.
Familial Alzheimer’s disease; Arctic AβPP mutation; β-amyloid peptide; Mass spectrometry; Truncation of Aβ; Topography of Aβ; Hyperphosphorylated tau; Neuronal damage
The amyloid hypothesis in Alzheimer disease (AD) considers amyloid β peptide (Aβ) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated Aβ peptides especially N-truncated pyroglutamate AβpE3-42 have been extensively studied. Together with full-length Aβ1–42 and Aβ1–40, N-truncated AβpE3-42 and Aβ4–42 are major variants in AD brain. Although Aβ4–42 has been known for a much longer time, there is a lack of studies addressing the question whether AβpE3-42 or Aβ4–42 may precede the other in Alzheimer’s disease pathology.
Using different Aβ antibodies specific for the different N-termini of N-truncated Aβ, we discovered that Aβ4-x preceded AβpE3-x intraneuronal accumulation in a transgenic mouse model for AD prior to plaque formation. The novel Aβ4-x immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from N-truncated Aβ species. While NT4X-167 significantly rescued Aβ4–42 toxicity in vitro no beneficial effect was observed against Aβ1–42 or AβpE3-42 toxicity. Phenylalanine at position four of Aβ was imperative for antibody binding, because its replacement with alanine or proline completely prevented binding. Although amyloid plaques were observed using NT4X-167 in 5XFAD transgenic mice, it barely reacted with plaques in the brain of sporadic AD patients and familial cases with the Arctic, Swedish and the presenilin-1 PS1Δ9 mutation. A consistent staining was observed in blood vessels in all AD cases with cerebral amyloid angiopathy. There was no cross-reactivity with other aggregates typical for other common neurodegenerative diseases showing that NT4X-167 staining is specific for AD.
Aβ4-x precedes AβpE3-x in the well accepted 5XFAD AD mouse model underlining the significance of N-truncated species in AD pathology. NT4X-167 therefore is the first antibody reacting with Aβ4-x and represents a novel tool in Alzheimer research.
Pyroglutamate Abeta; Abeta oligomer; Toxicity; Arctic; Swedish; Presenilin-1; 5XFAD; Transgenic mouse model; Familial Alzheimer’s disease; Sporadic Alzheimer’s disease; Abeta 4-40; Abeta 4–42
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.
frontotemporal lobar degeneration; frontotemporal dementia; progranulin; ubiquitin; TDP-43
Recent genome-wide association studies have described many loci implicated in type 2 diabetes (T2D) pathophysiology and beta-cell dysfunction, but contributed little to our understanding of the genetic basis of insulin resistance. We hypothesized that genes implicated in insulin resistance pathways may be uncovered by accounting for differences in body mass index (BMI) and potential interaction between BMI and genetic variants. We applied a novel joint meta-analytical approach to test associations with fasting insulin (FI) and glucose (FG) on a genome-wide scale. We present six previously unknown FI loci at P<5×10−8 in combined discovery and follow-up analyses of 52 studies comprising up to 96,496non-diabetic individuals. Risk variants were associated with higher triglyceride and lower HDL cholesterol levels, suggestive of a role for these FI loci in insulin resistance pathways. The localization of these additional loci will aid further characterization of the role of insulin resistance in T2D pathophysiology.
Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer’s 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-β peptide, implicated in Alzheimer’s 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-β 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 Alzheimer’s disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-012-0997-1) contains supplementary material, which is available to authorized users.
Neuroinflammation; Heparan sulfate; Heparanase; Amyloid-β; Clearance; Alzheimer’s disease
Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ 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 Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.
Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-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 α-synuclein fused to hemi:GFP constructs to study the effects of α-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 α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-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 α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.
The only established genetic determinant of non-Mendelian forms of Alzheimer’s disease (AD) is the ε4 allele of the apolipoprotein E gene (APOE). Recently, it has been reported that the P86L polymorphism of the calcium homeostasis modulator 1 gene (CALHM1) is associated with the risk of developing AD. In order to independently assess this association, we performed a meta-analysis of 7,873 AD cases and 13,274 controls of Caucasian origin (from a total of 24 centres in Belgium, Finland, France, Italy, Spain, Sweden, the UK and the USA). Our results indicate that the CALHM1 P86L polymorphism is likely not a genetic determinant of AD but may modulate age at onset by interacting with the effect of the ε4 allele of the APOE gene.
Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2) and v-myc myelocytomatosis viral oncogene homolog (MYC), were increased in Alzheimer's disease (AD) (P < .05). Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis.
Increasing age is associated with a poor prognosis following traumatic brain injury (TBI). Central nervous system axons may recover poorly following TBI due to expression of myelin-derived inhibitors to axonal outgrowth such as Nogo-A. To study the role of Nogo-A/B in the pathophysiological response of the elderly to TBI, 1-year old mice deficient in Nogo-A/B (Nogo-A/B homozygous −/− mice), Nogo-A/B heterozygous −/+ mice, and age-matched wild-type (WT) littermate controls were subjected to a controlled cortical impact (CCI) TBI. Sham-injured WT mice (7 months old) and 12 months old naïve Nogo-A/B−/− and Nogo-A/B−/+ served as controls. Neurological motor function was evaluated up to 3 weeks, and cognitive function, hemispheric tissue loss, myelin staining and hippocampal β-amyloid (Aβ) immunohistochemistry was evaluated at 4 weeks post-injury. In WT littermates, TBI significantly impaired learning ability at 4 weeks and neurological motor function up to 2 weeks post-injury and caused a significant loss of hemispheric tissue. Following TBI, Nogo-A/B−/− mice showed significantly less recovery from neurological motor deficits compared to brain-injured WT mice. Naïve Nogo-A/B−/−and Nogo-A/B−/+ mice quickly learned the MWM task in contrast to brain-injured Nogo-A/B−/− mice who failed to learn the MWM task at 4 week post-injury. Hemispheric tissue loss and cortical lesion volume was similar among the brain-injured genotypes. Neither TBI nor the absence of NogoA/B caused an increased Aβ expression. Myelin staining showed a reduced area and density in the corpus callosum in brain-injured Nogo-A/B−/−animals compared to their littermate controls. These novel and unexpected behavioral results demonstrate that the absence of Nogo-A/B may negatively influence outcome, possibly related to hypomyelination, following TBI in mice and suggest a complex role for this myelin-associated axonal growth inhibitor following TBI.
Traumatic brain injury; Nogo-A; Nogo-B; Morris Water Maze; neurological motor deficits
Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's 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 Aβ 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.
For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.
The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.
Recently, the P86L alteration in CALHM1 (calcium homeostasis modulator-1) was reported to be associated with Alzheimer’s disease (AD). Moreover, the risk allele increased amyloid-β (Aβ) levels in conditioned media from cultured cells. Therefore, we hypothesized that CALHM1 P86L may modulate Aβ 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 Aβ42, 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.
CALHM1; Calcium homeostasis modulator-1; Amyloid-β; Alzheimer’s disease; Cerebrospinal fluid; Biomarker; Total tau; Phospho-tau; Genotyping SNP
Amyloid β-peptide (Aβ) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Aβ deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Aβ plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Aβ40 dense cores of neuritic plaques, but was largely absent from diffuse Aβ42 plaques, suggesting that Aβ42 deposition may occur independently of HS. A codeposition pattern of HS with Aβ deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Aβ deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Aβ stimulation. These results suggest that HS codeposits with Aβ40 in neuritic plaques and is mainly derived from glial cells.
b-Amyloid; glial cells; heparan sulfate
Oligomeric assemblies of Amyloid-β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease, since levels of soluble Aβ much better correlate with the extent of cognitive dysfunctions than senile plaque counts do. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit maintenance of hippocampal long term potentiation. The tg-ArcSwe model, transgenic mice with the Arctic and Swedish Alzheimer mutations, expresses elevated levels of Aβ protofibrils in the brain, making tg-ArcSwe a highly suitable model to investigate the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ 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, as compared to several transgenic models lacking the Arctic mutation. In aged tg-ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50 percent higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg-ArcSwe mice showed deficits in spatial learning and individual performance in Morris water maze correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age-dependent manner in tg-ArcSwe mice, although to a far less extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.
Alzheimer’s disease; amyloid-β protofibrils; Arctic mutation; transgenic mice; spatial learning
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.
The FTO rs9936609 was genotyped using an Illumina golden gate assay. BMI was calculated using standard methods and body fat was estimated by measuring skinfold thickness using a Harpenden caliper. Physical activity was assessed using a four question medical questionnaire.
FTO rs9939609 was genotyped in 1153 elderly Swedish men taking part of a population-based cohort study, the ULSAM cohort. The risk of obesity and differences in BMI according to genotype at the ages of 50, 60, 70, 77 and 82 were investigated. We found no increased risk of obesity and no association with BMI at any age with the FTO rs9939609 variant. We found however interaction between physical activity at the age of 50 years and genotype on BMI levels (p = 0.039) and there was a clear trend towards larger BMI differences between the TT and AA carriers as well as between AT and AA carriers in the less physically active subjects.
Here we found that the well established obesity risk allele for a common variant in FTO does not associate with increased BMI levels in a Swedish population of adult men which reached adulthood before the appearance of today's obesogenic enviroment. There is an interaction between physical activity and the effect of the FTO genotype on BMI levels suggesting that lack of physical activity is a requirement for an association of FTO gene variants to obesity.
The presence of AβpE3 (N-terminal truncated Aβ starting with pyroglutamate) in Alzheimer’s disease (AD) has received considerable attention since the discovery that this peptide represents a dominant fraction of Aβ peptides in senile plaques of AD brains. This was later confirmed by other reports investigating AD and Down’s syndrome postmortem brain tissue. Importantly, AβpE3 has a higher aggregation propensity, and stability, and shows an increased toxicity compared to full-length Aβ. We have recently shown that intraneuronal accumulation of AβpE3 peptides induces a severe neuron loss and an associated neurological phenotype in the TBA2 mouse model for AD. Given the increasing interest in AβpE3, we have generated two novel monoclonal antibodies which were characterized as highly specific for AβpE3 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 AβpE3 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 AβpE3 plaque load with increasing age, while the density for Aβ1-x plaques declined with aging. We therefore assume that, in particular, the peptides starting with position 1 of Aβ are N-truncated as disease progresses, and that, AβpE3 positive plaques are resistant to age-dependent degradation likely due to their high stability and propensity to aggregate.
Transgenic mice; Arctic mutation; Swedish mutation; Presenilin-1 mutation; Sporadic Alzheimer’s disease; Pyroglutamate Abeta; Biacore; Antibody specificity