RoseMarie Brundin

Interference from Heterophilic Antibodies in Amyloid-β Oligomer ELISAs

Amyloid-β (Aβ) oligomers of different sizes and forms have recently been the focus formany Alzheimers disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Aβ species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Aβ oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples.

Association study of cholesterol-related genes in Alzheimer’s disease

Alzheimer’s disease (AD) is a genetically complex disorder, and several genes related to cholesterol metabolism have been reported to contribute to AD risk. To identify further AD susceptibility genes, we have screened genes that map to chromosomal regions with high logarithm of the odds scores for AD in full genome scans and are related to cholesterol metabolism. In a European screening sample of 115 sporadic AD patients and 191 healthy control subjects, we analyzed single nucleotide polymorphisms in 28 cholesterol-related genes for association with AD. The genes HMGCS2, FDPS, RAFTLIN, ACAD8, NPC2, and ABCG1 were associated with AD at a significance level of P ≤ 0.05 in this sample. Replication trials in five independent European samples detected associations of variants within HMGCS2, FDPS, NPC2, or ABCG1 with AD in some samples (P = 0.05 to P = 0.005). We did not identify a marker that was significantly associated with AD in the pooled sample (n = 2864). Stratification of this sample revealed an APOE-dependent association of HMGCS2 with AD (P = 0.004). We conclude that genetic variants investigated in this study may be associated with a moderate modification of the risk for AD in some samples.

Frontotemporal dementia-amyotrophic lateral sclerosis complex is simulated by neurodegeneration with brain iron accumulation.

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.

The Arctic AβPP mutation leads to Alzheimer's disease pathology with highly variable topographic deposition of differentially truncated Aβ

BackgroundThe 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.ResultsAβ 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.ConclusionsIn 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.

New Alzheimer’s disease locus on chromosome 8

Background: Family history is one of the most consistent risk factors for dementia. Therefore, analysis of families with a distinct inheritance pattern of disease can be a powerful approach for the identification of previously unknown disease genes. Objective: To map susceptibility regions for Alzheimer’s disease. Methods: A complete genome scan with 369 microsatellite markers was carried out in 12 extended families collected in Sweden. Age at disease onset ranged from 53 to 78 years, but in 10 of the families there was at least one member with age at onset of ⩽65 years. Mutations in known early-onset Alzheimer’s disease susceptibility genes have been excluded. All people were genotyped for APOE, but no clear linkage with the ε4 allele was observed. Results: Although no common disease locus could be found in all families, in two families an extended haplotype was identified on chromosome 8q shared by all affected members. In one of the families, a non-parametric multimarker logarithm of the odds (LOD) score of 4.2 (p = 0.004) was obtained and analysis based on a dominant model showed a parametric LOD score of 2.4 for this region. All six affected members of this family shared a haplotype of 10 markers spanning about 40 cM. Three affected members in another family also shared a haplotype in the same region. Conclusion: On the basis of our data, we propose the existence of a dominantly acting Alzheimer’s disease susceptibility locus on chromosome 8.

CALHM1 P86L polymorphism does not alter amyloid-beta or tau in cerebrospinal fluid

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.

No evidence of PGRN or MAPT gene dosage alterations in a collection of patients with frontotemporal lobar degeneration.

Background/Aims: Alterations in gene dosage have recently been associated with neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s 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. Methods: Gene dosage analysis of PGRN and MAPT was performed using multiplex ligation-dependent probe amplification. Results: We did not identify any PGRN or MAPT gene dosage variations in the 39 FTLD patients investigated. Conclusion: We therefore conclude that alterations in gene copy number of PGRN and MAPT are not a cause of disease in this collection of FTLD patients.

Increased Number of Plasma B Cells Producing Autoantibodies Against Aβ42 Protofibrils in Alzheimer’s Disease

Abstract The Alzheimer’s disease (AD)-related peptide amyloid-β (Aβ) has a propensity to aggregate into various assemblies including toxic soluble Aβ protofibrils. Several studies have reported the existence of anti-Aβ antibodies in humans. However, it is still debated whether levels of anti-Aβ antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma Aβ makes it difficult to reliably measure the concentration of circulating anti-Aβ antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-Aβ antibody production on a cellular level by measuring the amount of anti-Aβ antibody producing cells instead of the plasma level of anti-Aβ antibodies. To our knowledge, this is the first time the anti-Aβ antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding Aβ40 monomers, whereas the number of cells producing antibodies toward Aβ42 protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic Aβ protofibrils, which is significantly increased in AD patients.

Genetic associations with the pattern of amyloid beta-protein pathology point to subtypes of sporadic Alzheimer's disease

Chronic neurodegenerative diseases that involve the accumulation of misfolded proteins, such as Alzheimer’s disease, Parkinson’s disease and prion disease, proceed at variable rates in different patients. The environmental factors that might contribute to the variable rates of disease progression are poorly understood. We have been interested to learn how systemic infections, common co-morbidities in the elderly, contribute to disease progression. In a mouse model of prion disease the microglia take on an activated morphology early in disease evolution but have an anti-inflammatory phenotype characterized by the presence of TGFb, PGE2 and CCL2. We have suggested that although the microglia are ‘primed’ by the ongoing pathology they do not appear to significantly contribute to disease progression (Perry et al., 2007). Following an intraperitoneal challenge with endotoxin the systemic inflammatory response is communicated to the brain via both neural and humoral routes that leads to switching of the microglia from an anti-inflammatory phenotype to an aggressive pro-inflammatory phenotype. This pro-inflammatory phenotype is associated with exacerbation of symptoms of sickness behaviour, acceleration of the onset of behavioural deficits and increased neuronal degeneration (Cunningham et al., 2005, 2009). We investigated how systemic inflammation impacts on individuals with Alzheimer’s disease and found that those who have raised systemic levels of TNF and suffer from acute infections have accelerated cognitive decline. It appears that the communication of systemic inflammation to the brain, a normal homeostatic mechanism in the healthy brain, which has no long-term consequences, becomes maladaptive in the diseased brain and contributes to disease progression.

O3-02-04: Increased levels of CSF Aβ oligomers in Alzheimer's disease: Combination of techniques allows indirect estimates

Background: Soluble oligomeric amyloid(A ) is expected to be involved in the pathogenesis of Alzheimer’s disease (AD) as they mediate neurotoxicity both in vitro and in vivo. Oligomeric A in CSF have been suggested as a possible marker of AD and is thought to reflect the ongoing neurodegenerative process. Among the several oligomeric species described, the Arctic mutation (A E22G) provides clinical support for A protofibrils as a pathogenic species. The objective of this study was to evaluate the oligomer quotient as a diagnostic marker in CSF using samples from patients with different dementia diagnoses. Methods: TBS-soluble extracts from human and transgenic mouse brains and human CSF were analyzed with Western blot and C-terminal-specific ELISAs. Brain extracts were also analyzed with the protofibril specific mAb158 ELISA (Englund et al, 2007). Results: The oligomer quotient was determined with respect to total A content and C-terminal A signal, and based on a combination of well established denaturing and non-denaturing methods. For evaluation of this measure, transgenic AD mouse models were analyzed for both their oligomer quotient and their protofibril levels, specifically measured by the mAb158 sandwich ELISA. tg-ArcSwe mice (Lord et al, 2006) displayed A protofibrils in brain before onset of plaque pathology and the oligomer quotient agreed well with protofibril content. These results are now being confirmed in human brain samples. The oligomer quotient in 43 CSF samples from AD, MCI and FTD patients as well as healthy controls was determined and the quotient was highest for AD patients. It also distinguished AD from FTD and healthy controls (p 0.05). These results indicate that A oligomers are present in CSF and of possible diagnostic value. Additionally, results from this study imply that the reduced levels of A 42 in CSF found in AD could, at least partially be due to oligomerization of A . Conclusions: The oligomer quotient is an indirect measurement of oligomeric A in CSF and in tg-ArcSwe mouse brain. The quotient correlates with A protofibril levels, and offers opportunities to expand the panel of early markers for AD.