Dickkopf-related protein 3 is a potential Aß-associated protein in Alzheimer's Disease.

Amyloid-ß (Aß) is the most prominent protein in Alzheimer's disease (AD) senile plaques. In addition, Aß interacts with a variety of Aß-associated proteins (AAPs), some of which can form complexes with Aß and influence its clearance, aggregation or toxicity. Identification of novel AAPs may shed new light on the pathophysiology of AD and the metabolic fate of Aß. In this study, we aimed to identify new AAPs by searching for proteins that may form soluble complexes with Aß in CSF, using a proteomics approach. We identified the secreted Wnt pathway protein Dickkopf-related protein 3 (Dkk-3) as a potential Aß-associated protein. Using immunohistochemistry on human AD brain tissue, we observed that (i) Dkk-3 co-localizes with Aß in the brain, both in diffuse and classic plaques. (ii) Dkk-3 is expressed in neurons and in blood vessel walls in the brain and (iii) is secreted by leptomeningeal smooth muscle cells in vitro. Finally, measurements using ELISA revealed that (iv) Dkk-3 protein is abundantly present in both cerebrospinal fluid and serum, but its levels are similar in non-demented controls and patients with AD, Lewy body dementia, and frontotemporal dementia. Our study demonstrates that Dkk-3 is a hitherto unidentified Aß-associated protein which, given its relatively high cerebral concentrations and co-localization with Aß, is potentially involved in AD pathology. In this study, we propose that Dickkopf-related protein-3 (Dkk-3) might be a novel Amyloid-ß (Aß) associated protein. We demonstrate that Dkk-3 is expressed in the brain, especially in vessel walls, and co-localizes with Aß in senile plaques. Furthermore, Dkk-3 levels in cerebrospinal fluid strongly correlate with Aß40 levels, but were not suitable to discriminate non-demented controls and patients with dementia.

Amyloid-ß oligomer detection by ELISA in cerebrospinal fluid and brain tissue.

Amyloid-ß (Aß) deposits are important pathological hallmarks of Alzheimer's disease (AD). Aß aggregates into fibrils; however, the intermediate oligomers are believed to be the most neurotoxic species and, therefore, are of great interest as potential biomarkers. Here, we have developed an enzyme-linked immunosorbent assay (ELISA) specific for Aß oligomers by using the same capture and (labeled) detection antibody. The ELISA predominantly recognizes relatively small oligomers (10-25 kDa) and not monomers. In brain tissue of APP/PS1 transgenic mice, we found that Aß oligomer levels increase with age. However, for measurements in human samples, pretreatment to remove human anti-mouse antibodies (HAMAs) was required. In HAMA-depleted human hippocampal extracts, the Aß oligomer concentration was significantly increased in AD compared with nondemented controls. Aß oligomer levels could also be quantified in pretreated cerebrospinal fluid (CSF) samples; however, no difference was detected between AD and control groups. Our data suggest that levels of small oligomers might not be suitable as biomarkers for AD. In addition, we demonstrate the importance of avoiding HAMA interference in assays to quantify Aß oligomers in human body fluids.

Inhibition of α-synuclein aggregation by small heat shock proteins.

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.

Amyloid-ß oligomers relate to cognitive decline in Alzheimer's disease.

BACKGROUND: Amyloid-ß (Aß)-oligomers are neurotoxic isoforms of Aß and are a potential diagnostic biomarker for Alzheimer's disease (AD). OBJECTIVES: 1) Analyze the potential of Aß-oligomer concentrations in cerebrospinal fluid (CSF) to diagnose and predict progression to AD in a large clinical study sample. 2) Monitor Aß-oligomer concentrations over-time, both in early and advanced stages of AD. 3) Examine the relation between Aß-oligomer levels in CSF and cognitive functioning. METHODS: 24 non-demented, 61 mild cognitive impairment (MCI), and 64 AD patients who underwent lumbar puncture and cognitive testing at baseline and follow-up were selected from the memory clinic based Amsterdam Dementia Cohort. CSF samples were analyzed for standard AD-biomarkers and Aß-oligomer levels using a validated in-house Aß-oligomer specific enzyme-linked immunosorbent assay. Aß-oligomer levels were analyzed as indicators of disease progression (follow-up AD diagnosis) and cognitive decline, respectively. RESULTS: Patient groups did not differ in Aß-oligomer concentrations at baseline or follow-up. Baseline CSF Aß-oligomer levels were similar in MCI patients that develop AD as in stable MCI patients. MCI and AD patients showed an annual decrease in Aß-oligomer levels of 9.4% and 6.8%, respectively. A decrease in Aß-oligomer levels over time was strongly associated with more severe cognitive decline in AD patients. CONCLUSION: Despite the limited diagnostic potential of Aß-oligomer levels in CSF to differentiate between patient groups, and between MCI-AD and MCI-stable patients, changes in CSF Aß-oligomer levels were related to cognitive decline. Therefore, CSF Aß-oligomers may aid in the selection of patients with a more aggressive disease course.

The diagnostic value of CSF amyloid-ß(43) in differentiation of dementia syndromes.

Amyloid-ß (Aß) is known as the most prominent core protein in Alzheimer's Disease (AD) senile plaques. Although research has focused mainly on Aß40 and Aß42 as potential cerebrospinal fluid (CSF) biomarkers, a range of Aß peptides with variable lengths has been demonstrated in the brains and CSF of AD patients. Recently, it has been found that the Aß43 peptide may be more abundant than previously assumed, could therefore play an important role in AD pathophysiology, and hence also function as putative biomarker. In this study the value of CSF Aß43 in AD diagnosis was investigated. Aß43 levels in CSF were highly correlated with Aß42 levels. Furthermore, in differentiation of AD from nondemented controls and from patients with Lewy body dementia and frontotemporal dementia, Aß43 had an equal diagnostic value as Aß42, both as a single biomarker and in combination with total and phosphorylated tau. In conclusion, quantification of Aß43 in CSF does not add novel diagnostic information to the differential diagnosis of AD compared to existing biomarkers.

Methods for analysis of amyloid-ß aggregates.

Amyloid-ß protein (Aß) accumulation is one of the major hallmarks of Alzheimer's disease and plays a crucial role in its pathogenesis. Aß aggregates into fibrils, but rather than these end-products of the aggregation process, intermediate species, referred to as oligomers, have been identified as the most neurotoxic Aß aggregates. To characterize the different Aß species and to study the aggregation process, a wide range of techniques has been applied over the past years. These techniques aim to visualize the different Aß species and study their structure, to separate them, and to quantify the aggregated Aß forms by immunology-based methods. In this review, we provide an overview and discussion of the most important techniques used for these aims. Often a combination of techniques will be appropriate to obtain the most optimal information.