There are a number of ways in which a clinical diagnosis of dementia of the Alzheimer type can be made – the application of clinical criteria is the commonest but ancillary techniques such as neuroima

Clinical Diagnosis of Alzheimer's Disease

Oligomeric and Fibrillar Species of Amyloid-β Peptides Differentially Affect Neuronal Viability

Genetic factors play a major role in determining a person's risk to develop Alzheimer's disease (AD). Rare mutations transmitted in a Mendelian fashion within affected families, for example, APP, PSEN1, and PSEN2, cause AD. In the absence of mutations in these genes, disease risk is largely determined by common polymorphisms that, in concert with each other and nongenetic risk factors, modestly impact risk for AD (e.g., the e4-allele in APOE). Recent genome-wide screening approaches have revealed several additional AD susceptibility loci and more are likely to be discovered over the coming years. In this chapter, we review the current state of AD genetics research with a particular focus on loci that now can be considered established disease genes. In addition to reviewing the potential pathogenic relevance of these genes, we provide an outlook into the future of AD genetics research based on recent advances in high-throughput sequencing technologies.

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The genetics of Alzheimer's disease.

Alzheimer’s amyloid fibrils: structure and assembly

Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells Similar-sized vesicles can be further classified (eg, exosomes, microvesicles) based on their biogenesis, size, and biophysical properties Although initially thought to be cellular debris, and thus under-appreciated, EVs are now increasingly recognized as important vehicles of intercellular communication and circulating biomarkers for disease diagnoses and prognosis Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for EVs poses a barrier to clinical translation New analytical platforms including molecular ones are thus actively being developed to address these challenges Recent advances in the field are expected to have far-reaching impact in both basic and translational studies This article aims to present a comprehensive and critical overview of emerging analytical technologies for EV detection and their clinical applications

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New Technologies for Analysis of Extracellular Vesicles

Developing effective treatments for neurodegenerative diseases is one of the greatest medical challenges of the 21st century. Although many of these clinical entities have been recognized for more than a hundred years, it is only during the past twenty years that the molecular events that precipitate disease have begun to be understood. Protein aggregation is a common feature of many neurodegenerative diseases, and it is assumed that the aggregation process plays a central role in pathogenesis. In this process, one molecule (monomer) of a soluble protein interacts with other monomers of the same protein to form dimers, oligomers, and polymers. Conformation changes in three-dimensional structure of the protein, especially the formation of β-strands, often accompany the process. Eventually, as the size of the aggregates increases, they may precipitate as insoluble amyloid fibrils, in which the structure is stabilized by the β-strands interacting within a β-sheet. In this review, we discuss this theme as it relates to the two most common neurodegenerative conditions—Alzheimer’s and Parkinson’s diseases.

Protein aggregation in the brain: the molecular basis for Alzheimer's and Parkinson's diseases.

The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3-18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Circular dichroism indicates that none of the peptides displays beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.

Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity.

Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan® Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

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Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Review: formation and properties of amyloid-like fibrils derived from alpha-synuclein and related proteins.

Examination of the N-terminal sequence of non-A beta component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid beta peptide (A beta), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N-terminal half was likely to form a beta-structure whereas the C-terminal half was likely to form an alpha-helix. NAC in solution altered from random coil to beta-sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC-(1-18)-peptide and NAC-(19-35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1-18)-peptide became congophilic and aggregated to form fibrils of diameter 5-10 nm, whereas NAC-(19-35)-peptide did not bind Congo Red and remained in solution. Circular dichroism spectroscopy was used to study the secondary structure of NAC and the two fragments. In trifluoroethanol/water mixtures, NAC and NAC-(19-35)-peptide adopted alpha-helical structure but NAC-(1-18)-peptide did not. NAC-(1-18)-peptide and NAC formed beta-sheet in acetonitrile/water mixtures more readily than did NAC-(19-35)-peptide. CD spectra of NAC or NAC-(1-18)-peptide in aqueous solution indicate the formation of beta-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles A beta, and other amyloidogenic proteins, in that aggregation is dependent upon beta-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.

The N-terminal region of non-A beta component of Alzheimer's disease amyloid is responsible for its tendency to assume beta-sheet and aggregate to form fibrils.

G. Brent Irvine

Papers

Protein aggregation in the brain: the molecular basis for Alzheimer's and Parkinson's diseases.

Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity.

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Review: formation and properties of amyloid-like fibrils derived from alpha-synuclein and related proteins.

The N-terminal region of non-A beta component of Alzheimer's disease amyloid is responsible for its tendency to assume beta-sheet and aggregate to form fibrils.