Alzheimer's Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aβ species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aβ x-40 and x-42 peptide (hereafter Aβ40 and Aβ42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aβ40 in the CSF of AD patients. Together with measurements of total Aβ42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aβ40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD.

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP Sc ), we have been interested in how these peptides interact with PrP Sc . After screening peptides from the entire human PrP sequence, we found two peptides (PrP 19 -30 and PrP100-111) capable of binding full-length PrP Sc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP C ). The limit of detection for captured PrP Sc was calculated to be 8 amol from a Ϸ10 5 -fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP Sc over PrP C . Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP Sc . Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP Sc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP Sc , is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP C may play a role in the recruitment of PrP C to PrP Sc .plasma ͉ Creutzfeldt-Jakob disease ͉ detection ͉ cationic interaction ͉ diagnostic

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer’s disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

BackgroundPrion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrPC. Little is known about the details of the structural rearrangement of physiological PrPC into a still-elusive disease-associated conformation termed PrPSc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.Methodology/Principal FindingsHere, we report that trypsin digestion of PrPSc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrPSc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrPSc are exposed in PrPC.Conclusions/SignificanceWe conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrPSc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrPC participates in PrPSc conversion, and may also be exploited for diagnostic purposes.

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