SynopsisAlzheimer's disease (AD) affects millions worldwide. Currently, there are no treatments that prevent or slow AD. Like other neurodegenerative diseases, AD is characterized by protein misfolding in the brain. This process and associated brain damage begins years prior to the substantial neurodegeneration that accompanies dementia. Studies utilizing new neuroimaging techniques and fluid biomarkers suggest that AD pathology can be detected pre-clinically. These advances should enable novel clinical trial design and early mechanism-based therapeutic intervention. AD begins as a pathological process years before the onset of dementiaWith the emergence of disease-modifying strategies for the treatment of AD, impetus to diagnose the condition in its early 'preclinical' stages -before significant brain damage occurs -has intensified. Fortunately, advances in technology and in our perspective on what defines AD may soon make such antecedent diagnosis possible.Since their first description in 1907 1 , 'senile' plaques and neurofibrillary tangles (NFT) have remained the hallmark histopathological features of AD, and are employed by three sets of diagnostic histological criteria (Khachaturian, CERAD, and NIA/Reagan) 2-4. Historically, they have also been associated with the dementia caused by the disease. It is clear, however, that these lesions begin to accrue in significant amounts in many 'cognitively normal' elderly individuals 5 . To reconcile these incongruent inferences/observations, several tacit hypotheses have been spawned that persist even today: (1) that AD cannot be diagnosed in the absence of cognitive impairment/dementia, (2) that plaques and NFT increase in healthy aging, and (3) that AD and aging can be distinguished by quantitative (rather than qualitative) assessment of plaque and NFT burden 6 . Nevertheless, a growing body of evidence now supports a different philosophy regarding the onset of AD: independent of cognitive status, amyloid plaques and NFT actually define (but do not fully represent) the disease process, which also involves inflammation as well as neuronal, axonal, and synaptic loss and dysfunction.All correspondence should be addressed to David M. Holtzman. holtzman@neuro.wustl.edu. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 October 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptConsistently, neuropathological studies involving large numbers of non-demented subjects have identified significant AD pathology in the brains of older individuals 7,8 . Neocortical cerebral amyloid deposits have been identified in approximately 50% of brains from individuals over 75 8 . In contrast, the prevalence of AD dementia does not reach 50% until age 85 or more 9 . It appears that the onset of very mild dementia is correlated best not with plaque or NFT burden, but with significant synaptic and neuronal loss 6,10 . Together, these data support the concept of 'pre-clinical' AD, a phase during which plaques, and subsequently, NFT,...

␣-Synuclein has been centrally implicated in neurodegenerative disease, and a normal function in developmental synaptic plasticity has been suggested by studies in songbirds. A variety of observations suggest the protein partitions between membrane and cytosol, a behavior apparently conferred by a conserved structural similarity to the exchangeable apolipoproteins. Here we show that the capacity to bind lipids is broadly distributed across exons 3, 4, and 5 (encoding residues 1-102). Binding to phosphatidylserine-containing vesicles requires the presence of all three exons, while binding to phosphatidic acid can be mediated by any one of the three. Consistent with a "class A2" helical binding mechanism, lipid association is disrupted by introduction of charged residues along the hydrophobic face of the predicted ␣-helix and also by biotinylation of conserved lysines (which line the interfacial region). Circular dichroism spectroscopy reveals a general correlation between the amount of lipid-induced ␣-helix content and the degree of binding to PS-containing vesicles. Two point mutations associated with Parkinson's disease have little (A30P) or no (A53T) effect on lipid binding or ␣-helicity. These results are consistent with the hypothesis that ␣-synuclein's normal functions depend on an ability to undergo a large conformational change in the presence of specific phospholipids.

Background Disease-modifying therapies for Alzheimer’s disease (AD) would be most beneficial if applied during the ‘preclinical’ stage (pathology present with cognition intact) before significant neuronal loss occurs. Therefore, biomarkers that can detect AD pathology in its early stages and predict dementia onset and progression will be invaluable for patient care and efficient clinical trial design. Methods 2D–difference gel electrophoresis and liquid chromatography tandem mass spectrometry were used to measure AD-associated changes in cerebrospinal fluid (CSF). Concentrations of CSF YKL-40 were further evaluated by enzyme-linked immunosorbent assay in the discovery cohort (N=47), an independent sample set (N=292) with paired plasma samples (N=237), frontotemporal lobar degeneration (N=9), and progressive supranuclear palsy (PSP, N=6). Human AD brain was studied immunohistochemically to identify potential source(s) of YKL-40. Results In the discovery and validation cohorts, mean CSF YKL-40 was higher in very mild and mild AD-type dementia (Clinical Dementia Rating [CDR] 0.5 and 1) vs. controls (CDR 0) and PSP. Importantly, CSF YKL-40/Aβ42 ratio predicted risk of developing cognitive impairment (CDR 0 to CDR>0 conversion) as well as the best CSF biomarkers identified to date, tau/Aβ42 and p-tau181/Aβ42. Mean plasma YKL-40 was higher in CDR 0.5 and 1 vs. CDR 0 groups, and correlated with CSF levels. YKL-40 immunoreactivity was observed within astrocytes near a subset of amyloid plaques, implicating YKL-40 in the neuroinflammatory response to Aβ deposition. Conclusions These data demonstrate that YKL-40, a putative indicator of neuroinflammation, is elevated in AD, and that, together with Aβ42, has potential prognostic utility as a biomarker for preclinical AD.

E.M. designed the study and wrote the initial draft of the manuscript. All authors collected samples and data, helped to interpret the results and reviewed drafts of the manuscript.Competing interests R.J.B. has equity ownership interest in C2N Diagnostics and receives royalty income based on technology (stable isotope labeling kinetics and blood plasma assay) licensed by Washington University to C2N Diagnostics. R.J.B. receives income from C2N Diagnostics for serving on the scientific advisory board. Washington University, with R.J.B., E.M. and N.R.B. as co-inventors, has submitted the US nonprovisional patent application 'Cerebrospinal fluid (CSF) tau rate of phosphorylation measurement to define stages of Alzheimer's disease and monitor brain kinases/phosphatases activity'. R.J.B. has received honoraria from Janssen and Pfizer as a speaker, and from Merck and Pfizer as an advisory board member. E.M. has received royalty payments for an educational program supported by Eli Lilly and as a member of a scientific advisory board for Eli Lilly.

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BackgroundClinicopathological studies suggest that Alzheimer's disease (AD) pathology begins ∼10–15 years before the resulting cognitive impairment draws medical attention. Biomarkers that can detect AD pathology in its early stages and predict dementia onset would, therefore, be invaluable for patient care and efficient clinical trial design. We utilized a targeted proteomics approach to discover novel cerebrospinal fluid (CSF) biomarkers that can augment the diagnostic and prognostic accuracy of current leading CSF biomarkers (Aβ42, tau, p-tau181).Methods and FindingsUsing a multiplexed Luminex platform, 190 analytes were measured in 333 CSF samples from cognitively normal (Clinical Dementia Rating [CDR] 0), very mildly demented (CDR 0.5), and mildly demented (CDR 1) individuals. Mean levels of 37 analytes (12 after Bonferroni correction) were found to differ between CDR 0 and CDR>0 groups. Receiver-operating characteristic curve analyses revealed that small combinations of a subset of these markers (cystatin C, VEGF, TRAIL-R3, PAI-1, PP, NT-proBNP, MMP-10, MIF, GRO-α, fibrinogen, FAS, eotaxin-3) enhanced the ability of the best-performing established CSF biomarker, the tau/Aβ42 ratio, to discriminate CDR>0 from CDR 0 individuals. Multiple machine learning algorithms likewise showed that the novel biomarker panels improved the diagnostic performance of the current leading biomarkers. Importantly, most of the markers that best discriminated CDR 0 from CDR>0 individuals in the more targeted ROC analyses were also identified as top predictors in the machine learning models, reconfirming their potential as biomarkers for early-stage AD. Cox proportional hazards models demonstrated that an optimal panel of markers for predicting risk of developing cognitive impairment (CDR 0 to CDR>0 conversion) consisted of calbindin, Aβ42, and age.Conclusions/SignificanceUsing a targeted proteomic screen, we identified novel candidate biomarkers that complement the best current CSF biomarkers for distinguishing very mildly/mildly demented from cognitively normal individuals. Additionally, we identified a novel biomarker (calbindin) with significant prognostic potential.

Four plasma analytes were consistently associated with the diagnosis of very mild dementia/MCI/AD in 3 independent clinical cohorts. These plasma biomarkers may predict underlying AD through their association with CSF AD biomarkers, and the association between plasma and CSF amyloid biomarkers needs to be confirmed in a prospective study.

Code availabilityAll code for data cleaning and analysis associated with the current submission is available upon request to the corresponding author and is provided as part of the replication package.