Patent Application: US-201414896018-A

Abstract:
the present invention provides a method for diagnosing or assessing a neurodegenerative disease in a test subject , comprising : providing a protein - containing sample that has been obtained from the test subject ; determining the concentration , amount or degree of expression of at least one specific protein isoform and / or glycoform derived from a protein biomarker selected from the group consisting of : clusterin precursor ; apolipoprotein a - iv precursor ; apolipoprotein c - iii precursor ; transthyretin ; galectin 7 ; complement c4 precursor ; alpha - 2 - macroglobulin precursor ; ig alpha - 1 chain c ; histone 2b ; ig lambda chain c region ; fibrinogen gamma chain precursor ; complement factor h ; inter - alpha - trypsin heavy chain h4 precursor ; complement c3 precursor ; gamma or beta actin ; haptoglobin precursor ; and the serum albumin precursor , or a fragment thereof ; comparing said concentration , amount or degree determined in with a reference from a control subject with a specific neurodegenerative disease , dementia or stage of disease , or from a control subject that does not have a neurogenerative disease or dementia ; and based on the level of the at least one specific protein isoform and / or glycoform of the protein biomarker in the test subject relative to the reference , making a diagnosis or assessment as to the presence of and / or stage of neurodegenerative disease or dementia of the test subject . also provided are related products and systems for use in such a method .

Description:
in describing the present invention , the following terms will be employed , and are intended to be defined as indicated below . the term “ subject ” includes a human or an animal . in accordance with certain embodiments of the present invention , the subject may have been previously diagnosed with ad and / or previously diagnosed with mild cognitive impairment ( mci ). the subject is preferably a human . the subject may be a human of at least 60 years of age , optionally at least 70 or at least 80 years of age . the term “ diagnosis ”, as used herein , includes the provision of any information concerning the existence , non - existence or probability of the disorder in a patient . it further includes the provision of information concerning the type or classification of the disorder or of symptoms which are or may be experienced in connection with it . this may include , for example , diagnosis of the severity of the disorder . it encompasses prognosis of the medical course of the disorder , for example its duration , severity and the course of progression from mci to alzheimer &# 39 ; s disease . currently disease status is assessed by duration of disease from inception to present ( longer duration equals more severe disease ) and clinical assessment measures . these assessment measures include clinical tests for memory and other cognitions , clinical tests for function ( abilities of daily living ) and clinical assessments of global severity . trials of potential therapies in ad are currently evaluated against these measures . the fda and other medicines approval bodies require as part of these assessments measures of both cognition and global function . the global dementia scale is one such measure of global function . it is assessed by later assessment of severity including cognition and function against a standardised set of severity criteria . the term “ alleviate ”, as used herein , in relation to alzheimer &# 39 ; s disease means any form of reducing one or more undesired symptoms or effects thereof . any amelioration of alzheimer &# 39 ; s disease of the patient falls within the term “ alleviation ”. amelioration may also include slowing down the progression of the disease . as used herein “ assessing ” ad includes the provision of information concerning the type or classification of the disease or of symptoms which are or may be experienced in connection with it . this specifically includes prognosis of the medical course of the disease , for example its duration , severity and the course and rate of progression from e . g . mci or pre - symptomatic ad to clinical ad . this also includes prognosis of ad - associated brain pathology such as fibrillar amyloid burden , cortical and hippocampal atrophy and accumulation of neurofibrillary tangles . the assessment may be of an aggressive form of ad and / or a poor prognosis . as used herein “ biological sample ” refers to any biological liquid , cellular or tissue sample isolated or obtained from the subject . in accordance with the present invention the “ protein - containing sample ” may be any biological sample as defined herein . the biological sample may , in certain cases , comprise blood plasma , blood cells , serum , saliva , urine , cerebro - spinal fluid ( csf ) or a tissue biopsy . the biological sample may have been stored ( e . g . frozen ) and / or processed ( e . g . to remove cellular debris or contaminants ) prior to determining the amount ( e . g . concentration ) of the at least one protein isoform and / or glycoform in question that is found in the sample . clusterin ( apolipoprotein j ; sp - 40 , 40 ; trpm - 2 ; sgp - 2 ; padhc - 9 ; clj ; t64 ; gp iii ; xip8 ) is a highly conserved disulfide - linked secreted heterodimeric glycoprotein of 75 - 80 kda but truncated forms targeted to nucleus have also been identified . the protein is constitutively secreted by a number of cell types including epithelial and neuronal cells and is a major protein in physiological fluids including plasma , milk , urine , cerebrospinal fluid and semen . preferably , clusterin comprises or consists of an amino acid sequence having at least 70 %, 80 %, 90 %, 95 %, 99 % or 100 % identity to the human clusterin sequence disclosed in uniprot accession no . p10909 , sequence version 1 and gi no . 116533 ( seq id no : 1 ) ( incorporated herein by reference in its entirety ), calculated over the full length of said human clusterin sequence ; or a fragment thereof comprising at least 5 , 10 , 15 , 20 , 30 , 50 , 100 , 150 , 200 , 250 , 300 , 350 , 400 , 425 or 449 contiguous amino acids . expression of the clusterin gene is significantly elevated in alzheimer &# 39 ; s disease ( ad ) brain ( may et al ., 1990 ) and levels of plasma clusterin have also been shown to correlate with ad progression ( thambisetty et al ., 2010 ). the inventors have previously identified several plasma clusterin isoforms as candidate biomarkers for ad using 2 - dimensional gel electrophoresis ( 2de ). however , the use of immunoassays and unmodified peptides in selected reaction monitoring ( srm ) experiments did not fully replicate the regulation seen in 2de . the inventors hypothesised that this disconnect is perhaps due to alterations in specific post - translational events that were not being replicated in the validation studies . clusterin is a highly - glycosylated secreted protein and because glycosylation plays an important role in physiological functions of clusterin ( stuart et al ., 2007 ) the inventors proposed that the detailed profiling of plasma clusterin and comparison of glycosylation profiles observed in distinct clinically classified subjects , for example patients with low or high atrophy of the hippocampus , may reveal more potent biomarker isoforms . guided by the observations relating to the clusterin glycoforms , which demonstrate a β - n - acetyl - glucosaminidase activity in plasma , the inventors also devised a novel assay using a defined substrate to measure this specific activity . human clusterin , was enriched by immunoprecipitation ( ip ) from albumin / igg - depleted plasma , using a monoclonal anti - clusterin antibody ( millipore ). immunoprecipitated proteins were first analysed by western blotting as a quality control , then separated by either two - dimensional electrophoresis ( 2de ) or sds - page . the spots and single band (# 3 ) of interest were excised , reduced , alkylated and digested in - gel with trypsin prior to analysis by mass spectrometry ( ms ). samples were analysed via lc - ms / ms using nanoflow reverse phase chromatography ( easy - nlc ii , thermofisher scientific ) and a top20 collision induced dissociation ( cid ) method ( orbitrap velos , thermofisher scientific ). glycopeptides were manually identified by the presence of glycan - specific oxonium ion fragments , m / z 204 . 08 for n - acetylhexosamine , [ hexnac ] + , m / z 366 . 14 for hexose - n - acetylhexosamine , [ hex - hexnac ] + , and m / z 657 . 24 for n - acetylneuraminic acid - hexose - n - acetylhexosamine [ neuac - hex - hexnac ] + in the ms / ms spectra . initially , mathematical modelling was used to create an artificial map of the various clusterin glycoforms for the separate alpha and beta chains ( fig1 a ). further refinement of this approach was used to classify individual clusterin related glycoforms using simple ( x , y ) coordinates . in this way , the inventors were able to predict the content of the individual 2de spots , and demonstrate that discrete coordinates are shared by multiple glycoforms . hence , 2de spots are likely to be composite mixtures containing several glycoforms . this information was then used to aid the interpretation of complex lc / ms / ms data , which subsequently provided some rather useful insights . firstly , it became apparent that the major components within each of the 2de spots were , without exception , always fully sialylated forms , being either tetra , tri or biantennary structures ( fig1 b ). this was surprising because it had originally been predicted that differences in sialic acid were the primary cause of the separation of distinct forms during electrophoresis , with loss of 291 da concurrent with a decreasing charge thus resulting in a shift towards a more basic iso - electric point . however , the lc / ms / ms results ( fig2 ) indicated a trend towards lower number of antennae and this suggested the successive removal of whole antennae as a more pronounced effect on the location of the 2de spots . a basic vector was devised to illustrate this phenomena ( fig3 ) and it transpires that the detected clusterin glycoforms actually now indicate evidence to support both the removal of sialic acids alone as well as removal of full antennae suggesting distinct neurominidase and β - n - acetyl - glucosaminidase activity respectively . design of a substrate assay to measure specific n - acetyl - glucosaminidase activity in plasma the results of glycan analysis of 16 different clusterin isoforms visible on 2 - dimensional gel electrophoresis showed a sequential removal of sialic acids and entire antennae . several of the truncated glycoforms appeared to correlate with clusterin protein spots previously identified as candidate biomarkers of ad and mci . until now no detailed analysis of glycosylation of these clusterin isoforms has been performed and it was surprising to discover that the majority of the disease associated modification in plasma clusterin could be accounted for by the activity of a single glycosidase , namely β - n - acetyl - glucosaminidase . the inventors thus set up a specific assay method to determine the activity of β - n - acetyl - glucosaminidase in tissue or bodily fluid samples taken from subjects suspected of having , or previously diagnosed with mci , ad or other dementia . the artificial glycan na3 substrate ( fig4 ) contains both β1 , 2 and β1 , 4 linkages between adjoining β - n - acetyl - glucosamine and mannose subunits and is a preferred substrate to differentiate β1 , 2 and β1 , 4 n - acetyl - glucosaminidase activity in plasma . the molecular weight of the substrate is 2006 . 82 da and an [ m + 2h ] 2 + ion is detected at m / z 1003 . 8 using an esi - tof mass spectrometer ( fig6 ). na3 substrate is added to an appropriate sample of tissue or body fluid from a subject suspected of having , or previously diagnosed with dementia to achieve a final concentration of 300 - 1 , 000 pg / μl and incubated at 37 ° c . for 4 - 24 hours . the test sample is then centrifuged to remove debris and an aliquot submitted to lc - ms / ms analysis . the measurement of molecular ions corresponding to loss of either two or one antennae indicate β1 , 2 and β1 , 4 n - acetyl - glucosaminidase activity respectively . analysis of immuno - precipitated clusterin to create a unique glycopeptide reference resource for clusterin : _the clusterin glycomod database v1 . 0 a representative pooled clinical plasma sample was used to develop methodology and to assemble an “ observation - based ” database containing 41 distinct glycoforms associated with anticipated glycosylation consensus sites within the amino acid sequence . for each glycopeptide the m / z charge state and retention time ( rt ) of the analyte was tabulated ( see table 1a ). unambiguous annotation of the glycopeptide required the detection of the [ peptide + hexnac ] + fragment ion in the corresponding ms / ms spectra and interpretation of additional fragment ions relating to the sequential dissociation of the individual glycan subunits . an example ms / ms spectrum is shown ( fig7 ) and the current iteration of the clusterin glycomod database is provided in table 1a . an updated iteration of the clusterin glycomod database is provided in table 1b . using immuno - precipitation and lc / ms / ms we have characterised 41 glycopeptides encompassing 5 of 6 anticipated n - linked glycosylation consensus sites in plasma clusterin . in total 41 different n - linked glycopeptides have been characterised and are listed herein . the glycan distribution at these 5 sites was consistent with a cv of & lt ; 15 % ( n = 3 from two plasma samples ) indicating the technical and biological reproducibility of the method . the inventors have previously demonstrated 5 of 6 predicted n - linked glycosylation sites within human plasma clusterin ( glycomod database v1 . 0 ). it would be understood by the skilled practitioner that expansion of the glycomod database to cover all n - linked and o - linked sites of all the protein biomarkers is within the scope of the present invention . indeed , the inventors have subsequently completed mapping of the sixth n - linked site in human clusterin as set out in table 1b ( fig1 ), showing clusterin glycopeptides of version 1 . 1 of the clusterin glycomod database ( mci / ad plasma ). analysis of immuno - precipitated clusterin to compare individuals with low and high atrophy the inventors have identified certain isoforms of clusterin as differentially regulated in the plasma of patients with ad relative to non - demented controls . furthermore , it has also been shown that certain spots comprising clusterin on 2de gels correlate with the level of hippocampal atrophy , whilst yet other isoforms correlated with the subsequent rate of disease progression in ad . the inventors obtained plasma samples from four subjects previously diagnosed with ad who had a low level of hippocampal atrophy and from four subjects previously diagnosed with ad with high hippocampal atrophy . clusterin was enriched using immunoprecipitation , and subjected to the lc - ms / ms method described above . surprisingly , they identified that the extent of glycan pruning correlated with hippocampal atrophy . in patients with low levels of hippocampal atrophy there was little evidence of pruning of plasma clusterin . conversely , plasma clusterin from subjects with high levels of hippocampal atrophy was typically pruned to remove one or more complete antennae within the n - linked glycans . as an example , two sialylated forms of the tetra - antennary glycopeptide hn * stgclr ( seq id no : 2 ) are observed as triply charged molecular ions at m / z 1391 and m / z 1294 . 17 but only in individuals with low atrophy ( fig7 ). these moieties are therefore potential alternative biomarkers concordant with the extent of hippocampal atrophy . using data from all n - linked glycans monitored by the lc - ms / ms method the inventors saw a consistent reduction in the level of tetra - antennary glycans in subjects with high levels of hippocampal atrophy compared to those with low levels . based on the total glycoform signal for the n - linked glycosylation site on the tryptic peptide hn * stgclr ( seq id no : 2 ) of the clusterin beta chain ( fig8 ). validation analysis of immuno - precipitated clusterin to compare individuals with low and high atrophy having identified that changes in clusterin glycosylation patterns correlate to the extent of atrophy within a small cohort of clinical samples we performed a further validation study on an additional cohort of alzheimer &# 39 ; s disease patients with known levels of hippocampal atrophy . additional bioinformatics approaches were also assessed for their impact on class segregation based on glycoform profiles and a new , higher sensitivity mass spectrometer was employed in the expectation of identifying additional diagnostic glycoforms of clusterin . to ensure correlation with earlier data , samples from the original 4 × 4 cohort ( discovery cohort ) used in example 4 were re - analysed using the new methods alongside a separate cohort of 20 new samples from ad ( n = 10 ) and matched controls ( n = 10 )( replication cohort ). all sample details are provided in table 2 . clusterin was enriched from each sample , as described above . the relevant protein band was excised , reduced , alkylated , and digested with trypsin . after clean up , the clusterin digests were split into two aliquots and each tested by nanoflow high performance liquid chromatography and orbitrap velos pro or ultra - high performance liquid chromatography and orbitrap fusion tribrid lc - ms / ms systems ( all equipment from thermo scientific , hemel hempstead , uk ). data were ostensibly similar but , as expected , more glycosylated clusterin peptides were identified on the fusion and so all subsequent analysis was performed on the fusion dataset . mass spectrometer raw data were processed using proteome discoverer software ( thermo scientific ). ion intensities for the glycosylated clusterin peptides and their fragments described in tables 1a and 1b were exported into an excel ( microsoft corp ) spreadsheet . we employed a sum scaling technique to normalise the data and calculated significance values ( p ) for each glycopeptide by comparing the median values between the low and high atrophy groups in the discovery cohort , replication cohort and a combined analysis of both cohorts as a single group . student &# 39 ; s t test was used to identify peptide - associated glycoforms that change significantly between high and low atrophy , resulting in one - tailed p - values for each glycopeptide ( see tables 3a , 3b and 3c ). using our ip - lc / ms / ms workflow on the orbitrap fusion tribrid we were able to extend coverage to all six known n - glycosylation sites of clusterin : α64n , α81n , α123n , β64n , β127n , and β147n . by monitoring the glycan specific fragments we were also able to assign various antennary structures at all six sites and to perform relative quantification based on total ion counts . in total 42 different glycan structures were detected . whilst most glycosylation sites showed no regulation in glycan structures between high and low levels of hippocampal atrophy , two sites — β64n and β147n — showed significant regulations between the clinical groups . the specific glycan structures showing significant ( p ≦ 0 . 05 ) changes between the clinical groups in the discovery , replication and combined cohort analyses are indicated in table 3a , 3b , and 3c respectively . box plots for each glycopeptide were created to illustrate the separation achieved between the two groups ( fig9 - 11 ). interestingly , six glycoforms at β64n glycosylation site hn * stgclr ( seq id no : 2 ) were found significantly decreased in the 4 high atrophy samples ( alzheimer &# 39 ; s ) compared to the 4 low atrophy samples ( mild cognitive impairment ) of the discovery cohort when measured on the orbitrap fusion . this included the sialylated forms of the tetra - antennary glycopeptide observed as triply charged molecular ions at m / z 1391 . 54 which was consistent with the previous velos data analysis , confirming the robustness of this glycoform as a diagnostic marker to differentiate mild cognitive impairment from alzheimer &# 39 ; s disease when measured on a different lc - ms / ms platform . in the larger replication cohort , three glycoforms of β64n glycopeptides were significantly reduced in high atrophy samples . these include the sa1 -( hexnac - hex ) 2 , sa1 -( hexnac - hex ) 3 and sa2 -( hexnac - hex ) 3 glycoforms seen at m / z 953 . 71 , 1075 . 42 , 1172 . 45 in the spectra . as all of these glycoforms were also seen reduced in high atrophy patients in the discovery cohort this further supports their utility as prognostic biomarkers in patients with confirmed alzheimer &# 39 ; s disease . when the results of the two cohorts were combined we again , saw that changes in glycoforms found at site β64n correlated with atrophy , with four glycoforms significantly reduced over high atrophy , e . g . sa1 -( hexnac - hex ) 2 , sa2 -( hexnac - hex ) 2 , sa1 -( hexnac - hex ) 3 , and sa2 -( hexnac - hex ) 3 at m / z 953 . 71 , 1050 . 74 , 1075 . 42 , and 1172 . 45 respectively . use of the orbitrap fusion increased total glycoform coverage from 4 to 6 n - linked sites . several β64n site glycoforms are significantly reduced in plasma of patients with alzheimer &# 39 ; s disease compared to individuals with mild cognitive impairment . four of these glycoforms are also reduced in alzheimer &# 39 ; s patients with high levels of hippocampal atrophy . in combination this confirms the utility of clusterin isoforms as diagnostic and prognostic markers for alzheimer &# 39 ; s disease . development and preliminary testing of a selective reaction monitoring ( srm ) method for 8 glycoforms of clusterin in human plasma in readiness for higher throughput measurements within much larger numbers of clinical samples , we have also developed a targeted selective reaction monitoring ( srm ) method to measure specific glycopeptides of clusterin . this newly established tsq - srm workflow used eight glycoforms of clusterin β64n glycopeptides as precursors , and two glycan - specific oxonium ion fragments at m / z 366 . 14 and m / z 657 . 24 as transitions ( see table 4 ). additionally , the peptide ion at m / z 574 . 56 representing [ hn * stgclr ] 2 + ( seq id no : 2 ) where n *= asparagine residue + hexnac , was included to serve as the third transition ion providing confirmation of site - specific information . details of each monitored transition is provided in table 4 . in previous studies ( data not shown ), we were able to extract clusterin glycopeptides from human serum without prior immunoprecipitation . given the potential sensitivity gains offered by srm methods we followed a more straightforward gelc method for clusterin enrichment which would be more compatible with high throughput analysis such as would be required for a clinical diagnostic . initially , to identify the location of clusterin in a one dimensional sds - polyacrylamide gel electrophoresis ( sds - page ) experiment normal human plasma ( dade - behring , germany ) was depleted of albumin and igg and proteins extracted in laemmli buffer and subjected to sds - page . fig1 shows the gel - 10 analysis of albumin / igg - depleted plasma . all ten bands were excised , reduced , alkylated , and trypsin - digested prior to ms analysis on an orbitrap velos pro . glycopeptides of clusterin were identified in the tryptic digests of bands # 5 , # 6 , and # 7 ( data not shown ) with the majority seen in band # 7 with 45 % of peptide coverage . all ten tryptic - digested gel bands were also submitted for analysis using the newly - developed clusterin glycoform srm method to confirm the suitability of the gelc method for sample preparation . fig1 shows clusterin glycopeptides were found in the band # 5 , # 6 and # 7 , and majority of clusterin was identified at band # 7 , suggesting these glyco - srm results were consistent with the previous orbitrap velos pro discovery data . when the srm data files were examined for the extracted ion chromatograms ( xic ) of eight β64n glycopeptide precursors ( fig1 ), we were able to determine that the majority of them eluted between 7 - 8 minutes . precise identification of elution time allows subsequent scheduling of srm or adjustment of elution buffers to improve separation of closely related species if more complex methods should be developed subsequently . it is a particular advantage of the present method that the integrated peak area for each monitored species can be used for label - free quantification using a sum - scaled approach . furthermore , since this gel10 - glyco - srm method does not require immunoprecipitation and clusterin glycopeptides can be detected within 30 minutes by tsq , instead of one hour by orbitrap , this newly - established method provides a more efficient and faster way to verify the potential biomarker glycopeptide of clusterin . the same method may also be used for clinical assessment of patient samples to aid the diagnosis of mci and alzheimer &# 39 ; s disease as well as providing prognostic information on the rate of hippocampal atrophy and cognitive decline . it would also be understood that the same srm method may be applied with little further optimisation to digested human plasma , serum , saliva , urine or cerebrospinal fluid without the need for prior sds - page separation . it is also possible to employ the same srm method for the analysis of clusterin enriched from human plasma by immunoprecitpitation . thus , to further validate our targeted biomarker glycopeptides , the clusterin glycol - srm method was applied to evaluate immunoprecipitated clusterin from the discovery cohort . as expected , the srm method gave tighter quantitative results and this improved precision resulted in higher levels of significance for the reduction in specific glycoforms in alzheimer &# 39 ; s patients with higher levels of hippocampal atrophy ( table 5 ). in total , five of the eight monitored glycopeptides at β64n were significantly reduced in high atrophy cases . a selection of box plots for srm quantification of individual clusterin glycoforms is provided in fig1 . the eight clusterin glycopeptide gel10 - glyco - srm assay developed in example 6 was applied to the analysis of the validation cohort of alzheimer &# 39 ; s disease patient plasma samples comprising 9 cases with [ high ] level of hippocampal atrophy and 10 cases with [ low ] level of hippocampal atrophy . samples were as described in table 2 and all sample preparation and analytical methods are as described in example 6 . across this cohort three specific β64n site - specific glycoforms showed a statistically significantly difference between patients with high levels of hippocampal atrophy and those with lower rates of hippocampal atrophy ( table 6 ). when the results for the discovery and validation cohorts were combined , surprisingly all eight glycoforms attained statistical significance for reduced concentrations in the high atrophy group compared to those with low hippocampal atrophy ( table 7 ). the power of these eight clusterin glycopeptides to differentiate patients based on their hippocampal volume provides a minimally invasive means to diagnose and predict the progression of alzheimer &# 39 ; s disease and will be applicable to the analysis of other neurodegenerative diseases characterized by the aggregation of proteins leading to neuronal damage including parkinson &# 39 ; s disease , huntington &# 39 ; s disease , and frontotemporal dementia . a high sensitivity srm method for eight specific n - linked glycopeptides at β64n of human clusterin can differentiate between alzheimer &# 39 ; s disease cases with high hippocampal atrophy and mild cognitive impairment cases with low hippocampal atrophy . this method may provide the basis for a routine clinical test to assess hippocampal atrophy based on the detection of the level of specific glycoforms in an individual patient and comparing this to levels known to represent specific levels of hippocampal atrophy . the same method may be expanded to incorporate other clusterin peptides or indeed ( glyco ) peptides from other plasma proteins that act as diagnostic or prognostic biomarkers of any neurodegenerative disease the foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention . the present invention is not to be limited in scope by examples provided , since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments , including application to the homologous protein biomarkers in different species are within the scope of the invention . various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims . the advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention . all references , including patent documents , disclosed herein are incorporated by reference in their entirety for all purposes , particularly for the disclosure referenced herein . andreasen , n ., c . hesse , et al . 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