Validation of amyloid-b peptides in CSF diagnosis of neurodegenerative dementias

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1 ORIGINAL ARTICLE (2007) 12, & 2007 Nature Publishing Group All rights reserved /07 $ Validation of amyloid-b peptides in CSF diagnosis of neurodegenerative dementias M Bibl 1,6, B Mollenhauer 2,6, P Lewczuk 3, H Esselmann 3, S Wolf 1, C Trenkwalder 4, M Otto 5, G Stiens 1, ERüther 1, J Kornhuber 3 and J Wiltfang 3 1 Department of Psychiatry, University of Goettingen, Goettingen, Germany; 2 Center for Neurologic Diseases, Brigham and Women s Hospital, Harvard Medical School, Boston, MA, USA; 3 Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, Germany; 4 University of Goettingen, Paracelsus-Elena Klinik, Kassel, Germany and 5 Department of Neurology, University of Ulm, Ulm, Germany Biomarkers for differential diagnosis of the three most frequent degenerative forms of dementia, Alzheimer s disease (AD), dementia with Lewy bodies (DLB) and frontotemporal dementias (FTD), are currently under intensive investigation, but disease-specific biomarkers for FTD and DLB are still lacking. We analyzed 303 cerebrospinal fluid (CSF) samples of 71 AD, 32 DLB and 36 FTD patients in comparison to 93 various other dementias (OD), 20 peripheral neurologic disease (PND) controls, 25 neurodegenerative disorders without dementia (ND) and 26 depressive cognitive complainers (DCC) for distinct CSF amyloid-b (Ab) peptide patterns, using the quantitative Ab-SDS-PAGE/immunoblot. Additionally, the novel electrochemiluminescence technique (MSD) was used to validate the measures on Ab1-38. The main outcome measures were a striking decrease of Ab1-42 in AD (P = ), and most interestingly a pronounced decrease of Ab1-38 in FTD (P = ). Moreover, a novel peptide that most probably represents an oxidized a-helical form of Ab1-40 (Ab1-40 ox ) displayed a highly significant increase in DLB (P = ) as compared to non-demented disease controls. The overall diagnostic accuracy of percentage Ab peptide abundances (Ab1-X%) was clearly superior to absolute CSF Ab levels. Ab1-42% and Ab1-38% enabled contrasts of 85% or beyond to distinguish AD and FTD, respectively, from all other investigated subjects. Ab1-40 ox % yielded a diagnostic sensitivity and specificity of 88 and 73% for the detection of DLB among all other investigated patients. We found a strong correlation between Ab1-38 levels as measured by the Ab-SDS-PAGE/immunoblot and MSD, respectively. CSF Ab peptides may reflect disease-specific impact of distinct neurodegenerative processes on Ab peptide metabolism and represent a potential diagnostic biomarker for AD, FTD and DLB. (2007) 12, ; doi: /sj.mp ; published online 6 March 2007 Keywords: Alzheimer s disease; frontotemporal dementia; dementia with Lewy bodies; cerebrospinal fluid; amyloid-b peptides; biomarkers Introduction Amyloid plaques, one of the major neuropathological hallmarks of Alzheimer s disease (AD), 1 mainly consist of aggregated amyloid-b (Ab) peptides and also occur in other neurodegenerative diseases, such as dementia with Lewy bodies (DLB), 2 but are rarely found in frontotemporal dementias (FTD). 3 Correspondence: Dr J Wiltfang, Molecular Neurobiology Laboratory, Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6, Erlangen, Germany. Jens.Wiltfang@psych.imed.uni-erlangen.de or Dr M Bibl, Neurobiology Laboratory, Department of Psychiatry and Psychotherapy, University of Goettingen, von-siebold-str. 5, Göttingen, Germany. mbibl@gwdg.de 6 These authors contributed equally to this work. Received 24 October 2006; revised 14 December 2006; accepted 3 January 2007; published online 6 March 2007 The recently established Ab-SDS-PAGE/immunoblot enables measurement of a pattern of the carboxyterminally truncated (Ct-truncated) Ab peptides 1 37, 1 38 and 1 39 in addition to 1 40 and 1-42 (Ab peptide pattern) in cerebrospinal fluid (CSF). This approach can be assumed to reflect most adequately disease-specific changes of APP metabolism during ongoing neurodegenerative processes and has shown diagnostic value for differentiation of AD among other dementias (OD) or organic brain diseases. 4,5 In particular, a novel peptide that most probably represents an oxidized a-helical form of Ab1-40 (Ab1-40 ox ) was significantly increased in DLB in a smaller pilot study. 5 Some of our own data already suggested a specific reduction of Ab1-38 in FTLD as measured by Ab-SDS-PAGE/ immunoblot. However, these studies lack proper comparison groups for estimating test accuracies in a larger collective of differential diagnostic diagnoses.

2 672 To the best of our knowledge, the present study is the largest CSF-based biomarker investigation on Ab peptide patterns directly comparing the three major degenerative dementias AD, DLB and FTD among various other dementia patients as well as peripheral neurologic diseases (PND), neurodegenerative disorders without dementia (ND) and depressive cognitive complainers (DCC). Methods Participants We prospectively investigated 303 consecutive CSF samples referred to our laboratory between 1999 and Seventy-four of these patients had been investigated in previous studies under another objective and the results have been published. 5,6 The diagnoses thereof were non-demented disease controls (NDC) (n = 15), AD (n = 21), DLB (n = 20) and OD (n = 18). CSF of AD, FTD and OD as well as three patients with depression came from the memory clinic of the University of Goettingen, other came from wards. Seven AD patients came from the dementia outpatient clinic of the University of Erlangen. The CSF of hospitalized patients with Parkinson s disease (PD), Parkinson s disease dementia (PDD) and DLB patients was recruited in the Paracelsus-Elena Klinik, Kassel, specialized in the diagnosis and treatment of movement disorders. Diagnoses were rendered by a psychiatrist, a neurologist and a neuropsychologist, all very experienced in clinical differential diagnosis of dementias, on the basis of thorough anamnesis, clinical examination, results of neuropsychological assessment, clinical records and best clinical judgment. All three investigators were blinded to the neurochemical outcome measures. Investigations were carried out with the informed consent of patients or their authorized caregiver. If possible, neuropsychological assessment (Mini-Mental-Status Examination (MMSE) at minimum) was performed on patients suffering from cognitive impairments at the time of lumbar puncture. The study was conducted under the guidelines of the Declaration of Helsinki 7 and approved by the ethics committee of the University of Goettingen, Erlangen-Nuremberg and Hessen. Test methods The preanalytical handling of CSF samples followed a standardized protocol according to previously published data. 8 Ab-SDS-PAGE/immunoblot. For analysis of Ab peptides, 10 ml of CSF were boiled in SDS-PAGE sample buffer 4,8 and Ab-SDS-PAGE/immunoblot was conducted as published elsewhere. 4,8 CSF samples of each individual patient were run as triplicates. Bands were quantified from individual blots of each patient relative to a four-point dilution series of synthetic Ab peptides 4 using a chargecoupled device camera. The detection sensitivity was 0.6 pg (Ab1-38, Ab1-40) and 1 pg (Ab1-37, Ab1-39, Ab1-42), respectively. The inter- and intra-assay coefficients of variation for pg of synthetic Ab peptides were below 10%. 4,8 All neurochemical measurements and quantifications were performed in the neurobiology laboratory of the University of Goettingen between 2003 and 2006 by two very experienced technical assistants blinded to clinical diagnosis. Electrochemiluminescence detection of Ab1-38. The novel electrochemiluminescence detection technology (MSD) was applied to determine CSF Ab1-38 levels independently of the Ab-SDS-PAGE/ immunoblot. It was conducted according to the manufacturer s recommendations (Meso Scale Discovery). In brief, Multi-Spot 4 96 well plates precoated with the N-terminal-specific anti-ab antibody 6E10 were blocked with solution A for 1 h. The plates were then incubated with peptide dilution series or 100 ml CSF sample, followed by C-terminal SULFO-TAG Ab 1 38 detection antibody and Read Buffer, 1 h each. Washing with 1 Tris buffer was performed between incubation steps. The measurement of emitted light was performed at B620 nm. Statistical analysis Patient groups were characterized by mean and standard deviation. Ab peptide values are given in absolute (ng/ml) and percentage values relative to the sum of all investigated Ab peptides (Ab1-X%). The Mann Whitney U-test was employed to determine significant differences of diagnostic groups (unpaired samples). Comparisons of multiple groups (age, MMSE) were evaluated by the Kruskal Wallis test. Correlations of measured values were estimated by Spearman s r. The two-sided level of significance was taken as P < The global diagnostic accuracies were assessed by the area under the curve (AUC) of receiver operating characteristic curve. Cut-off points were determined at the maximum Youden index, providing a sensitivity of X85%. The statistical software packages SPSS, version 10.0 and SAS, version 8.2 served for computations. Results Participants Non-demented disease controls. Patients with history of persistent cognitive decline for more than 6 months, MMSE score below 26 or clear focal atrophy in brain imaging, were excluded. The NDC group consisted of three subgroups: peripheral neurological diseases without organic brain affection (PND). Twenty patients (13 men and 7 women) underwent lumbar puncture for exclusion of central nervous affection in case of polyneuropathy (n = 11), peripheral facial nerve palsy (n = 3), benign

3 paroxysmal positioning vertigo (n = 2), intervertebral disk herniation (n = 1), facial hemispasm (n = 1), autosomal-dominant hereditary spastic spinal palsy (n = 1) and Lyme disease without central nervous affection (n = 1). Age of this subgroup was years (mean7s.d.). Neurodegenerative diseases without dementia syndrome (ND). Twenty-five patients (14 men and 11 women) underwent lumbar puncture to exclude chronic inflammatory central nervous disease in case of genetically reconfirmed Huntington s disease (n = 10), PD (n = 7), multisystem atrophy (n = 5), spinocerebellar ataxia (n = 2) and amyotrophic lateral sclerosis (n = 1). The MMSE score in patients with cognitive complaints (n = 9) was (mean7s.d.). None of these patients displayed clinical features of dementia syndrome. Age of this subgroup was years (mean7s.d.). Depressive cognitive complainers (DCC). Twenty-six depressive patients (8 men and 18 women) underwent lumbar puncture for differential diagnosis of cognitive complaints during the course of disease. The diagnosis of depression was made according to the criteria of DSM IV and cognitive impairment was assessed by MMSE at minimum. The mean MMSE score was (mean7s.d.). Age of this subgroup was years (mean7s.d.). Patients with Alzheimer s disease (AD). Seventy-one patients (29 men and 42 women) fulfilled DSM IV criteria and NINCDS-ADRDA criteria for clinical diagnosis of AD. 9 Structural (CT or MRI) or functional (SPECT or PET) brain imaging, respectively, displayed global cortical atrophy or temporal, parietotemporal, frontotemporal focal atrophy or marked hypometabolism of these regions. Patients with dementia with Lewy bodies (DLB). Thirty-two patients (19 men and 13 women) fulfilled the DSM IV criteria for dementia and the McKeith criteria for clinical diagnosis of DLB. 10 Patients presented with at least two core features according to the criteria 10 and with parkinsonism less than 1 year before onset of dementia. Enrolled patients were hospitalized for several days to evaluate fluctuating cognition, extrapyramidal symptoms and visual hallucinations. Patients with frontotemporal dementias (FTD). All 36 patients (22 men and 14 women) of this group fulfilled the DSM IV and the consensus criteria for FTD. 11 Detailed neuropsychological testing additional to MMSE, including clock drawing and CERAD, was carried out on 23 patients. Neuropsychological assessment was hindered in five patients by severe lingual or cognitive deficits. Structural (CT or MRI) or functional (SPECT or PET) brain imaging revealed frontal or frontotemporal focal atrophy or marked hypometabolism. Patients with other dementias (OD). Ninety-three patients (57 men and 36 women) fulfilled the DSM IV criteria for dementia. Patients with primary progressive aphasia (n = 10) fulfilled the consensus criteria of Neary et al. 11 Structural or functional (SPECT or PET) brain imaging revealed left anterior temporal focal atrophy or marked hypometabolism. The diagnosis of vascular dementia (VAD) was made in 27 patients according to NINDS-AIREN criteria. 12 All patients exhibited signs of relevant vascular disease in structural brain imaging (CT or MRI). PDD was diagnosed in 21 patients according to UK Parkinson s Disease Society Brain Bank clinical diagnostic criteria for idiopathic PD 13 and the consensus criteria. 10 All patients presented parkinsonism at least 1 year before onset of dementia. Normal pressure hydrocephalus according to the proposed criteria of Ishikawa 14 was diagnosed in nine patients. All these patients exhibited at least two symptoms of the typical triad and improved after spinal tap. Six patients fulfilled the criteria of probable progressive supranuclear palsy according to established NINDS-SPSP criteria. 15 Six patients were diagnosed as corticobasal degeneration according to the established criteria. 15 Seven patients suffering from sporadic Creutzfeld- Jakob s disease (CJD) were evaluated according to established criteria 16 at the national surveillance unit for transmissible spongiform encephalopathies in Goettingen, Germany. Seven patients with Korsakow s syndrome were evaluated according to the criteria of Oslin et al. 17 The mean age and MMSE score of each patient group is given in Table 1. Test results The mean age of FTD and NDC was significantly younger than all other patient groups. The mean MMSE score did not significantly differ between the dementia groups. The Ab-SDS-PAGE/immunoblot revealed a regular abundant pattern of six peptides: Ab1-40, Ab1-38, Ab1-42, Ab1-39, Ab1-37 and Ab1-40 ox. All Ab peptides were strongly correlated to each other throughout the whole group of patients (P < ). A correlation of the investigated Ab peptides with age was not found in any of the diagnostic groups. Neurochemical phenotype in NDC. There was no significant difference among PND and ND. In contrast, DCC exhibited higher absolute levels of Ab1-37 (P = ), Ab1-38 (P = ) and Ab1-42 (P = ) than a combined group of PND and ND. In percentage terms, Ab1-37% (P = ) and Ab1-42% (P = ) were increased, paralleled by diminished Ab1-40% (P = ) and Ab1-40 ox %(P = ) values in DCC. 673

4 674 Table 1 Absolute (ng/ml) and percentage abundances of CSF Ab peptides in the diagnostic groups Diagnosis NDC (n = 71) AD (n = 71) DLB (n = 32) FTD (n = 36) OD (n = 93) MW 7s.d. MW 7s.d. MW 7s.d. MW 7s.d. MW 7s.d. Age MMSE Ab Ab Ab1-38 (MSD) a Ab Ab Ab1-40 ox Ab total Ab b Ab1-37 c Ab1-38 c Ab1-39 c Ab1-40 c Ab1-40 ox c Ab1-42 c Abbreviations: AD, Alzheimer s disease; DLB, dementia with Lewy bodies; FTD, frontotemporal dementias; NDC, nondemented disease controls; OD, other dementias. a Electrochemiluminescence detection (MSD), subgroup of patients: NDC (n = 37), AD (n = 31), DLB (n = 2), OD (n = 47) and FTD (n = 33). b Total Ab peptide concentration. c Percentage abundance of Ab peptides relative to the total Ab peptide concentration. Neurochemical phenotype in AD. AD versus NDC AD presented with clearly decreased Ab1-42 levels in absolute (P = ) and percentage terms (P = ), whereas Ab1-40 ox levels were increased in absolute (P = ) and percentage (P = ) terms. Additionally, there was a percentage increase of all peptides C- terminally shorter than Ab1-42, which failed the level of significance for Ab1-37% and Ab1-38%. The elevation of Ab1-38% was highly significant compared with the ND group (P = ). AD versus all other dementias A specific decrease of Ab1-42 was evident in AD, whereas other dementias with low-ab1-42 levels displayed an overall decrease of all Ab peptides. Correspondingly, Ab1-42 was reduced in absolute (P = ) and percentage (P = ) terms. In contrast, absolute levels of Ab1-37 (P = ), Ab1-38 (P = ), Ab1-39 (P = ) and Ab1-40 (P = ) were elevated. In percentage terms, the aforementioned alterations were only present for Ab 1 38%, 1 39% and 1 42%, respectively. Neurochemical phenotype in DLB. DLB versus NDC DLB presented with higher Ab1-40 ox absolute (P = ) and percentage (P = ) levels. Conversely, absolute levels of Ab1-37 (P = ), 1 38 (P = ), 1 39 (P = ), 1 40 (P = ) and 1 42 (P = ) were lowered, although not evident in percentage terms for Ab1-37 and Ab1-39. DLB versus all other dementias Ab1-40 ox was elevated in absolute (P = ) and percentage ( ) terms. The overall decrease of other Ab peptides was still evident, but less pronounced than in NDC. Neurochemical phenotype in FTD. FTD versus NDC FTD showed lower levels of Ab1-37 (P = ), Ab1-38 (P = ) and Ab1-42 (P =610 5 ). In percentage terms, there was an additional increase of Ab1-40% values (P = ). FTD versus all other dementias FTD presented lower Ab1-38 (P = ) and Ab1-40 ox (P = ) levels, whereas Ab1-42 levels were elevated (P = ). In percentage terms, there were drops in Ab1-37% (P = ), Ab1-38% (P = ), Ab1-39% (P = ) and Ab1-40 ox % (P = ), paralleled by elevated levels for Ab1-40% (P = ) and Ab1-42% (P = ). Compared to PPA, there were decreased absolute Ab1-37 (P = ), Ab1-38 (P =110 2 ) and Ab1-39 (P = ) levels in FTD. Additionally, Ab1-40% was elevated (P = ) in FTD (see Figures 1 3).

5 Ab1-38 in the Ab-SDS-PAGE/immunoblot and electrochemiluminescence detection. A total of 150 patients were reevaluated using novel electrochemiluminescence detection technology (MSD). Diagnoses thereof were NDC (n = 37), AD (n = 31), DLB (n = 2), OD (n = 47) and FTD (n = 33). Absolute levels of peptide concentration were considerably lower in MSD compared with the Ab-SDS-PAGE/ immunoblot (see Tables 1 and 2). Conversely, there was a strong correlation of values between the two independent methods of measurement (Spearman s r = 0.45, P = ) (see Figure 4). 675 Estimates Neurochemically supported differential diagnosis. Figure 1 Mean and 95% confidence interval (CI) of Ab1-42% for each diagnostic group. Figure 2 Mean and 95% confidence interval (CI) of Ab1-40 ox % for each diagnostic group. Diagnosis of AD The striking drop of Ab1-42% enabled contrasts beyond 85% for discrimination of AD among the total of all investigated patients. A sensitivity of 85% gave a specificity of 81% for exclusion of all non-alzheimer dementias. Owing to decreased values of Ab1-38 and a less marked drop of Ab1-42 in dementias other than AD, the ratio of Ab1-38 to Ab1-42 (Ab1-42/1-38) improved the test slightly to contrasts of 85% or beyond for all investigated differential diagnostic questions. Otherwise, the sole absolute values of Ab1-42 yielded a specificity of 50% for exclusion of non-alzheimer dementias, when the sensitivity for AD detection was set to a minimum of 85%. Diagnosis of DLB DLB could be detected with a sensitivity and specificity of 88 and 83% among NDC with the use of Ab1-40 ox % levels. The discrimination of DLB from all other dementias was less accurate mainly owing to an overlap with increased Ab1-40 ox % in PSP, CBD and CJD. The percentage portion of Ab1-40 ox relative to Ab1-40 levels exhibited similar test accuracies for detection of DLB among NDC and all other dementias. Diagnosis of FTD The pronounced percentage reduction of Ab1-38% in FTD exhibited satisfactory accuracies above 85% for discrimination among all other dementias and NDC. The combination of decreased Ab1-38 and elevated Ab1-40 (Ab1-38/ 1 40) levels just failed to fulfill the requirements for differentiation of FTD and all other dementias, but still exhibited contrasts above 85% for detection of FTD among NDC. The loss of accuracy of the Ab1-38/ 1-40 ratio as compared to Ab1-38% was mainly because of elevated Ab1-40 levels in single AD, DLB and PPA patients. Table 2 summarizes the diagnostic accuracies and cut off points of the afore mentioned peptides for each differential diagnostic testing. Figure 3 Mean and 95% confidence interval (CI) of Ab1-38% for each diagnostic group. Discussion The Ab-SDS-PAGE/immunoblot revealed the regular abundance of the Ab peptides Ab1-37, Ab1-38, Ab1-

6 676 Table 2 Cut-off points and specificities at a minimum sensitivity of 85% of the best discriminating factor for each differential diagnostic testing Differential diagnosis Parameter Cut-off Sensitivity (%) Specificity (%) Youden index AUC 95% CI AD (n = 71) versus Ab1-42% all other patients (n = 232) Ab1-42/ AD (n = 71) versus Ab1-42% NDC (n = 71) Ab1-42/ AD (n = 71) versus Ab1-42% all other dementias (n = 161) Ab1-42/ DLB (n = 32) versus Ab1-40 ox % all other patients (n = 271) Ab1-40 ox /1-40% DLB (n = 32) versus Ab1-40 ox % NDC (n = 71) Ab1-40 ox /1-40% DLB (n = 32) versus Ab1-40 ox % all other dementias (n = 200) Ab1-40 ox /1-40% FTD (n = 36) versus Ab1-38% all other patients (n = 267) Ab1-38/ FTD (n = 36) versus Ab1-38% NDC (n = 71) Ab1-38/ FTD (n = 36) versus Ab1-38% all other dementias (n = 196) Ab1-38/ Abbreviations: AD, Alzheimer s disease; AUC, area under the curve; DLB, dementia with Lewy bodies; FTD, frontotemporal dementias; NDC, non-demented disease controls. Figure 4 Correlation of Ab1-38 levels as measured by the Ab-SDS-PAGE/immunoblot and electrochemiluminescence detection (MSD). 39, Ab1-40, Ab1-40 ox and Ab1-42 in all investigated CSF samples. In line with previous studies, 18,4 the absolute abundances of these peptides were strongly correlated to each other, suggesting a close regulation of the Ab peptides enzymatic processing and their post-translational modification. The most prominent alterations of the investigated Ab patterns were the expected decrease of Ab1-42 in AD, the increase of Ab1-40 ox in DLB and the decrease of Ab1-38 in FTD. Ab1-42 levels in CSF: neurochemical detection of AD The CSF pattern of diminished Ab1-42 and elevated tau concentration can be considered as typical, and has recently been reviewed by experts as an applicable AD biomarker. 19,20 Nevertheless, levels of decreased Ab 1-42 and increased tau can also be found in other dementias, such as VAD, 21 DLB, 22 FTLD 23 and CJD. 24 At a sensitivity of 85% for AD detection, the diagnostic value of these biomarkers for the exclusion of non-alzheimer dementias was therefore limited to 58%. 21 The detailed characterization of CSF Ab peptides by the Ab-SDS-PAGE/immunoblot has shown additional diagnostic value to discriminate AD from DLB, PDD and CJD. 5,24 In AD, the pronounced decrease of Ab1-42 seems to be counteracted by an upregulation of Ct shortened Ab peptides, leading to a highly selective reduction of Ab1-42 in percentage terms. In contrast, decreased absolute CSF Ab1-42 levels, paralleled by an overall reduction of all investigated Ab peptides, could be found for patients with DLB, PDD, CJD, PSP, CBD and in part VAD. This may particularly contribute to the loss of accuracy, when absolute Ab1-42 values are employed to discriminate AD from these dementias. Moreover, the evaluation of Ab1-42 relative to the sum of all investigated Ab peptides led to a reduced interindividual variance of values. 24 This may be explained by the assumption that the abundances of single Ab peptide species are closely correlated to each other and are thus regulated in narrow limits, whereas the total amount of Ab peptides varies interindividually. 4,24 The discriminative power of the selective Ab1-42% decrease for AD in a combined non-demented control and non-

7 Alzheimer dementia group was similar to what was reported for the p-tau-elisa Moreover, it was superior to reported accuracies of the widely accepted and established measurement of tau and Ab ,5,24 Ab1-42 levels in CSF: pathophysiological considerations The relationship between decreased levels of Ab1-42 in CSF and the occurrence of senile amyloid plaques needs to be clarified. The reduction of Ab1-42 levels in AD has long been explained by an increased clearance of the peptide from CSF into plaques. 26 Otherwise, dementias, where plaque formation is a rare feature, also exhibit low absolute levels of Ab In addition, the amount of measured Ab1-42 levels strongly depended on the method of measurement and preanalytical sample handling. For example, denaturizing of samples before measurement (e.g., SDS-heat pretreatment or presence of detergents) led to higher absolute Ab1-42 levels and demonstrated a more pronounced reduction of Ab 1-42 in AD. 8,26 The freezing of samples before denaturizing resulted in an irreversible loss of Ab peptides, markedly in the case of Ab Accordingly, we hypothesize that Ab1-42 may be bound to a specific carrier in an either SDS-labile or SDS-stable manner. In AD, the portion of SDS-stable peptide bindings may be increased at the cost of both the SDS-labile bound and the free peptide. The mutual dysfunction of a specific carrier for the peptide could contribute to a loss of its solubility, sufficient metabolism and consequently promote its aggregation. Thus far, candidates reported for such a carrier include apolipoprotein E and J, 27 SDS-stable oligomers 28 and supramolecular aggregates of Ab peptides. 29 Whatever mechanisms are responsible for the reduction of CSF Ab peptides, they seem to be restricted to Ab1-42 only in AD. In case of an overall reduction of Ab, either they also apply to other Ab peptide species or a reduced production of Ab must be considered. Ab1-38 levels in CSF: neurochemically supported diagnosis of FTD and PPA For lack of promising disease-specific biomarkers for FTD, previous studies have mainly focused on pronounced alterations of Ab1-42 and tau in AD as compared to FTD. Results on the performance of these biomarkers for differential diagnosis of FTD and AD have been inconsistent. 30,31,32 However, the pattern of mildly increased tau and moderately decreased Ab 1-42 is not unique for FTD, and it can be assumed that the diagnostic value of these biomarkers to discriminate FTD among dementias other than AD is limited. 33 A disease-specific biomarker test for FTD among dementias other than AD and differential diagnostic non-dementive disorders, such as depressive patients with cognitive complaints, has not yet been established. Analogous to the selective decrease of Ab1-42 in AD, the decrease in Ab1-38 was unique for FTD, only after determination of its percentage value (Ab1-38%). Ab1-38 levels in CSF: pathophysiological considerations As little is known about the role of Ab peptides in FTD, the pathogenic relevance of the above findings remains speculative. CSF Ab1-42 is reduced in FTD, whereas amyloid plaques are uncommon. This phenomenon has recently contributed to a putative binding protein that masks its epitopes to antibodies during immunologic detection. 4,8,32 Given that similar mechanisms may apply to CSF Ab1-38, the postulated carrier may possess a disease-specific affinity to Ab1-38 in FTD. However, the analogy between the characteristics of the reduction of Ab1-42 and Ab1-38 in AD and FTD, respectively, points to similar diseasespecific mechanisms causing this phenomenon. Alternatively, an overall reduction of Ab peptides could be counteracted by upregulating other Ab peptides, such as Ab1-40. The mutual involvement of Ct-truncated Ab peptides in pathogenic events of FTD is supported by a previous publication reporting a correlation between CSF Ab1-40 levels and the degree of frontal lobe atrophy in FTD. 34 In line with their results, we have found elevated Ab1-40% levels in FTD, when compared to all other dementias, even to PPA. Most noteworthy, presenilin1 mutations have been shown to be associated with FTD-like clinical phenotypes, one of which causes a Pick-type tauopathy without the development of amyloid plaques in neuropathological confirmation. 35 The molecular impact of presenilin1 mutations on APP processing and the etiology of FTD has been attributed to an altered or dysfunctional Notch processing. 35 For further elucidation of this aspect, especially with regard to CSF neurochemical phenotypes, we propose to investigate Ab peptide patterns in affected families and transgenic cell culture models. Ab1-40 ox levels in CSF: neurochemical detection of DLB The combined assay of tau and Ab1-42 ELISA revealed sensitivities and specificities of around 75% for discrimination of DLB among AD and nondemented controls, respectively. 36 The discrimination of these two dementias is mainly based on strikingly elevated tau levels in AD as compared to mild elevations in probable DLB. 37 A disease-specific CSF biomarker for applicable diagnostic testing of DLB among other dementias is not yet available. In a pilot study, we have shown promising results about discrimination of DLB from PDD using a pronounced increase of an oxidized and presumably a-helical form of Ab1-40 (Ab1-40 ox ) in DLB. 5 Likewise, the increase of Ab1-40 ox % was most specific for DLB in contrast to all other investigated patients in the present study. This enabled a detection of DLB with a sensitivity of 87% at a specificity of 70%. A prominent overlap of values with PSP, CBD and CJD hampered a more accurate discrimination. 677

8 678 Ab1-40 ox levels in CSF: pathophysiological considerations Given an oxidized and a-helical structure of the peptide, this would imply a particular relevance of oxidation and hydrophobic interactions in the pathogenesis of DLB. The occurrence of oxidative stress and amyloid pathology is well documented for DLB, and oxidized Ab comprises a major component of amyloid plaques. 38 a-synuclein facilitates the aggregation of Ab owing to hydrophobic interactions, 39 which have been shown to induce an a-helical structure of Ab in vitro. 40 Thus, a-synuclein may also induce an enhanced conformational shift of Ab into an a-helix in vivo. The a-helical Ab is specifically prone to aggregation, as the assembly of toxic oligomers 41 and b-sheet formation of Ab involve the transient formation of an a-helix, suggesting it to be an on-pathway to aggregation. 42 Moreover, the a-helix predisposes the peptide to oxidation. 43 The oxidized Ab is more hydrophilic 44 and less toxic 43 to neurons than the reduced peptide. Otherwise, metal ions may particularly trigger its aggregation owing to a facilitated release from the cell membrane. 44 This may result in Ab precipitation as a kind of seed around synapses, a quite sensitive site of the neuron. 44 Furthermore, one-electron oxidation of Ab by metal ions at met-35 produces a sulfuramyl free radical on the sulfur atom. 43 Taken together, the hydrophobic interaction between Ab peptides and a-synuclein may cause an overproduction of a-helical Ab that is prone to both fibril formation in its reduced and metal-dependent precipitation in its oxidized form. Additionally, a- helical Ab may undergo facilitated one-electron oxidation resulting in a toxic sulfuramyl radical. Thus, the increased abundance of Ab1-40 ox may indicate a disease-specific mechanism of amyloid toxicity in DLB. Ab-peptide patterns in differential diagnosis of dementias Tau, Ab1-42 and p-tau may differentiate AD from DLB, FTD and controls, but a specific biomarker for diagnosis of DLB or FTD among other dementias and differential diagnostic non-dementive disorders is still lacking. The expression of CSF Ab peptides varies among AD, DLB and FTD, resulting in unique peptide patterns as compared to NDC and other dementias. Ab peptide patterns came closest to the requirements for a useful biomarker in these dementias. 20 Previous reports of our group investigated considerably smaller groups of patients and focused on one or two crucial differential diagnoses. First, AD patients were evaluated in comparison to various non-dementive neuropsychiatric disorders and chronic inflammatory diseases (CID) of the central nervous system. 4 Although AD and CID shared a percentage increase of Ab1-38, AD could be separated from the other groups without any overlap by a selective decrease of Ab With respect to the differentiation of AD from non-dementive neuropsychiatric disorders, we were able to reproduce this in several subsequent studies, 5,6,8,24,36 including the present one. The examination of CJD in comparison to AD revealed a more pronounced selective drop of Ab1-42 in AD and the ratio of Ab1-42/1-39 yielded a sensitivity and specificity of 100% and 79%, respectively, in differentiating the two disorders. 24 Accordingly, the overlap of absolute Ab1-42 values could largely be reduced by the introduction of a ratio of Ab1-42 relative to Ct-truncated Ab peptides. 24 A similar finding was obtained in comparing Alzheimer s to Huntington s disease. 6 Next, the evaluation of DLB in comparison to AD and PDD revealed a novel potential biomarker (i.e., Ab1-40 ox ), which was markedly elevated in DLB and to a lesser degree in AD. 5 In fact, there was an overlap among the latter two disorders, but the sensitivity for detection of DLB and specificity for exclusion of all other investigated disorders were still 81 and 70%, respectively. 5 The slight improvement of accuracy of this marker for DLB in the present study may rely on (i) the larger study size, especially for DLB and AD, and (ii) the inclusion of other differential diagnostically relevant disorders that do not share the elevation of Ab1-40 ox in CSF (e.g., FTD). Two of the studies published so far have reported a direct comparison between absolute CSF values of Ab1-42 as measured by ELISA and the Ab-SDS-PAGE/ immunoblot and consistently found higher levels using the latter method. 8,37 This has led to the assumption that Ab1-42 may be bound to specific carrier proteins in CSF. In the present study, Ab1-38 levels were determined by both Ab-SDS-PAGE/immunoblot and electrochemiluminescence detection. The principle of detection in the latter method is comparable to a sandwich ELISA, as it relies on antibody-mediated binding of Ab. Analogous to Ab1-42, the Ab-SDS-PAGE/immunoblot yielded considerably higher values for Ab1-38 than electrochemiluminescence detection, suggesting that there might also be a portion of CSF Ab1-38, which is not freely accessible to antibodies. Obviously, SDS-heat denaturizing of samples increases the accessibility of their epitopes to respective antibodies in the immnuoblot, probably by stripping off the peptide from a putative binding protein. 8 Up to now, we have not directly compared the amounts of Ab1-40 in the same CSF samples as obtained by the Ab-SDS-PAGE/immunoblot and ELISA, respectively. However, the values measured by ELISA (The Genetics, Salzburg, Austria) in a previous study 45 are comparable to what the Ab-SDS-PAGE/immunoblot revealed in the present report. Given the above considerations, it can be assumed that, in comparison to Ab1-42 and Ab1-38, a larger portion of Ab1-40 is freely accessible to antibodies during the ELISA. In contrast, studies of others have shown discrepant results with regard to Ab1-40 and Ab1-38, respectively. Using their ELISA, Schoonenboom et al. 46 found higher absolute values for Ab1-40 in non-

9 demented control subjects and AD. Furthermore, they were the first to report CSF Ab1-38 levels as measured by ELISA. 46 Their results for Ab1-38 largely match what we found using the Ab-SDS-PAGE/immunoblot, but levels were considerably higher as compared to electrochemiluminescence detection. The reason for these discrepancies may include the application of different Ct and/or N-terminally specific anti-ab antibodies with distinct affinities and specificities, respectively, in each assay. However, whereas the measured absolute values of Ab peptides may depend on preanalytical sample handling and the method of measurement, their percentage amount has been widely found to be stable among these factors. 8 Once more, this argues in favour of percentage Ab peptide values instead of absolute ones for neurochemical dementia diagnostic use. Limitations of the study Although the Ab-SDS-PAGE/immunoblot can be considered as a quantitative and highly reproducible method 4,5,8,24 and has meanwhile also been used successfully by other groups, 47 the method is time consuming and requires specially trained personnel. This limits its application for high throughput screening (HTS) in routine neurochemical work-up to specialized centers. However, HTS-capable platforms and multiplex assays are upcoming neurochemical diagnostic tools that may include measurement of different Ab peptide species in addition to other biomarker candidates. In this respect, it is of tremendous value that we could demonstrate a good correlation between the measurement of Ab1-38 by Ab-SDS-PAGE/immunoblot and electrochemiluminescence detection. Further limitations of the study arise from the reliance on clinical diagnosis, which is claimed to misclassify 15 20% of dementia cases. The study size is limited for some rarer dementia forms, namely PSP, PPA, CBD, CJD and Korsakow s syndrome, but the high level of the AUCs shows the global diagnostic accuracy of the investigated factors. Conclusions The patterns of differentially expressed distinct Ab peptide species represent a biomarker candidate with disease-specific alterations among the three most frequent neurodegenerative causes of dementia, AD, DLB and FTD. The combined evaluation of the species Ab1-42, Ab1-38 and Ab1-40 ox relative to the total CSF Ab amount came close to the accuracy recommendations of an international consensus work group on applicable biomarkers for dementias and could be demonstrated to be clearly superior to absolute peptide values. Nevertheless, this study awaits confirmation from other work groups and validation by independent methods of measurement. In particular, HTS-capable methods will have to be evaluated for a future application of Ab peptide patterns in routine diagnostic neurochemical workup. Moreover, the data will have to be correlated with neuropathological data to draw more exact conclusions on their relationship to the specific pathogenic events in the respective dementias. Acknowledgments MB, PL, HE, SW, JK, MO and JW were supported by grants from the German Federal Ministry of Education and Research (Competence Net Dementia, grant O1 GI 0420); MB was supported by the Research Program, Faculty of Medicine, Georg-August-Universität Göttingen; JK, JW and PL were supported by grants from the German Federal Ministry of Education and Research CJK (01 GI 0301) and HBPP-NGFN2 (01 GR 0447). MO was supported by grants from the German Federal Ministry of Education (German CJD therapy study FK 01KO0506), EU (anteprion ) and Landesstiftung Baden Württemberg). References 1 Glenner GG, Wong CW. 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