Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 https://doi.org/10.1186/s13195-020-00696-1 RESEARCH Open Access Cerebrospinal fluid A beta 1–40 peptides increase in Alzheimer’s disease and are highly correlated with phospho-tau in control individuals Sylvain Lehmann1* , Julien Dumurgier2, Xavier Ayrignac3, Cecilia Marelli3, Daniel Alcolea4, Juan Fortea Ormaechea4, Eric Thouvenot5, Constance Delaby1, Christophe Hirtz1, Jérôme Vialaret1, Nelly Ginestet1, Elodie Bouaziz-Amar2, Jean-Louis Laplanche2, Pierre Labauge3, Claire Paquet2, Alberto Lleo4, Audrey Gabelle6 and for the Alzheimer’s Disease Neuroimaging Initiative (ADNI) Abstract Background: Amyloid pathology, which is one of the characteristics of Alzheimer’s disease (AD), results from altered metabolism of the beta-amyloid (Aβ) peptide in terms of synthesis, clearance, or aggregation. A decrease in cerebrospinal fluid (CSF) level Aβ1–42 is evident in AD, and the CSF ratio Aβ42/Aβ40 has recently been identified as one of the most reliable diagnostic biomarkers of amyloid pathology. Variations in inter-individual levels of Aβ1–40 in the CSF have been observed in the past, but their origins remain unclear. In addition, the variation of Aβ40 in the context of AD studied in several studies has yielded conflicting results. Methods: Here, we analyzed the levels of Aβ1–40 using multicenter data obtained on 2466 samples from six different cohorts in which CSF was collected under standardized protocols, centrifugation, and storage conditions. Tau and p-tau (181) concentrations were measured using commercially available in vitro diagnostic immunoassays. Concentrations of CSF Aβ1–42 and Aβ1–40 were measured by ELISA, xMAP technology, chemiluminescence immunoassay (CLIA), and mass spectrometry. Statistical analyses were calculated for parametric and non-parametric comparisons, linear regression, correlation, and odds ratios. The statistical tests were adjusted for the effects of covariates (age, in particular). (Continued on next page) * Correspondence: s-lehmann@chu-montpellier.fr Data used in preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in the analysis or writing of this report. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how_to_apply/ ADNI_Acknowledgement_List.pdf 1 Univ Montpellier, CHU Montpellier (LBPC-PPC), INSERM (IRMB, INM), Montpellier, France Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 2 of 12 (Continued from previous page) Results: Regardless of the analysis method used and the cohorts, a slight but significant age-independent increase in the levels of Aβ40 in CSF was observed in AD. We also found a strong positive correlation between the levels of Aβ1–40 and p-tau (181) in CSF, particularly in control patients. Conclusions: These results indicate that an increase in the baseline level of amyloid peptides, which are associated with an increase in p-tau (181), may be a biological characteristic and possibly a risk factor for AD. Further studies will be needed to establish a causal link between increased baseline levels of Aβ40 and the development of the disease. Keywords: Alzheimer’s disease, Amyloid peptides, Tau proteins, Biomarkers, Cerebrospinal fluid (CSF) Background especially indicative of an amyloidogenic process, while Alzheimer’s disease (AD) neuropathological brain lesions an increase in tau proteins (total tau and its phosphory- consist of aggregates of hyper-phosphorylated tau proteins, lated form p-tau (181)) is known to be associated with which have also been called neurofibrillary tangles (NFTs), axonal loss and tau pathology in AD [9, 10]. Tests on and extracellular deposits of amyloid precursor protein these two biomarkers are being included nowadays in (APP) derived amyloid-beta (Aβ) peptides, which are the international clinical research guidelines [11, 12], known as amyloid plaques. Much research has been focus- and many centers [11–15], and ourselves [13–15] have ing recently on the molecular mechanisms underlying integrated them into daily clinical practice. Importantly, these pathological events as it has become essential to these biochemical CSF measurements are concordant develop preventive and therapeutic strategies for AD. For a with the results of the PET imaging approaches which long time, the main explanation for the pathogenesis of were initially developed to determine the brain amyloid AD was that amyloidogenesis was the primum movens of load [16], and now also serve to measure tau accumula- the affection, which led to the concept of the amyloid tion [17]. These data are in line with hypotheses put cascade [1]. According to this picture of the disease, the forward by Jack et al. [18] about the chronology of the alteration of APP metabolism (increasing amyloid produc- evolution of biomarkers during the pathophysiological tion, decreasing clearance rates), the aggregation of Aβ process, and the relevance of amyloid markers in peptides, and the formation of amyloid plaques might particular at a very early stage, probably as early as 10 to result in microglial and astrocyte activation, local in- 15 years before the onset of clinical symptoms. flammatory responses, oxidative stress, and eventually Under non-pathological conditions, Aβ40 is highly the hyper-phosphorylation of tau proteins and second- correlated with Aβ42 [19]. The computation of the ratio arily the formation of NFTs [2]. Aβ42/40 is now being used in routine clinical practice The idea that amyloid peptides contribute importantly on AD patients in some centers [20–22]. This is a useful to the etiology of AD is supported by cases of AD who approach for reducing pre-analytical Aβ42 biases [23–25] carry presenilins (1 or 2) or APP mutations [3]. These and improving the diagnostic performances of CSF bio- gene mutations trigger the overproduction of Aβ markers [26], especially in discordant cases [27]. This ratio peptides or the preferential production of Aβ42, which can also be used to account for inter-individual amyloid is the most amyloidogenic of all the peptides. An APP variations in the baseline CSF level [28]. Low CSF Aβ40 gene dose effect triggering AD development, as occurs levels might also be indicative of frontotemporal dementia in Down syndrome [4] and in gene duplication processes (FTD) [29, 30], cerebral amyloid angiopathy (CAA) [31], [5], is a further/an additional potential factor contributing HIV [32], multiple sclerosis [33], or normal pressure to amyloid pathogenesis. Other genetic factors have been hydrocephalus (NPH) [34]. described, such as apolipoprotein E4 allele, in particular [6]. In their meta-analysis of AD biomarkers, Olsson et al. Studies on cerebrospinal fluid (CSF) biomarkers in AD [35] observed the existence of a negligible difference in have greatly improved our understanding of the patho- CSF Aβ40 between AD and control patients. Most of the physiology of this disease. The production of amyloid 32 studies considered had a limited number of patients peptides following the neuronal processing of APP has included in each group (the median number of subjects been involved in the response to physiological challenge per group was less than 30, and the maximum number with neurotrophic, anti-microbial, tumor suppression, or of subjects was 137 and 328 in the AD and non-AD synaptic function regulation activities [7]. Regarding tau groups, respectively). The focus of these studies was also proteins which are associated to microtubules, their quite different, looking at the diagnostic interest of physiological secretion by neuronal cells is a recent Aβ42/40 in AD and of Aβ peptides in other neurodegen- discovery which physiological relevance and benefit are erative diseases, or being more interested in patho- still matter of debate [8]. A decrease in CSF Aβ42 is physiological mechanisms. Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 3 of 12 The rationale for this study is therefore based on such as MRI. The possible AD category without amyloid or several elements: first, to resolve the controversy tau biomarker evidence of AD corresponds to the mild cog- conflicting studies showing or not an increase in Aβ40 nitive impairment (MCI) in the absence of core features of in AD; second, to provide explanations for the very good synucleinopathies, frontotemporal dementia (FTD), primary diagnostic performance of the Aβ42/40 ratio calculation; progressive aphasia, or evidence for another concurrent, ac- and finally, in the context of the pathophysiology of the tive neurological disease, or a non-neurological medical co- disease, to provide elements linking, in sporadic AD, the morbidity or use of medication that could have a level of expression of amyloid peptides and the disease. substantial effect on cognition. Subjective cognitive impair- We revisited the issue of the Aβ40 levels using large ment (SCI) corresponds to a situation where a patient re- series of multicenter data. The results show the occur- ports an alteration of their cognitive functions, including rence of a significant age-independent increase in CSF memory, but this cannot be documented by clinical, neuro- Aβ40 in AD. Another noteworthy finding was the exist- psychological, imaging, or biological tests. ence of a strong positive correlation between CSF Aβ40 The NAD diagnosis included SCI, CAA, NPH, FTD and the p-tau (181) concentration, even in patients with- based on relevant criteria [38], dementia with Lewy out Alzheimer’s disease (NAD). These findings suggest bodies (based on the McKeith criteria [39]), corticobasal that the baseline amyloid peptide level may constitute a degeneration (based on the criteria defined by Boeve risk factor contributing to sporadic AD, which is associ- et al. [40]), progressive supranuclear palsy, amyotrophic ated with p-tau (181) production. lateral sclerosis, and vascular dementia (based on the usual consensus diagnostic criteria). All the patients at Methods each clinical center gave their written informed consent Study design and subjects to participating in clinical research on CSF biomarkers, Patients with cognitive impairments were recruited which was approved by the respective Ethics Commit- and followed at the Montpellier and Paris Memory tees. The committee responsible in Montpellier was the Resources Center (CMRR). The Montpellier partici- regional Ethics Committee of the Montpellier University pants were subdivided into two cohorts which were Hospital and Montpellier CSF-Neurobank #DC-2008- recruited during different periods: Montpellier 1 (Mtp-1) 417 at the certified NFS 96-900 CHU resource center (recruited from July 2015 to May 2017) and Montpellier 2 BB-0033-00031, www.biobanques.eu. Authorization to (Mtp-2) (recruited from September 2009 to June 2015). handle personal data was granted by the French Data Pro- These two periods corresponded to the use of different tection Authority (CNIL) under the number 1709743 v0. ELISA kits (SupTable 1). The cohort Mtp-1 consisted of Two sets of data originating from the analysis of CSF 400 patients (126 AD, 274 NAD), and the cohort Mtp-2 samples at the Alzheimer’s Disease Neuroimaging Initia- consisted of 504 patients (220 AD, 284 NAD). The Paris tive (ADNI) database (www.loni.ucla.edu/ADNI) were cohort consisted of 624 patients (299 AD, 325 NAD) from used after the agreement of the scientific committee. the Centre de Neurologie Cognitive, Groupe Hospitalier ADNI UPEN-RESULTS, UPEN-ELYCYS (n = 311), and Lariboisière Fernand-Widal (recruited from March 2012 MS UPENNMSMSABETA (n = 400) data were also used. to May 2017). The Barcelona SPIN (Sant Pau Initiative on In the ADNI cohorts, which included many patients with Neurodegeneration) cohort (79 AD, 148 NAD) consisted mild cognitive impairments (MCI), we had to rely on the of patients who had undergone lumbar puncture for CSF biological PLM (Paris-Lille-Montpellier) scale [41] to AD biomarkers at the Sant Pau Memory Unit [36, 37] define populations with a low (ADNI(−)) and high (recruited from May 2009 to December 2017). All the (ADNI(+)) prevalence of AD. This scale combines the patients underwent a thorough clinical examination concentration of the three CSF biomarkers [Aβ42, tau, including biological lab tests, neuropsychological assess- p-tau (181)] into a probability scale for AD. The score ments, and brain imaging. The same diagnostic procedure ranges from 0 to 3 based on the number of abnormal [27] and AD diagnostic criteria [11] were used at all the CSF biomarkers. ADNI(−) population corresponded to clinical centers which participated. We included in the PLM scale of 0 or 1 with less than 25% of AD, while AD group patients which, in the absence of substantial ADNI(+) corresponded to PLM scale of 2 and 3 with concomitant cerebrovascular disease, meet the AD core more than 75% of AD. Importantly, the PLM score used clinical criteria and are considered as probable AD or was not based on the Aβ40 values so as to prevent circular possible AD with biomarker evidence of AD (CSF amyl- reasoning. This way of stratifying patients in the ADNI oid, tau, FDG-PET, or structural MRI) [11]. Although CSF cohort represents anyway a limitation of our study. amyloid biomarkers were included in the diagnostic criteria for AD, we observed that between 2 and 7% of AD CSF samples and assays cases, depending on the cohort, were not based on evi- CSF was collected using standard conditions of collection, dence of amyloid biomarkers but rather on other elements centrifugation, and storage [42, 43]. CSF tau and p-tau Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 4 of 12 (181) concentrations were measured using the standardized were adjusted to account for the effects of covariates (age, commercially available INNOTESTR sandwich ELISA, in particular). Odds ratios corresponded to the presence of Luminex® xMAP technology (x = analyte, MAP = Multi-An- AD in the various percentile groups, based on the distribu- alyte Profiling) assays in line with the manufacturer’s in- tion of Aβ40. The 95% confidence odds ratio intervals were structions (Fujirebio-Europe). The consistency of the p-tau computed along with the z-statistics and the associated (181) detection using the ELISA assays is ensured by its P values. comparison with the mass spectrometry detection per- formed in this fluid [44]. In the Mtp-1 cohort, CSF Aβ1–42 Results and Aβ1–40 (denoted here by Aβ42 and Aβ40) were CSF Aβ42 and Aβ40 in AD and NAD populations measured with Euroimmun kits (EQ-6511-9601 (Aβ1–40); CSF data on 2466 samples originating from six different EQ-6521-9601 (Aβ1–42)). In the Mtp-2 and Paris cohorts, cohorts were included in the present study. The AD and CSF Aβ42 and Aβ40 were measured using INNOTESTR NAD populations were defined based on clinical criteria sandwich ELISA from IBL and Fujirebio, respectively, as in the Montpellier 1 (Mtp-1), Montpellier 2 (Mtp-2), recommended by the manufacturer. Roche Elecsys auto- Paris, and Barcelona cohorts. For the ADNI cohort, we mated chemiluminescence immunoassay (CLIA) and mass relied on the PLM score to distinguish ADNI(+) and spectrometry were used on the ADNI cohorts to measure ADNI(−) population. Differences were observed in terms CSF Aβ1–42 and Aβ1–40 as previously described [45, 46]. of age and CSF biomarker profiles in the overall popula- Detection limits of these kits are compatible with CSF clin- tion, as well as in each clinical cohort (Table 1). As was ical ranges. Average concentration of analytes may differ to be expected, AD patients obtained lower MMSE between kits in relation with standard value assignments by scores and showed higher CSF tau and p-tau (181) levels the vendors in the absence of certified reference materials. than NAD patients/participants. A significant decrease The pre-analytical procedure was standardized [43] in the CSF Aβ42 concentrations was observed in the AD but differed, depending on the type of collection tubes population (Fig. 1a), regardless of the cohort tested. used [36, 47]. This explains the differences observed Noteworthy differences were also observed in the Aβ40 between cohorts in the mean Aβ40 and Aβ42 values levels between all the cohorts (Fig. 1b): the values re- measured with the same detection kit (SupTable 1). The corded in the AD population were significantly higher quality of the results was ensured by using validated than in the NAD group regardless of analytical method, standard operating procedures and internal quality the sex, or the age as covariate. We then conducted a controls (QCs). The QC coefficient of variation obtained meta-analysis study including the four independent on the CSF analytes in each batch and between batches cohorts using the Hedges g statistic as a formulation for ranged consistently below 15%. In addition, external QC the standardized mean difference (SMD) under the fixed procedures were used to confirm the quality/accuracy of effects model (Fig. 2). The overall SMD with 95% CI is the results [42]. In the case of the ADNI cohorts, CSF at 0.5, which is greater than 0.2, the significance level. samples were deep frozen after the lumbar puncture The difference between AD and NAD population for without performing any centrifugation or aliquoting, and Aβ40 was however limited with an important overlap shipped to the UPENN ADNI Biomarker Laboratory resulting in AUCs lower than 0.8 (Sup Figure 2). The in Philadelphia on dry ice, where they were thawed, stratification of the ADNI population using the PLM aliquoted, and re-frozen. scale (combining Aβ42, tau, and p-tau (181)) limits the interpretation of the Aβ40 results of this cohort in our Statistical analysis study. We have nevertheless observed that the ADNI(+) Statistical analyses were computed with the MedCalc group, which by definition has less Aβ42, did indeed software program (18.11.3). Data tested for normality have more Aβ40 (Fig. 1c). The physiological correlation were expressed either as means ± SDs or as median between CSF Aβ40 and Aβ42 [19] is therefore not well 25th/75th percentile, and differences between groups preserved in AD. were taken to be significant in the Student t tests or the When we stratified and combined the clinical cohort non-parametric Mann-Whitney U test at P < 0.05. Linear populations into AD, MCI, FTD, Control (SCI), and regression was computed between continuous biomarkers, other neurological diseases (Other) groups, the difference and the corresponding Pearson correlation coefficients and between AD and the other clinical groups was eventually statistical significance have been specified in the tables. For confirmed (Fig. 3a). We also performed a more in-depth meta-analysis of the study, we used the Hedges g statistic comparison, stratified by clinical groups. Importantly, the as a formulation for the standardized mean difference Paris and SPIN-Barcelona cohorts are composed of only a (SMD) under the fixed effects model. The SMD Hedges g small number of pathologies whereas the Montpellier is the difference between the two means divided by the cohorts have a wide range of diagnoses corresponding to pooled standard deviation. When indicated, statistical tests the Memory consultations (see the “Methods” section). As Table 1 Demographical and cerebrospinal fluid (CSF) biomarkers’ characteristics of the six cohorts: Montpellier 1 (Mtp-1), Montpellier 2 (Mtp-2), Paris, SPIN-Barcelona, ADNI-MS, and ADNI-Elecsys Cohort/analyte NAD AD P Cohort/analyte NAD AD P Mtp-1 Mean SD Mean SD SPIN-Barcelona Mean SD Mean SD Aß40 7035 2800 8579 3274 < 0.0001 Aß40 6799 3633 7985 2891 0.0128 Aß42 909 399 561 237 < 0.0001 Aß42 793 288 440 81 < 0.0001 Tau 343 279 739 267 < 0.0001 Tau 284 223 727 392 < 0.0001 p-Tau 42 16 96 36 < 0.0001 p-Tau 47 22 103 47 < 0.0001 Age 67.0 11.4 70.7 9.2 0.0015 Age 66.1 10.1 70.8 8.3 0.0004 MMSE 22.1 6.2 19.8 7.5 0.1392 MMSE 26.6 6.2 23.5 4.6 0.0097 Sex (%M) 53.6% – 38.1% – 0.0040* Sex (%M) 57.4% – 38.0% – 0.0055* Lehmann et al. Alzheimer's Research & Therapy ApoE (% E4) NA ApoE (% E4) 63.3% – 49.4% – 0.1910* Cohort/analyte NAD AD P Cohort/analyte ADNI(−) ADNI(+) P Mtp-2 Mean SD Mean SD ADNI-MS Mean SD Mean SD Aß40 12,720 6046 16,802 6473 < 0.0001 Aß40 7387 2517 8101 2167 0.0032 (2020) 12:123 Aß42 821 369 700 334 0.0002 Aß42 1076 547 620 231 < 0.0001 Tau 321 248 655 302 < 0.0001 Tau 66 24 140 58 < 0.0001 p-Tau 42 18 89 37 < 0.0001 p-Tau 23 8 50 18 < 0.0001 Age 66.0 12.7 70.0 9.3 0.0001 Age 75.4 6.7 74.0 7.5 0.0430 MMSE 21.9 7.3 19.6 5.4 0.0031 MMSE 27.3 2.4 25.9 2.6 < 0.0001 Sex (%M) 51.4% – 50.9% – 0.9114* Sex (%M) 61.0% – 58.6% – 0.6349* ApoE (% E4) ND ApoE (% E4) 53.8% – 89.3% – < 0.001* Cohort/analyte NAD AD P Cohort/analyte ADNI(−) ADNI(+) P Paris Mean SD Mean SD ADNI-Elecsys Mean SD Mean SD Aß40 10,767 4468 13,149 5850 < 0.0001 Aß40 15,865 5265 19,417 5224 < 0.0001 Aß42 925 277 574 200 < 0.0001 Aß42 1039 610 673 269 < 0.0001 Tau 236 134 609 280 < 0.0001 Tau 241 68 433 148 < 0.0001 p-Tau 42 16 90 37 < 0.0001 p-Tau 21 6 45 18 < 0.0001 Age 65.2 9.4 70.7 8.1 < 0.0001 Age 72.9 7.3 72.8 6.3 0.9155 MMSE 23.0 5.2 21.8 5.8 0.0069 MMSE 27.1 2.4 25.4 3.6 < 0.0001 Sex (%M) 51.9% – 38.5% – 0.0008* Sex (%M) 59.8% – 49.7% – 0.0816* ApoE (% E4) NA ApoE (% E4) 49.7% – 38.6% – 0.1538* Results are expressed as the mean ± standard deviation (SD). Abbreviation: MMSE Mini-Mental State Examination, AD Alzheimer’s disease, NAD non-Alzheimer’s disease, ADNI(−) cognitive patients with non-Alzheimer’s disease PLM profile, ADNI(+) cognitive patients with Alzheimer’s disease PLM profile, P significance level of the Student t test and *chi-squared test for the comparison of two proportions. Values of Aβ40, Aβ42, tau, and p-tau (181) are in picograms per milliliter Page 5 of 12 Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 6 of 12 not unexpected since the MCI group is itself heteroge- neous, with some patients progressing to AD in subse- quent years. As expected from previous studies [29, 30, 48], lowered Aß40 values for CAA and FTD were found. This is even more pronounced for NPH, as expected [34]. Based on this stratification, we can conclude that the in- crease in Aβ40 in the AD population is not a bias related to the presence of certain diagnoses possibly present in the NAD group. The influence of APOE status, which was available in the case of 983 samples (524 NAD with 36.6% E4+; 459 AD with 58.4% E4+), was also assessed with respect to the Aβ levels (SupFigure 1A-F). As previously reported [49, 50], the presence of ApoE4 was significantly associ- ated with lower Aβ42 levels, as well as lower Aβ40 levels. The difference in Aβ40 values between NAD and AD patients was also observed in both ApoE4 positive and negative populations (SupFigure 1G-I). To investigate more closely the relationship between Aβ40 and AD diagnosis, the total population was sorted into four percentile classes based on the value of this biomarker in the CSF (< 25th, 25th–50th, 50th–75th, and > 75th percentiles). The percentage of AD patients in each cohort clearly increased along with the Aβ40 percentiles (SupTable2). To account for the differences in AD prevalence between the cohorts, the odds ratios for AD were plotted in the case of increasing Aβ40 percentile classes, and a significant increase ranging from 0.4 to 1.8 was observed (Fig. 3b). To establish whether the difference in age observed between NAD and AD patients (SupTable 1) might be a significant determinant here, the age distribution between percentile classes was also plotted (Fig. 3c). A significant difference in age distribution was observed only between the 50th/ 75th and the > 75th Aβ40 percentile classes (Fig. 3c). Age cannot therefore account for the association between Aβ40 levels and AD prevalence. Nevertheless, further statistical tests were adjusted using age as covariate. Fig. 1 CSF Aβ42 and Aβ40 in non-AD and AD populations. CSF Correlations between Aβ40 and the other CSF biomarkers concentration of Aβ42 (a) and Aβ40 (b) in four independent cohorts The correlation between Aβ40 and the other CSF (Montpellier 1 (Mtp-1), Montpellier 2 (Mtp-2), Paris, SPIN-Barcelona) biomarkers was computed in global, NAD, and AD pop- confirmed the significant difference between NAD and AD patients ulations, for each cohort, and in the overall population for both analytes (t test). Differences in CSF Aβ40 measured in two (Table 2). As was to be expected [19], a correlation was ADNI cohorts (c) were also significant between ADNI(+) and ADNI(−) patients stratified using the PLM scale (see the “Methods” section). found to exist between Aβ40 and Aβ42, especially in the Note that Aβ has been assessed using five different detection NAD group. Aβ40 was also correlated with the tau methods (supTable 1) levels, and it was striking that the highest correlation coefficients were obtained with p-tau (181) rather than with t-tau, especially in the Mtp-1 cohort (a significant illustrated in the Sup Figure 3, we observed that AD difference was observed between the correlation coeffi- patients have significantly higher levels of Aß40 than most cients at P = 0.02). The correlation was clearly visible other diagnostic groups, with the exception of MCI (in when the mean-centered p-tau (181) values were plotted two out of three cohorts, there was no statistical difference in the various Aβ40 percentile classes (Fig. 3d), showing between AD and MCI patients). However, this result is significant differences between classes. This correlation Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 7 of 12 many groups are now also measuring Aβ40 in their patients. A meta-analysis was however not conclusive regarding its differential levels in AD [35]. Looking back in detail at various studies, Aβ40 either was lower or showed no significant changes [21, 29, 30, 53], or appar- ently increased in AD in comparison with other forms of dementia [54, 55]. In a recent report, the increase in Aβ40 was clearly identified as one of the reasons for the good performances of the Aβ42/Aβ40 ratio as an index [20], while another study on PET amyloid findings also established that CSF Aβ40 increased in the PIB+ popula- tion [56]. These discrepancies might be linked to differ- ences in cohort composition, since the Aβ40 levels may be affected by various pathological conditions [29–31]. The stage of AD, corresponding to various levels of cere- bral atrophy probably reducing amyloid production [18], may also account for differences between studies. This is coherent with a recent study confirming the increase of Fig. 2 Meta-analysis including the four independent cohorts. We used Aβ40 in prodromal AD [57]. Differences in the precision with to compare the Aβ40 means between AD and NAD populations of the analytical methods used, combined with the size the Hedges g statistic as a formulation for the standardized mean difference (SMD) under the fixed effects model. The SMD Hedges g is of the cohorts, might also explain why only a small, non- the difference between the two means divided by the pooled significant difference between AD and NAD patients has standard deviation. The plot has marker sizes relative to study weight. been observed in some cases. The results of the different studies, with 95% CI, and the overall Nevertheless, the present study on CSF Aβ40 which standardized mean difference with 95% CI are shown. Cohen’s rule of confirms it increased in AD can be considered a “surprise.” thumb for interpretation of the SMD statistic is that a value of 0.2 indicates a small effect, a value of 0.5 indicates a medium effect, and a The fact that this result has not been clearly identified value of 0.8 or larger indicates a large effect previously is due to several factors, the first being that since the differences between populations are small, statistical significance requires a larger number of samples and meas- had to be put in perspective with the fact that both ana- urement methods that are as accurate as possible. Using lytes increased in AD with the patients’ age (SupTable 1), large cohorts, we were able to confirm the existence of an which justifies the adjustments made for age in our stat- age- and ApoE-independent increase in CSF Aβ40 in AD istical analysis. We illustrated graphically the correlation compared with other diagnostic consisting mostly of con- between p-tau (181) and Aβ40 in the all NAD and the trols and patients with other neurodegenerative diseases AD populations (Fig. 4a, b). We noticed that p-tau (181) and dementia. This observation is valid in different analyt- values were higher and more widely distributed in the ical contexts despite differences in the threshold or range AD population with a regression slope that was higher for biomarker measurement. Our finding provides add- than in the NAD population. To document further the itional explanations for the very good diagnostic perform- relationship between Aβ40 and p-tau (181) outside the ance of the Aβ42/40 ratio calculation [20]. Interestingly, context of AD, the correlation was tested in a series of Janelidze et al. [58] observed that some Aβ42 assays were clinically defined patients with multiple sclerosis [51] partly quenched by Aβ40. An increase of Aβ40 in AD as and FTD [52] (Fig. 4c, d). The corresponding correlation suggested by our study may therefore also indirectly con- coefficients were both significantly higher in these tribute to the diagnostic performance of Aβ42. It is worth groups than in the AD population (P < 0.001). mentioning that in the blood, where the amyloid peptide 42/40 ratio could well indicate the presence of brain amyl- Discussion oidosis [59, 60], it has been established that high Aβ40 The decrease in Aβ42 observed in the CSF of AD levels are associated with greater mortality rate in the eld- patients has attracted considerable attention in clinical erly [61]. The fact that the CSF and blood amyloid levels and research communities. This decrease is attributable are poorly correlated, however, makes it difficult at this to the accumulation of Aβ42 in the brain parenchyma, stage to extend the present conclusions to this fluid. along with a decrease in the rates of CSF clearance and The overlap in the CSF Aβ40 values between the AD an increase in the production of oligomeric/multimeric and NAD populations is worth noting, and the area forms. Since determining the CSF Aβ42/Aβ40 ratio under the receiver operating characteristic curve (AUC) provides a useful means of improving AD diagnosis [26], for AD diagnosis was under 0.8 in all the cohorts tested Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 8 of 12 Fig. 3 Aβ40 in different diagnoses; representation in percentile; AD odds ratio, age, and p-tau (181) distribution. The Montpellier, Paris, and SPIN- Barcelona cohorts displayed a large range of pathological samples from patients with Alzheimer’s disease (AD), mild cognitive impairment (MCI), frontotemporal dementia (FTD), Control (subjective cognitive impairment), and the other neurological diseases (Other) (see also Sup-Figure 3). Mean-centered Aβ40 values in these cohorts were combined and compared in the different clinical groups confirming the significant increase of the peptides in AD (a). The four cohorts (Montpellier 1 (Mtp-1), Montpellier 2 (Mtp-2), Paris, SPIN-Barcelona) have been sorted in four classes based on their Aβ40 percentile values as follows: p25, < 25th percentile; p25–50, 25th–50th percentile; p50–75, 50th–75th percentile; and p75, > 75th. The odds ratio for AD (b), the age of the patients (c), and the concentration of CSF p-tau (181) (d) were then plotted in each percentile class. Significant differences between classes are indicated Table 2 Age-adjusted Pearson’s correlation between Aβ40 and Aβ42, tau, or p-tau (181) values in the six cohorts (Montpellier 1 (Mtp-1), Montpellier 2 (Mtp-2), Paris, SPIN-Barcelona, ADNI-MS, ADNI-Elecsys), in the overall population using mean-centered values to account for level differences between analytical methods Aß40 All NAD or ADNI(−) AD or ADNI(+) correlation p-Tau Tau Aß42 p-Tau Tau Aß42 p-Tau Tau Aß42 Mtp-1 0.501 0.377 0.520 0.557 0.235* 0.764 0.504 0.443 0.613 Mtp-2 0.514 0.443 0.407 0.430 0.244 0.533 0.304 0.213* 0.469 Paris 0.419 0.36 0.188 0.421 0.145* 0.571 0.288 0.245 0.247 SPIN-Barcelona 0.468 0.396 0.161 0.483 0.348 0.419 0.355 0.256* 0.308* ADNI-MS 0.254 0.413 0.505 0.257 0.496 0.705 0.223* 0.470 0.574 ADNI-Elecsys 0.536 0.567 0.449 0.487 0.624 0.782 0.502 0.496 0.580 Overall 0.445 0.418 0.368 0.455 0.318 0.615 0.339 0.318 0.426 Computation has been done in the All population and in the NAD, AD, ADNI(−), and ADNI(+) groups. Correlation coefficient statistical value P < 0.001 for all but *P < 0.01 Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 9 of 12 Fig. 4 Correlation between Aβ40 and p-tau (181) in different clinical populations. To illustrate the correlation between Aβ40 and p-tau (181) (Table 2), the mean-centered concentrations of the two analytes in the total study population were plotted in NAD (a) and AD populations (b). Aβ40 and p-tau (181) concentrations were also plotted in a selection of multiple sclerosis (c) and FTD patients (d) (SupFigure2). CSF Aβ40 cannot therefore be used as a study between Aβ40 and p-tau (181) in AD is an add- diagnostic biomarker but could be taken to be a itional argument supporting this pathophysiological feature “risk factor” in view of the odds ratio of model. Tau has many phosphorylated isoforms [62, 63], almost 2 recorded on the population having the some of them believed to be more specific for AD than highest CSF Aβ40 concentration. The increased Aβ40 p-tau (181), highlighting the pathophysiological role might be a consequence of a reduced clearance of and therapeutic interest of kinases like PKA, CAMkII, amyloid peptides in sporadic cases and/or a higher or Cdk5. This isoform is however one of the best indi- production-lower degradation. This matches the fact cators of AD pathology in the CSF where it begins to that in autosomal dominant forms of AD linked to increase as two decades before the development of APP or presenilin mutations [3, 5] and in Down aggregated tau pathology [44]. In this work, we had to syndrome [4], an overproduction of amyloid peptides rely only on the correlation with p-tau (181) because it is thought to trigger the AD process, along with all its con- is the only isoform with in vitro diagnostic (IVD) certi- sequences, including tau protein hyper-phosphorylation, in fication and has been measured in large clinical co- particular. horts. The fact that this correlation was also present in In this context, baseline Aβ40 concentration could a control population including a subgroup of well- indicate subjects with risk of early AD development. defined FTD [52] and multiple sclerosis patients [51] The positive correlation found to exist in the present raises many questions, however. It is tempting to take Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 10 of 12 this relationship to confirm that Aβ peptides may degeneration; MCI: Mild cognitive impairment; MMSE: Mini-Mental State induce the phosphorylation of tau, as observed both Examination; MRI: Magnetic resonance brain imaging; NAD: Non-Alzheimer’s disease; NFT: Neurofibrillary tangle; NPH: Normal pressure hydrocephalus; in vitro and in vivo [64]. PET: Positron emission tomography; PiB: Pittsburgh compound B; PLM: Paris- Lille-Montpellier; QC: Quality control; ROC: Receiver operating characteristic; SD: Standard deviation; SCI: Subjective cognitive impairment Limitation Since the present study was based on a cross-sectional Acknowledgements design, and without neuropathological confirmation, Not applicable. further studies involving a longitudinal design are now Authors’ contributions required to confirm the idea that high baseline CSF Lehmann, Dumurgier, Lleo, and Gabelle had full access to all the data in the levels of Aβ peptides may have prejudicial effects, lead- study and take responsibility for their presentation and the accuracy of the ing to AD. analysis. Concept and design: Dumurgier, Gabelle, and Lehmann. Acquisition of the data (biological and clinical): Ayrignac, Marelli, Alcolea, Fortea, Thouvenot, Delaby, Hirtz, Vialaret, Ginestet, Bouaziz-Amar, Laplanche, Conclusions Labauge, and Paquet. Drafting of the manuscript: Dumurgier, Gabelle, and Lehmann. Administrative, technical, and material support: Hirtz and Delaby. In conclusion, our results indicate that an increase in The authors read and approved the final manuscript. the baseline level of amyloid peptides, which are associ- ated with an increase in p-tau (181), could be a bio- Funding logical characteristic of AD. Further studies will be This work was supported by “France Alzheimer” and via the French National Alzheimer effort (“Le Plan Alzheimer”). Data collection and sharing for this needed to establish a causal link between increased base- project was also funded by the Alzheimer’s Disease Neuroimaging Initiative line levels of Aβ40 and the development of the disease. (ADNI) (National Institutes of Health Grant U01 AG024904) and DOD ADNI (Department of Defense award number W81XWH-12-2-0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Im- Supplementary information aging and Bioengineering, and through generous contributions from the fol- Supplementary information accompanies this paper at https://doi.org/10. lowing: AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery 1186/s13195-020-00696-1. Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly Additional file 1 : SupTable 1. Number of patients/samples in each and Company; EuroImmun; F. Hoffmann-La Roche Ltd. and its affiliated clinical group and Aβ40 detection method used. company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC; Johnson & Additional file 2 : Sup-Table 2. Percentage of AD/ADNI(+) patients in Johnson Pharmaceutical Research & Development LLC; Lumosity; Lundbeck; the four classes based on the Aβ40 percentile values recorded in the Merck & Co., Inc.; Meso Scale Diagnostics, LLC; NeuroRx Research; Neurotrack various cohorts; p25: 25th percentile, p25-50: 25th-50th percentile; p50- Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal 75: 50th-75th percentile; p75: 75th. Imaging; Servier; Takeda Pharmaceutical Company; and Transition Additional file 3 : Sup-Figure 1. CSF Aβ42 and Aβ40 in Non-AD and Therapeutics. The Canadian Institutes of Health Research is providing funds AD populations with regards to ApoE status Mean-centered values of CSF to support ADNI clinical sites in Canada. Private sector contributions are Aβ42 (panels A-C) and Aβ40 (panels D-F) in total (all), NAD and AD popu- facilitated by the Foundation for the National Institutes of Health (www.fnih. lation with regards to ApoE4 allele presence (+) or absence (-). Panels G-I org). The grantee organization is the Northern California Institute for illustrate the difference in Aβ40 between AD and NAD in the population Research and Education, and the study is coordinated by the Alzheimer’s with the ApoE status determine, in the total (all), ApoE4 allele presence Therapeutic Research Institute at the University of Southern California. ADNI (+) or absence (-). data are disseminated by the Laboratory for Neuro Imaging at the University Additional file 4 : Sup-Figure 2. ROC curves of Aβ40, Aβ42 and Aβ42/ of Southern California. 40 ROC curves of Aβ40, Aβ42 and Aβ42/40 for the detection of AD in different cohorts. AUC of Aβ40 had lower values than the other Availability of data and materials biomarkers (Mtp-1 0.686 (0.638 to 0.731); Mtp-2 0.751 (0.711 to 0.788); The datasets used for the analyses are available from the corresponding Paris 0,679 (0.641 to 0.716); SPIN-Barcelona 0.730 (0.667 to 0,787)). author on reasonable request. Additional file 5 : Sup-Figure 3. Aβ40 in different clinical groups. The Ethics approval and consent to participate Montpellier, Paris and SPIN-Barcelona cohorts displayed a large range of All the patients at each clinical center gave their written informed consent pathological samples from patients with Alzheimer’s disease (AD), cere- to participating in clinical research on CSF biomarkers, which was approved bral amyloid angiopathy (CAA), frontotemporal degeneration (FTD), mild by the respective Ethics Committees. The committee responsible in cognitive impairment (MCI), normal pressure hydrocephalus (NPH), vascu- Montpellier was the regional Ethics Committee of the Montpellier University lar dementia (VASC), other neurological diseases (amytrophic lateral scler- Hospital and Montpellier CSF-Neurobank #DC-2008-417 at the certified NFS osis, Parkinson’s disease, Lewy Body dementia..) (Other) and control 96-900 CHU resource center BB-0033-00031, www.biobanques.eu. (subjective cognitive impairment). Box plots with median and 25th/75th Authorization to handle personal data was granted by the French Data percentile of CSF Aβ40 values are plotted for the four cohorts (panels A- Protection Authority (CNIL) under the number 1709743 v0. D). Note that the distribution of the diagnoses differ in the cohorts (see Methods). Non parametric Mann–Whitney U test were performed be- tween the AD and the other clinical groups. P values below statistical sig- Consent for publication nificance are in red. Not applicable. Competing interests Abbreviations The authors report no conflict of interest to disclose. AD: Alzheimer’s disease; ADNI: Alzheimer’s Disease Neuroimaging Initiative; APP: Amyloid precursor protein; AUC: Area under the curve; Aβ: Amyloid β; Author details 1 CAA: Cerebral amyloid angiopathy; CI: Confidence interval; Univ Montpellier, CHU Montpellier (LBPC-PPC), INSERM (IRMB, INM), CLIA: Chemiluminescence immunoassay; CMRR: Centre Mémoire de Montpellier, France. 2Centre de Neurologie Cognitive et Service de Biochimie Ressources et de Recherche; CSF: Cerebrospinal fluid; FTD: Frontotemporal et de Biologie Moléculaire, Groupe Hospitalier Lariboisière Fernand-Widal, Lehmann et al. Alzheimer's Research & Therapy (2020) 12:123 Page 11 of 12 INSERMU942, Université Paris Diderot, Paris, France. 3CHU de Montpellier, 21. Lewczuk P, Esselmann H, Otto M, Maler JM, Henkel AW, Henkel MK, et al. Département de Neurologie, INSERM, Univ Montpellier, Montpellier, France. 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