Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 https://doi.org/10.1186/s13195-021-00861-0 RESEARCH Open Access VAMP-2 is a surrogate cerebrospinal fluid marker of Alzheimer-related cognitive impairment in adults with Down syndrome Alberto Lleó1,2, Maria Carmona-Iragui1,2,3, Laura Videla1,2,3, Susana Fernández3, Bessy Benejam1,3, Jordi Pegueroles1,2, Isabel Barroeta1,2, Miren Altuna1,2, Silvia Valldeneu1,2, Mei-Fang Xiao4, Desheng Xu4, Raúl Núñez-Llaves1,2, Marta Querol-Vilaseca1,2, Sònia Sirisi1,2, Alexandre Bejanin1,2, M. Florencia Iulita1, Jordi Clarimón1,2, Rafael Blesa1,2, Paul Worley4,5, Daniel Alcolea1,2, Juan Fortea1,2 and Olivia Belbin1,2* Abstract Background: There is an urgent need for objective markers of Alzheimer’s disease (AD)-related cognitive impairment in people with Down syndrome (DS) to improve diagnosis, monitor disease progression, and assess response to disease-modifying therapies. Previously, GluA4 and neuronal pentraxin 2 (NPTX2) showed limited potential as cerebrospinal fluid (CSF) markers of cognitive impairment in adults with DS. Here, we compare the CSF profile of a panel of synaptic proteins (Calsyntenin-1, Neuroligin-2, Neurexin-2A, Neurexin-3A, Syntaxin-1B, Thy-1, VAMP-2) to that of NPTX2 and GluA4 in a large cohort of subjects with DS across the preclinical and clinical AD continuum and explore their correlation with cognitive impairment. Methods: We quantified the synaptic panel proteins by selected reaction monitoring in CSF from 20 non-trisomic cognitively normal controls (mean age 44) and 80 adults with DS grouped according to clinical AD diagnosis (asymptomatic, prodromal AD or AD dementia). We used regression analyses to determine CSF changes across the AD continuum and explored correlations with age, global cognitive performance (CAMCOG), episodic memory (modified cued-recall test; mCRT) and CSF biomarkers, CSF Aβ42:40 ratio, CSF Aβ1-42, CSF p-tau, and CSF NFL. P values were adjusted for multiple testing. Results: In adults with DS, VAMP-2 was the only synaptic protein to correlate with episodic memory (delayed recall adj.p = .04) and age (adj.p = .0008) and was the best correlate of CSF Aβ42:40 (adj.p = .0001), p-tau (adj.p < .0001), and NFL (adj.p < .0001). Compared to controls, mean VAMP-2 levels were lower in asymptomatic adults with DS only (adj.p = .02). CSF levels of Neurexin-3A, Thy-1, Neurexin-2A, Calysntenin-1, Neuroligin-2, GluA4, and Syntaxin-1B all strongly correlated with NPTX2 (p < .0001), which was the only synaptic protein to show reduced CSF levels in DS at all AD stages compared to controls (adj.p < .002). * Correspondence: obelbin@santpau.cat 1 Memory Unit and Biomedical Research Institute Sant Pau (IIB Sant Pau), Neurology Department, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, c/Sant Quintí, 77-79, 08025 Barcelona, Spain 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain Full list of author information is available at the end of the article © The Author(s). 2021 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. Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 2 of 10 Conclusion: These data show proof-of-concept for CSF VAMP-2 as a potential marker of synapse degeneration that correlates with CSF AD and axonal degeneration markers and cognitive performance. Keywords: Down syndrome, Alzheimer’s disease, Synapse, Biomarker, Cognitive decline Introduction of neurodegeneration in sporadic AD [18]. We also com- Alzheimer’s disease (AD) is the leading cause of death in pare the CSF profile of the synaptic panel proteins in adults with Down syndrome (DS), with a cumulative in- adults with DS to that of previously published data on cidence that exceeds 90% in the seventh decade [1–4]. NPTX2 and GluA4 in the same cohort [17]. Current standard cerebrospinal fluid (CSF) markers for AD in the DS population are restricted to surrogate Material and methods markers of amyloidosis (Aβ42:40 ratio, Aβ1-42) and tau- Objectives mediated neurodegeneration (p-tau) and neurofilament The primary objective of this study was to evaluate a light chain combined with neuropsychological assess- comprehensive panel of synaptic proteins as surrogate ment. However, neuropsychological assessment can be markers of early AD-related cognitive decline in adults confounded by substantial inter-individual variation in with DS from across the preclinical and clinical AD con- intellectual disability (ID). Therefore, there is an urgent tinuum, specifically exploring their relationship to cognitive need for objective markers of AD-related cognitive performance and AD biomarkers. impairment in people with DS to improve diagnosis, monitor disease progression, and assess response to disease-modifying therapies. Study design Synapse loss is an early event in AD [5] and one of the This is a single-center, cross-sectional study of CSF best pathological correlates of cognitive dysfunction levels of synaptic markers in adults with DS, sporadic [6–9]. As such, synaptic proteins that show AD-associated AD patients and cognitively normal controls. The study changes in biofluids are rapidly gaining attention as poten- (IIBSP-BMS-2018-103) was approved by the local ethics tial surrogate markers of AD-related synapse loss and may committee (Comité Ètic d’Investigació Clínica, Fundació be informative markers of early AD-related cognitive de Gestió Sanitària de l’Hospital de la Santa Creu i Sant dysfunction in adults with DS. Pau) and was conducted in accordance with the Declar- Neuronal pentraxin-2 (NPTX2), a protein involved in ation of Helsinki. All participants gave their written in- inhibitory circuit dysfunction [10], is a promising bio- formed consent to participate in the study. Non-trisomic fluid surrogate marker of inhibitory circuit dysfunction controls were selected from the Sant Pau Initiative in and cognitive decline in sporadic AD [11–13], vascular Neurodegeneration (SPIN) cohort, a prospective longitu- dementia [14], genetic frontotemporal dementia [15], dinal cohort at Hospital Sant Pau, Barcelona, Spain [19]. and Lewy body dementia [16]. We recently reported low Adults with DS were selected from the Down Alzheimer CSF NPTX2 concentrations in adults with DS across the Barcelona Neuroimaging Initiative (DABNI), a prospect- AD continuum, which correlated with cortical atrophy ive longitudinal cohort, linked to a population-based and reduced glucose metabolism. However, CSF NPTX2 health plan in Catalonia, Spain, led by the Fundació levels did not correlate with measures of cognitive de- Catalana Síndrome de Down and Hospital de Sant Pau cline in our DS cohort [17]. In the same study, we also [20]. Inclusion criteria for controls required the absence evaluated the glutamatergic receptor, GluA4, and found of a cognitive or neurological disorders and normal CSF no association with cognitive measures. core AD biomarker (Aβ1-42, Aβ42/40 ratio, t-tau, p-tau) The aim of this study was to evaluate a comprehensive concentrations using our validated cut-offs for sporadic panel of alternative synaptic proteins (Calsyntenin-1, AD [21]. For adults with DS, inclusion criteria for Neuroligin-2, Neurexin-2A, Neurexin-3A, Syntaxin-1B, participation in the study required that all participants Thy-1, VAMP-2) as surrogate markers of early AD- were over 18 years of age. Where consent was given, related cognitive decline in non-trisomic cognitively participants received a comprehensive neurological and normal controls (n = 20) and a large cohort of adults neuropsychological evaluation [22] and underwent a with DS (n = 80) from across the preclinical and clinical lumbar puncture to assess CSF biomarkers [20]. As in AD continuum, exploring their relationship to cognitive previous studies [4, 20], participants with DS were classi- performance. The panel comprises 8 proteins that were fied by neurologists and neuropsychologists, blind to shown to be specifically expressed at the synapse in biomarker data in a consensus meeting into asymptom- human frontal cortex postmortem tissue and show CSF atic AD (aDS), prodromal AD (pDS), and AD dementia alterations that precede clinical symptoms and markers (dDS) according to previously published criteria [20]. Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 3 of 10 Neuropsychological assessment peptides over the course of the experiment, we injected The level of ID in adults with DS was categorized a pool of all the samples over the duration of the mass according to the Diagnostic and Statistical Manual of spectrometric measurements and monitored the peak Mental Disorders (DSM), Fifth Edition, as mild, moder- area of the standard peptides. The GluA2 peptide was ate, severe, or profound ID, based on caregivers’ reports unstable and removed, thus resulting in the exclusion of of the individuals’ best-ever level of functioning and the GluA2 from the study. We processed the SRM transi- Kaufmann Brief Intelligence Test (KBIT) [23]. As tions using the dataProcess function of MSstats v3.5 previously described [20, 22], neurological and neuro- package in R [29] and removed transitions with psychological examination of the full range of cognitive between-run interference (betweenRunInterference- impairment included a semi-structured health question- Score< 0.8). One censored transition (VAMP-2 peptide) naire (Cambridge Examination for Mental Disorders of where endogenous log2 intensity was below the detec- Older People with Down Syndrome and others with tion cut-off designated by the MSstats package (8.49) intellectual disabilities [CAMDEX-DS]) [24] and a was removed. We used the EqualizeMedians function to neuropsychological battery including the Cambridge normalize the transitions and Tukey’s Median Polish to Cognition Examination (CAMCOG) adapted for intellec- generate a summarized value of transitions for each pro- tual disabilities in DS participants and was restricted to tein. Two peptides (Calsyntenin-1 and Neurexin-3A) those with mild and moderate ID. The Spanish version were excluded from the summarization as the endogen- of the cued recall test modified for use in people with ID ous peptide was not detected in all samples. The results (mCRT) [25] was used to evaluate episodic memory as for the two Tenascin-R peptides are not reported here previously described [26]. The total mCRT scores for due to the lack of synapse specificity of Tenascin-R [18]. immediate recall were calculated as free recall score + Data for the 3 GluA4 peptides from the same SRM cued recall score. experiment have been reported previously [17]. CSF collection, biomarker assessment Statistical analysis CSF samples were collected following international con- All statistical analyses were performed in R version 3.4.3 sensus recommendations [27] as previously described [30]. We excluded 1 data-point each for Neurologin-2, [28]. Samples had been previously stored at − 80 °C and Neurexin-3A, Syntaxin-1B, Thy-1, VAMP-2, NPTX2 p- had not been thawed prior to analysis. Commercially tau, and t-tau as outliers due to violation of the 3 × available fully automated immunoassays were used to interquartile range rule. The outlier values were from 3 determine levels of CSF Aβ1-42, Aβ1-40, NFL, total tau, different samples from the DS group. Group compari- and p-tau at threonine residue 181 (Lumipulse Aβ1-42 , sons were performed using χ2 test, t test, or linear Aβ1-40, total tau G, p-tau 181, Fujirebio-Europe, NFL regression. Where regression residuals deviated from a Simoa Quanterix, MA, USA) [21]. Gaussian distribution (Shapiro-Wilk p < 0.05), tests were performed on square root or log2 transformed values, Targeted liquid chromatography mass spectrometry (LC-SRM) which did not deviate from a Gaussian distribution We monitored a set of 22 proteotypic peptides corre- (Shapiro-Wilk p > 0.05). Raw values were used for those sponding to 10 proteins (Calsyntenin-1, GluA2, GluA4, sub-analyses. We used Pearson coefficients to assess Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, correlations. However, to account for the ceiling effect Tenascin-R, Thy-1 and VAMP-2) using the previously of cognitive tests we used Spearman coefficients on raw described selected reaction monitoring (SRM) method values to assess correlations with cognitive measures. [18]. Briefly, we digested individual CSF samples over- Linear regressions of cognitive data were performed on night and spiked isotopically labeled peptides (Pepotech raw data as transformations did not improve the distri- SRM custom peptides, grade 2, Thermo Fisher bution. When comparing the association of multiple Scientific) into each sample. We analyzed an equivalent synaptic proteins, p values were adjusted for multiple of 5 μl of each sample in a randomized order over a 120- testing using the Benjamini-Hochberg method. min gradient (0–35% ACN + 0.1% FA) in SRM mode using a triple quadrupole-Qtrap mass spectrometer Results (5500 QTrap, Sciex, Masachussetts) coupled to a nano- Demographics LC chromatography column (300 μl/min, 25-cm C18 Table 1 shows the demographic and clinical data for the column, 75 μm I.d., 2 μm particle size). We ran BSA participants included in the study, which included 20 technical controls between each sample. We used controls and 80 adults with DS from across the AD isotopically labeled peptides as internal standards. We continuum (40 aDS, 19 pDS, and 21 dDS). The mean visualized and analyzed transitions using Skyline 3.5 as age-at-analysis across the whole study was 44.5 years previously described [18]. To evaluate the stability of the (standard deviation; SD = 11.2). Compared to controls, Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 4 of 10 Table 1 Demographics and clinical data for study participants Controls aDS pDS dDS N 20 40 19 21 Age-at-analysis, years 47 (11, 24–64) 35 (9, 22–57)b 52 (4, 45–60) 52 (5, 42–62) % Female 60% 40% 42% 38% % Mild or moderate ID 0% 83% 79% 67% a c CAMCOG score NA 80/107 (11, 55–96, n = 31) 70/107 (13.8, 41–92, n = 11) 59/107 (13.9, 39–87, n = 10)c a c mCRT score (immediate) NA 35/36 (1.5, 30–36, n = 30) 20/36 (11.2, 0–36, n = 12) 15/36 (7.9, 0–32, n = 11)c mCRT score (delayed)a NA 12/12 (0.9, 8–12, n = 31) 6/12 (3.8, 0–12, n = 13)c 4/12 (3.3, 0–12, n = 11)c b b CSF Aβ42:40 ratio 0.11 (0.01, 0.08–0.12) 0.09 (0.02, 0.04–0.12) 0.05 (0.01, 0.03–0.08) 0.05 (0.01, 0.04–0.08)b CSF p-tau pg/ml 36 (8, 22–54) 35 (24, 10–122) 145 (86, 22–304)b 158 (82, 31–323)b b CSF t-tau pg/ml 243 (57, 167–366) 295 (166, 86–671) 936 (658, 118–2565) 959 (500, 212–1988) b CSF NFL pg/ml NA 355 (234, 65–1036) 1071 (767, 313–3123) 1387 (832, 627–3957) Mean values (standard deviation, range) are given for each variable across clinical groups. NA, not available. aIn individuals with mild/moderate intellectual disability (ID) only. bp < 0.05 compared to controls. cp < 0.05 compared to aDS the mean age was comparable in pDS (+ 5 years, p = .20) controls (all p < .0001). CSF NFL levels were available and dDS (+ 5 years, p = .13) but lower in aDS (− 12 for DS only and were elevated in pDS and dDS com- years, p < .0001). The male to female proportion was pared to aDS (p < .00001). comparable across clinical groups (p = .45). The level of ID in the adults with DS was classified as either mild/ CSF VAMP-2 levels show a distinct profile to other moderate (78% of cases) or severe/profound (22% of synaptic proteins in adults with DS cases), a proportion that was comparable across clinical The synaptic panel analyzed here includes 7 synaptic groups (p = .37). Cognitive tests were restricted to indi- proteins previously unpublished in this cohort (Calsyntenin- viduals with mild or moderate intellectual disability. As 1, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, would be expected, cognitive scores were sequentially Thy-1, and VAMP-2) and their comparison to 2 synaptic lower in pDS (CAMCOG; − 11, p = .02, mCRT immedi- proteins, NPTX2 and GluA4, previously reported in the ate; − 15, p < .0001, mCRT delayed; − 5, p < .0001) and same cohort [17]. We first sought to determine the degree of dDS (CAMCOG − 21 p < .0001, mCRT immediate; − correlation between CSF levels of the 9 synaptic proteins. 20. p < .0001, mCRT delayed; − 7, p < .0001) compared Figure 1 shows that in adults with DS, synaptic proteins, in- to aDS. As previously reported [20], the mean Aβ42:40 cluding Neurexin-3A, Thy-1, Neurexin-2A, Calysntenin-1, ratio (all p < .0001) was lower in all DS groups com- Neuroligin-2, GluA4, and Syntaxin-1B, were all correlated pared to controls, while mean CSF p-tau and t-tau (pair-wise r = .70 to .96, n = 78-80, p < .0001). They also all levels were higher in pDS and dDS compared to correlated with NPTX2 (pair-wise r = .56 to .84, n = 78-79, p Fig. 1 Pair-wise correlation coefficients of CSF levels of 9 synaptic proteins in DS and controls. r coefficients resulting from statistical tests performed in the DS group (red) and controls (blue) for the 8 synaptic panel proteins and NPTX2 are shown. Degree of shading is relative to size of r coefficients, which are shown in bold where p < .05 and italicized where p > .05. NPTX2 and GluA4 data for these samples are published in [17] Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 5 of 10 < .0001). VAMP-2 showed the weakest correlation with all (.73-fold, p = .01) and compared to the symptomatic other proteins (r = .47 to.69, n = 78-80, p < .0001). In con- group (pDS and dDS combined; .67-fold, p = .007). CSF trols, all proteins showed weaker pair-wise correlations than VAMP-2 intensities were comparable to controls in pDS in the DS group, although NPTX2, Neurexin-3A, Thy-1, (.98-fold, p = .52) and dDS (.93-fold, p = .52). This rela- Neurexin-2A, Calysntenin-1, Neuroligin-2, GluA4, and tive increase in CSF VAMP-2 at late AD stages in adults Syntaxin-1B were moderately correlated in at least one pair- with DS is supported by Fig. 2b, which shows that CSF wise combination (pair-wise r = .46 to .87, n = 20, p < .04). VAMP-2 directly correlated with age in DS (r = .43, n = VAMP-2 did not correlate with any other protein in controls 79, p < .0001). This association was also observed in a (pair-wise r = -.34 to .29, n = 20, p > .14). We took VAMP-2 linear regression analyses adjusting for degree of ID forward for further analyses due to its relative independence (adj.r2 = .16, n = 79, p < .002). Conversely, CSF VAMP-2 from NPTX2. inversely correlated with age in controls (r = −.51, n = 20, p = .02). The control and DS regression lines for CSF VAMP2 changes over the course of AD and with age VAMP-2 were non-overlapping at the earliest age in- in adults with DS cluded in the study (22 years old) and did not intercept Figure 2a shows that mean CSF VAMP-2 SRM inten- until the age of 42. CSF VAMP-2 was not associated sities were lower in individuals with DS compared to with AD diagnosis when controlling for age p = .61). controls (.84-fold, p = .04). Mean CSF VAMP-2 inten- Figure 2c shows the correlation between CSF VAMP-2 sities were lower in the aDS group compared to controls and CSF biomarkers of brain amyloid and tau pathology Fig. 2 CSF levels of VAMP-2 in non-trisomic controls and DS. a Violin plots show the distribution of SRM intensities for VAMP-2 quantified in CSF for non-trisomic cognitively normal subjects (controls) and adults with DS across AD stages; asymptomatic AD (aDS), prodromal AD (pDS) or AD dementia (dDS). The horizontal dotted line represents the mean value in controls. *p < .05, **p < .01 for linear regression using square root transformed VAMP-2 levels in 1 DS vs controls, 2 aDS vs controls and 3 pDS/dDS vs aDS. b Age-at-analysis (years) is plotted against VAMP-2 SRM intensities in controls and adults with DS. c CSF biomarkers; Aβ42:40 ratio, p-tau and NFL are plotted against VAMP-2 SRM intensities in adults with DS. Linear regression lines in b and c are shown for each group (see legends). Shaded areas represent standard error of the regression lines. The vertical dotted lines in c represent the validated cut-offs for biomarker positivity in sporadic AD Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 6 of 10 and axonal degeneration in adults with DS. VAMP-2 in- associated with any of the cognitive measures when versely correlated with the Aβ42:40 ratio (r = − .47, n = age was included as a covariate (all p > .43). 79, p < .0001) and directly correlated with p-tau, (r = .56, n = 78, p < .0001) and NFL (r = .57, n = 78, p < Compared to other synaptic proteins, VAMP-2 is the best .0001). To determine whether low CSF VAMP-2 is re- correlate of cognitive performance, age, and CSF amyloid lated to AD biomarker positivity in asymptomatic DS, and neurodegeneration markers in adults with DS we compared CSF VAMP-2 SRM intensities in the aDS Finally, we compared these findings for VAMP-2 to the group stratified by positivity for CSF Aβ1-42 using our other synaptic panel proteins and to NPTX2 and applied validated in-house cut-offs for sporadic AD. Compared a strict adjustment of p values to account for multiple to controls, CSF VAMP-2 SRM intensities were lower in testing. Calsyntenin-1 (p = .03), Neuroligin-2 (p = .02), individuals positive for CSF Aβ1-42 (0.67-fold, n = 17, p Neurexin-2A (p = .02), Neurexin-3A (p = .02), and Thy- = .009) but not in individuals negative for CSF Aβ1-42 1 (p = .03) were associated with ID in individuals aged < (0.78-fold, n = 23, p = .30). Thus, low CSF VAMP-2 is 35, suggesting some influence of neurodevelopmental related to AD biomarker positivity and changes over the factors on CSF concentrations of these proteins, albeit course of AD and with age in adults with DS. that the associations did not survive adjustment for mul- tiple testing (all adj.p < .14). The correlation of VAMP-2 CSF VAMP2 is associated with cognitive performance in with immediate recall (adj.p = .17) and association with adults with DS CAMCOG (adj.p = .41) did not survive adjustment for We next explored the relationship between CSF multiple testing. Variables associated with at least one VAMP-2 and measures of intellectual and cognitive synaptic protein (adj.p < .05) in DS are shown in Fig. 4. impairment in adults with DS. Mean CSF VAMP-2 The associations of CSF NFL and p-tau with each vari- SRM intensities were comparable across individuals able are also shown for comparison. VAMP-2 was the with, mild, moderate, and severe intellectual disability only synaptic protein to correlate with mCRT delayed (Fig. 3a) in aDS p = .31), pDS (p = .71), and dDS (p = recall (adj.p = .04) and age (adj.p = .0008) and was the .73). To determine whether neurodevelopmental best correlate of CSF Aβ42:40 (adj.p = .0001), CSF p-tau factors may influence CSF VAMP-2 concentrations, we (adj.p < .0001), and CSF NFL (adj.p < .0001). On the compared VAMP-2 across ID groups stratified by < 35 other hand, NPTX2 was the best correlate of CSF Aβ1-42 or 35+ (Fig. 3b). CSF VAMP-2 intensities were (r = .58, adj.p < .0001), showed the greatest fold-change comparable across individuals with mild, moderate, or across all AD stages (0.34 to 0.55-fold, adj.p < .002), and severe/profound ID in the younger (p = .15) and older was the only synaptic protein to show changes in pDS (p = .87) age groups. Furthermore, CSF VAMP-2 SRM (0.47-fold, adj.p = .002). NFL and p-tau remain the best intensities were not associated with K-bit score (Fig. correlates of cognitive performance in this population 3c) when analyzed independently (r2 = .02, p = .11) or and were not altered in the aDS group compared to when controlling for age (r2 = .19, p = .77). While Fig. controls (1.03 fold-change, p = .56 and 0.96 fold- 3d shows a similar regression line for VAMP-2 with change, p = .93). CAMCOG and mCRT scores, correlation analyses showed that VAMP-2 SRM intensities were associated Discussion with immediate (r = − .32, p = .02) and delayed (r = − Here, we report a comprehensive evaluation of synaptic .36, p = .007) recall in the mCRT test but not with proteins in CSF from adults with DS across the whole CAMCOG scores (r = − .19, p = .17). However, since clinical continuum of AD. We show that of the 9 synap- ID had a greater impact on CAMCOG score than on tic proteins evaluated, VAMP-2 is the only correlate of mCRT in our previous study [22], we performed re- cognitive performance and age in this relatively under- gression analysis of VAMP-2 and cognitive perform- studied population. We also show that while mean CSF ance including level of ID as a covariate. We found VAMP-2 levels were lower in asymptomatic adults with that both ID (t = − 4.04, p = .0002) and VAMP-2 (t = DS compared to cognitively normal controls, mean − 2.05, p = .04) were associated with CAMCOG score VAMP-2 levels were elevated at advanced AD stages. In- (model r2 = .27, p = .00002), while VAMP-2 (t = − creased CSF VAMP-2 correlated with low CSF Aβ42/40, 2.61, p = .01) but not ID (t = − 0.69, p = .49) was asso- increased CSF p-tau and NFL and worse cognitive ciated with immediate recall in the mCRT test (model performance. Thus, changes in CSF VAMP-2 are closely r2 = .10, p = .03). We observed a similar association related to CSF AD biomarkers and cognitive measures with delayed recall (VAMP-2; t = − 2.94, p = .005, ID t in adults with DS. = − 0.76, p = .45). Therefore, VAMP-2 was associated In controls, CSF VAMP-2 levels decreased with age with both CAMCOG and mCRT score in adults with and when compared across similar ages, VAMP-2 levels DS even when controlling for ID. VAMP-2 was not were lower in adults with DS compared to controls from Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 7 of 10 Fig. 3 Relationship between CSF VAMP-2 and measures of intellectual impairment and cognitive performance in DS. Violin plots show the distribution of CSF VAMP-2 SRM intensities in adults with DS grouped according to degree of intellectual disability and a AD diagnosis and b age group. Circles represent mean intensities and error bars represent standard error of the mean. VAMP-2 SRM intensities in adults with DS are plotted against quantitative measures of intellectual disability (c) and cognitive performance (d). Linear regression lines are shown for models in total DS dataset and standard error of the regression lines are shown as shaded region. Regression lines for individuals aged < 35 or aged 35+ (c) or with mild or moderate ID (d) are also shown (see legends) the earliest age included in the study (22 years old) and replicate the nonlinear CSF profile of the 8 synaptic did not converge until the age of 42. This finding sug- panel proteins previously reported across disease stages gests a distinct CSF profile of VAMP-2, and potentially a in sporadic AD [18], and (d) CSF VAMP-2 did not different mechanism of synaptic pruning, between correlate with K-bit score and was comparable between healthy aging and the presence of AD pathology and/or individuals classified as having mild, moderate, severe, or triplication of chromosome 21. It is possible that the profound intellectual impairment across all AD stages relatively lower CSF VAMP-2 levels in younger adults an in individuals aged < 35 (where AD pathology is less with DS compared to controls is a result of reduced likely to be a confounding factor), but did correlate with VAMP-2 expression from birth due to neurodevelop- age, AD biomarkers, and episodic memory performance. mental factors. However, several lines of evidence sug- Based on these findings, we propose that low VAMP-2 gest that CSF VAMP-2 levels change as a function of levels in these individuals may at least partially reflect AD as opposed to ID: (a) the association between CSF changes related to the preclinical phase of AD, similar to VAMP-2 and age was independent of ID, (b) CSF that previously report in sporadic AD where CSF VAMP-2 was comparable to controls in adults with DS VAMP-2 levels were nominally reduced in preclinical negative for the CSF amyloid marker, (c) the findings re- AD and significantly elevated in prodromal and demen- ported here for this genetically determined form of AD tia stages compared to cognitively normal controls [18]. Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 8 of 10 Fig. 4 Comparison of CSF profile of 9 synaptic proteins in adults with DS. Assessment of the 9 synaptic proteins and their association (adj. r2 or r coefficients) with immediate and delayed recall in the mCRT test (mCRTi and mCRTd), CAMCOG, age, CSF NFL, Aβ42:40, p-tau and Aβ1-42 and the fold-change (FC) compared to controls in adults with DS across clinical groups. Associations of NFL and p-tau with the same variables are shown for comparison (red for synaptic proteins, blue for NFL and p-tau). Degree of shading is relative to r coefficients or FC. Values are shown in bold where p < .05 and italicized where adjusted p > .05 (in the case of synaptic proteins this is Benjamini-Hochberg adjusted p). Quantification of NPTX2 and GluA4 and FC for NPTX2 have been published previously in [17] We have previously evaluated NPTX2 as a synaptic dysfunction in AD, DS, and other neurodegenerative marker in the same cohort reported here [17]. Similar to diseases, VAMP-2 may be a better surrogate marker of VAMP-2, CSF NPTX2 levels were lower in DS com- cognitive performance in adults with DS. pared to controls, albeit that NPTX2 was reduced at all A previous study reported that CSF NPTX2 correlates AD stages. In fact, here we report that compared to the well CSF levels of two other synaptic proteins, SNAP-25 other 8 synaptic proteins, NPTX2 was the only protein and neurogranin in sporadic AD CSF [12]. In this study, to be reduced at all AD stages compared to controls. we report that, with the exception of VAMP-2, CSF However, unlike VAMP-2, CSF NPTX2 did not correlate levels of synaptic proteins were highly inter-correlated in with cognition or age. The distinct expression and func- adults with DS and that VAMP-2 was the only synaptic tion of these two proteins could explain their distinct protein not to correlate with at least one other synaptic CSF profiles in adults with DS. VAMP-2 is expressed at protein in controls. the human cortical synapse with increased enrichment The novelty of VAMP-2 is that it was the only synap- compared not only to the other 7 synaptic panel proteins tic protein evaluated here to correlate with age (a surro- evaluated here but also to the widely used pre-synaptic gate measure of disease progression in DS) and mCRT marker, synaptophysin [18]. This high synapse specificity in the DS population. Similar to our previous study, we supports other studies that have shown that VAMP-2 is found that ID had a greater impact on CAMCOG score predominantly found at glutamatergic synapses [31] as than on mCRT [22] such that the association of VAMP- part of the synaptic exocytosis core vesicular complex 2 with mCRT score was evident without the need to [32] where it is necessary for regulating the releasable control for level of ID. The mCRT test is a version of pool of glutamate at the pre-synapse [33] and is also the CRT modified for use in DS and discriminates well critical for post-synaptic trafficking of glutamate between DS adults with and without dementia [26]. The receptor subunits, particularly in the CA1 region of the CRT test is considered a clinical marker of episodic hippocampus [34]. Reduced VAMP-2 brain expression memory disorders due to medial temporal damage, has been reported in AD [35]. NPTX2 is specifically especially in the CA1 field of the hippocampus [36], expressed by pyramidal neurons where it mediates which is consistent with the functional role of VAMP-2 activity-dependent strengthening of pyramidal neuron at CA1 synapses [34]. excitatory synapses on GABAergic parvalbumin inter- neurons [10]. Therefore, both VAMP-2 and NPTX2 are specifically expressed at distinct populations of synapses Study limitations where they have distinct functions that are critical for While DS and autosomal dominant cohorts with avail- synaptic transmission. We therefore propose that CSF able CSF are scarce, further replication of the findings levels of these 2 synaptic proteins may reflect degener- reported here in independent genetic AD and DS co- ation of distinct synapse populations. While NPTX2 re- horts would be valuable. A limitation of this study is the mains a promising surrogate marker of inhibitory circuit cross-sectional design, particularly in the analysis of Lleó et al. Alzheimer's Research & Therapy (2021) 13:119 Page 9 of 10 cognitive decline. Longitudinal studies are needed to Fellowhsip grant from the Institute of Health Carlos III (ISCIII, CP18/00011), fully establish the prognostic value of VAMP-2 in DS Spain, and M.F I is supported by the Jérôme Lejeune Foundation, France. PW, DS, and MX were supported by R35NS097966. cohorts. Availability of data and materials Conclusion The datasets used and/or analyzed during the current study are available NFL remains the best CSF correlate of cognition in this from the corresponding author on reasonable request. population and this work opens the door to future stud- Declarations ies exploring the prognostic capacity of CSF VAMP-2 in adults with DS and sporadic AD. The data reported in Ethics approval and consent to participate this manuscript show proof-of-concept for CSF VAMP- The study (IIBSP-BMS-2018-103) was approved by the local ethics committee (Comité Ètic d’Investigació Clínica, Fundació de Gestió Sanitària de l’Hospital 2 as a potential marker of synapse degeneration that cor- de la Santa Creu i Sant Pau) and was conducted in accordance with the relates with CSF AD and axonal degeneration markers Declaration of Helsinki. All participants gave their written informed consent and cognitive performance. Whether VAMP-2 could be to participate in the study. a useful addition to NFL to specifically monitor synapse Consent for publication engagement and therapeutic response, particularly in Not applicable. AD clinical trials would be an interesting avenue worth pursuing; as anti-tau and anti-Aβ are common therapies, Competing interests there is a need for an alternative surrogate measure of Drs Belbin, Lleó, Fortea, and Alcolea declare a filed patent application (WO2019175379 A1 Markers of synaptopathy in neurodegenerative disease) cognitive performance not directly affected by the drug. that includes the 9 synaptic proteins evaluated in this study. D.A. participated An ELISA-based immunoassay to facilitate the quantifi- in advisory boards from Fujirebio-Europe and Roche Diagnostics and re- cation of VAMP-2 in patient CSF is in development. ceived speaker honoraria from Fujirebio-Europe, Nutricia and from Krka Farm- acéutica S.L. J.F participated in advisory boards from AC-Immune and was Moreover, as VAMP-2 is also detectable in blood [37], consultant to Novartis. MX, DX and PW are co-founders of CogNext. whether plasma VAMP-2 can be used as a surrogate marker of brain VAMP-2 is also worth pursuing. Author details 1 Memory Unit and Biomedical Research Institute Sant Pau (IIB Sant Pau), Abbreviations Neurology Department, Hospital de la Santa Creu i Sant Pau, Autonomous AD: Alzheimer’s disease; aDS: Asymptomatic Alzheimer’s disease in Down University of Barcelona, c/Sant Quintí, 77-79, 08025 Barcelona, Spain. 2Centro syndrome; CAMCOG: Cambridge Cognition Examination; CSF: Cerebrospinal de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas fluid; DABNI: Down Alzheimer Barcelona Neuroimaging Initiative; (CIBERNED), 28031 Madrid, Spain. 3Barcelona Down Medical Center, Fundació dDS: Dementia due to Alzheimer’s disease in Down syndrome; DS: Down Catalana Síndrome de Down, Barcelona, Spain. 4Solomon H. Snyder syndrome; DSM: Diagnostic and Statistical Manual of Mental Disorders; Department of Neuroscience, Johns Hopkins University School of Medicine, KBIT: Kaufmann Brief Intelligence Test; ID: Intellectual disability; Baltimore, MD 21205, USA. 5Department of Neurology, Johns Hopkins mCRT: Modified cued recall test; NPTX2: Neuronal pentraxin-2; University School of Medicine, Baltimore, MD 21205, USA. pDS: Prodromal Alzheimer’s disease in Down syndrome; SD: Standard deviation; SPIN: Sant Pau Initiative in Neurodegeneration; SRM: Selected Received: 17 March 2021 Accepted: 14 June 2021 reaction monitoring; VAMP-2: Vesicle-associated membrane protein-2 Acknowledgements References We thank the participants and their families for their generosity. We also 1. McCarron M, McCallion P, Reilly E, Mulryan N. A prospective 14-year acknowledge the Fundació Catalana Síndrome de Down for global support; longitudinal follow-up of dementia in persons with Down syndrome. 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