Received: 30 March 2020 Revised: 28 July 2020 Accepted: 5 October 2020 DOI: 10.1002/alz.12229 F E AT U R E D A R T I C L E Nerve growth factor (NGF) pathway biomarkers in Down syndrome prior to and after the onset of clinical Alzheimer’s disease: A paired CSF and plasma study Rowan Pentz1 M. Florencia Iulita2,3,4 Adriana Ducatenzeiler2 Laura Videla3,4,5 Bessy Benejam3,4,5 María Carmona-Iragui3,4,5 Rafael Blesa3,4 Alberto Lleó3,4 Juan Fortea3,4,5 A. Claudio Cuello1,2,6,7 1 Department of Neurology and Neurosurgery, McGill University, Montreal, Canada 2 Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada 3 Sant Pau Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 4 Center of Biomedical Investigation Network for Neurodegenerative Diseases (CIBERNED), Madrid, Spain 5 Barcelona Down Medical Center, Fundación Catalana Síndrome de Down, Barcelona, Spain 6 Department of Anatomy and Cell Biology, McGill University, Montreal, Canada 7 Department of Pharmacology, Oxford University, Oxford, UK Correspondence A. Claudio Cuello, Department of Pharmacol- Abstract ogy and Therapeutics, Faculty of Medicine, McGill University, 3655 Sir-William-Osler Background: The discovery that nerve growth factor (NGF) metabolism is altered in Promenade, Room 1210, Montreal, QC H3G Down syndrome (DS) and Alzheimer’s disease (AD) brains offered a framework for the 1Y6, Canada. Email: claudio.cuello@mcgill.ca identification of novel biomarkers signalling NGF deregulation in AD pathology. Methods: We examined levels of NGF pathway proteins (proNGF, neuroserpin, tissue Rowan Pentz and M. Florencia Iulita con- plasminogen activator [tPA], and metalloproteases [MMP]) in matched cerebrospinal tributed equally to this study. fluid (CSF)/plasma samples from AD-symptomatic (DSAD) and AD-asymptomatic Funding information (aDS) individuals with DS, as well as controls (HC). National Institutes of Health, Grant/Award Number: RO1-AG056850; Canadian Consor- Results: ProNGF and MMP-3 were elevated while tPA was decreased in plasma from tium of Neurodegeneration in Aging; Fondo de individuals with DS. CSF from individuals with DS showed elevated proNGF, neuroser- Investigaciones Sanitario; Instituto de Salud Carlos III, Grant/Award Numbers: PI14/01126, pin, MMP-3, and MMP-9. ProNGF and MMP-9 in CSF differentiated DSAD from aDS PI17/01019, PI13/01532, PI16/01825, (area under the curve = 0.86, 0.87). NGF pathway markers associated with CSF amy- PI18/00335, PI14/1561, PI17/01896; CIBERNED; Fondo Europeo de Desarrollo loid beta and tau and differed by sex. Regional; Unión Europea; Una manera de Discussion: Brain NGF metabolism changes can be monitored in plasma and CSF, sup- hacer Europa; National Institutes of Health, Grant/Award Numbers: R21AG056974, porting relevance in AD pathology. These markers could assist staging, subtyping, or R01AG061566; Departament de Salut de la precision medicine for AD in DS. Generalitat de Catalunya; Pla Estratègic de Recerca i Innovació en Salut, Grant/Award This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2020 The Authors. Alzheimer’s & Dementia published by Wiley Periodicals, Inc. on behalf of Alzheimer’s Association Alzheimer’s Dement. 2021;17:605–617. wileyonlinelibrary.com/journal/alz 605 606 PENTZ ET AL . Number: SLT002/16/00408; Fundació la Marató de TV3, Grant/Award Numbers: KEYWORDS 20141210, 044412; Fundació Catalana Sín- Alzheimer’s disease, biomarkers, blood, cerebrospinal fluid, cholinergic, Down syndrome, metallo- drome de Down and Fundació Víctor Grífols i proteases, MMP-1, MMP-3, MMP-9, nerve growth factor, neuroserpin, NGF metabolic pathway, Lucas; Generalitat de Catalunya, Grant/Award plasma, proNGF, tissue plasminogen activator Number: SLT006/17/00119; Fundació Ban- caria La Caixa 1 INTRODUCTION our previous findings of dysregulation of NGF metabolism-associated proteins16,29 in a new, independent clinical cohort of individuals with Individuals with Down syndrome (DS) represent the largest geneti- DS, and extends it by showing that such changes also exist in CSF cally determined population at risk for Alzheimer’s disease (AD).1–6 and in fact better reflect the NGF metabolic changes observed in the A critical factor associated with cognitive decline in AD, both in the brain. We also demonstrate that CSF proNGF and MMP-9 can effec- general population and in DS, is the degeneration of the cortical fore- tively differentiate AD-symptomatic individuals with DS from those brain cholinergic system.5,7–14 Cholinergic neurodegeneration in these who were asymptomatic, and that proteins related to NGF metabolism disorders has been linked to dysregulation of the nerve growth fac- correlate to core biomarkers of AD, especially when measured in tor (NGF) metabolic pathway.15–19 This pathway controls the matu- CSF. ration and degradation of the neurotrophin NGF, on which forebrain cholinergic neurons depend.20 In this pathway, the tissue plasmino- gen activator (tPA)/plasminogen system is responsible for the matu- 2 MATERIALS AND METHODS ration of NGF from its precursor, proNGF, a process that is inhibited by neuroserpin.20,21 The extracellular degradation of mature NGF is 2.1 Study cases accomplished primarily by matrix metalloproteinase (MMP)-9,20 which can be activated by MMP-322 and is inhibited by tissue inhibitor metal- Adults with DS aged 18 years and older were recruited by the loproteinase (TIMP)-1.23 Alzheimer-Down Unit of the Fundació Catalana Síndrome de Down ProNGF levels have been found to be elevated in AD brains with and the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, total- advanced pathology.24 The NGF metabolic pathway has likewise been ing 37 cases who assented to blood and CSF extraction. Participants or found to be dysregulated in mild cognitive impairment (MCI), AD, and their legal representative gave written informed consent, as approved apparently healthy individuals with elevated brain amyloid17,25,26 as by the ethics committee of Hospital Sant Pau following the standards well as in the brains of people with DS and AD dementia.15 NGF dys- for medical research in humans recommended by the Declaration of regulation in the early phases of AD correlates to cholinergic dysfunc- Helsinki and reported to the Ministry of Justice according to the Span- tion and cognitive decline,26 and the ability to monitor this dysfunction ish law for research in people with intellectual disabilities. Subjects through biomarkers would have high potential utility in research and were required to understand and accept the study procedures to give clinical contexts.27,28 informed consent. Genetic screening of trisomy 21 aneuploidy was Correspondingly, an analysis of NGF metabolic pathway markers done as previously described, confirming full trisomy of chromosome in plasma samples from people with DS has previously revealed that 21 in 95% of participants.30 the levels of NGF metabolism-associated markers, including proNGF, Participants received a neurological and neuropsychological MMP-1, MMP-3, and MMP-9 were elevated in individuals with DS, examination which included the Cambridge Cognitive Examination with and without the cognitive symptoms of AD.16 Critically, it was (CAMCOG) and Cambridge Examination for Mental Disorders of demonstrated that longitudinal within-subjects increases in proNGF the Elderly-Down Syndrome (CAMDEX-DS) batteries, as previously over 1 year correlated with cognitive decline over the subsequent 2 described.30 Behavioral measures were assessed with the Neuropsy- years.16 chiatric Inventory.31 We stratified the level of intellectual disability While plasma is the most accessible body fluid for the measurement according to the Diagnostic and Statistical Manual of Mental Disor- of biomarkers, cerebrospinal fluid (CSF) can more faithfully reflect ders, Fifth Edition as mild, moderate, or severe or profound (which changes in the brain, as it is in direct contact with the extracellular were grouped together), based on the individuals’ best-ever level of central nervous system (CNS) milieu. Therefore, in this study, we ana- functioning as determined from carers’ reports. Participants were lyzed a unique set of matched plasma and CSF samples from adults classified as asymptomatic, without AD-related cognitive impairment with DS with and without AD dementia, as well as from healthy, non- (aDS; n = 14), prodromal AD (n = 10), or AD dementia (n = 12) in a DS controls. The use of matched samples offers a unique opportunity consensus meeting between the neurologist and the neuropsychol- to directly compare the performance of markers in the two most com- ogists who assessed them blind to the biomarker data, as previously monly analyzed biofluids as well as to assess their correspondence, described.30 One participant was classified as ’uncertain’ and excluded which could support the ability of plasma markers to reflect changes from analysis. Pilot studies with these samples indicated no significant in the CNS milieu. The present study confirms and elaborates upon differences in NGF biomarkers between the prodromal AD and AD PENTZ ET AL . 607 dementia groups, and these were therefore grouped together as a symptomatic AD group (referred to as DSAD) to conserve statistical RESEARCH IN CONTEXT power. 1. Systematic review: We used PubMed to review stud- Control subjects were healthy volunteers of both sexes (age range ies pertaining to the plasma/cerebrospinal fluid (CSF) 20 to 60 years, n = 16) recruited from the SPIN cohort (Sant Pau biomarkers of central nervous system cholinergic deficit Initiative on Neurodegeneration).32 Volunteers had no neurologi- and nerve growth factor (NGF) metabolism in Down cal deficits, a Clinical Dementia Rating (CDR) total score of zero, syndrome (DS). Basal forebrain cholinergic degeneration and with normal levels in core AD CSF biomarkers (amyloid beta and NGF metabolic deficits have been demonstrated in [Aβ]42/Aβ40, total tau [t-tau] and phosphorylated tau [p-tau]) assessed DS brains. One study in 2016 showed an altered NGF by Lumipulse (LUMIPULSE G600II automated platform, Fujirebio).33 metabolic signature in DS plasma. There were no reports For both cohorts (DS and controls), the period of recruitment was in CSF. between November 2013 and November 2015. Apolipoprotein E 2. Interpretation: Our findings indicate that NGF metabolic (APOE) allele genotyping was done as previously described.34 Table 1 alterations can be monitored in plasma and CSF in DS, illustrates the demographics of the populations included in this though CSF more closely reflects the changes observed study. in brain. Two CSF markers (proNGF 50 KDa and MMP-9) distinguished AD-symptomatic individuals from the over- all DS population. These markers could have relevance 2.2 Plasma collection for diagnosis, staging, subtyping, or precision medicine for AD in DS. Plasma samples were collected and processed following international 3. Future directions: NGF metabolic signatures should also protocols35 and established procedures at the Sant Pau Memory Unit be tested in biofluids from familial and sporadic AD, laboratory.30 Briefly, fasting venous blood was collected in lavender both pre- and post-symptomatically. In DS, associa- EDTA-K2 BD vacutainer tubes and centrifuged at 2000 g for 10 min- tions with cognitive decline, basal forebrain imaging, utes at 4◦ C for plasma separation. Plasma samples were separated and cholinergic positron emission tomography would be in aliquots of 100, 250, and 500 μl with protease inhibitors (cOm- revealing. plete, Mini Protease Inhibitors, Roche) and stored at –80◦ C. They were shipped on dry ice to McGill University and upon arrival were immedi- ately stored at –80◦ until analysis. Samples were analyzed within 12-18 months after collection. Freeze-thaw cycles were minimized to three. cialized neurologist at the Sant Pau Hospital (Barcelona, Spain), follow- ing international consensus recommendations for CSF collection.36–39 The procedure was performed either in sitting or lying position, using 2.3 CSF collection via lumbar puncture a pen-point atraumatic spinal needle (Whitacre-22G). CSF was col- lected by free-flow/dripping in 10 mL polypropylene tubes (Sarstedt, A plasma-paired CSF sample of every subject was obtained at the Ref# 62.610.018) and processed within 2 hours after acquisition and time of blood collection. Lumbar puncture was performed by a spe- aliquoted as previously described.33 The Memory Unit laboratory at TA B L E 1 Characteristics of the study cohort HC aDS DSAD Test for difference Number of cases 16 14 22 Age (± SEM) 52.8 ± 1.1 39.9 ± 2.9 52.9 ± 2.9 1 way ANOVA, P < .001 (aDS lower than HC/DSAD) Proportion female 0.44 0.50 0.50 Chi-square test of independence, Χ2 > 0.05 APOE allele frequency (proportion) ε4: 0.19 ε4: 0.11 ε4: 0.13 Chi-square test of ε3: 0.78 ε3: 0.82 ε3: 0.85 independence, Χ2 > 0.05 ε2: 0.03 ε2: 0.07 ε2: 0.02 Intellectual disability (proportion) N/A Mild: 0.29 Mild: 0.26 Chi-square test of Moderate: 0.57 Moderate: 0.48 independence, Χ2 > 0.05 Severe: 0.14 Severe: 0.26 Notes: Individuals with aDS were younger than those with DSAD as well as non-trisomic controls. Sex and APOE allele balance were equivalent between all groups. Underlying intellectual disability was equivalent between the aDS and DSAD groups. Abbreviations: aDS, Alzheimer’s disease asymptomatic; ANOVA, analysis of variance; APOE, apolipoprotein E; DSAD, Down syndrome Alzheimer’s disease; HC, healthy control; SEM, standard error of the mean. 608 PENTZ ET AL . the Sant Pau Hospital participates in the Alzheimer’s Association exter- 2.5 Analysis of metallo-proteases nal quality control program for CSF biomarkers. CSF aliquots were stored at –80◦ C, shipped on dry ice to McGill University, and upon The levels (pg/mL) of MMP-1, MMP-3, and MMP-9 in diluted plasma arrival were immediately stored at –80◦ C. Samples were analyzed and CSF samples (1:10) were measured with a Multi-Spot MMP 3- within 12-18 months after collection. Freeze-thaw cycles were mini- Plex Ultra-Sensitive kit (MesoScale Discovery, USA), following manu- mized to three. facturer’s instructions. Electrochemiluminiscent signals were read in a SECTOR Imager 2400 (MesoScale Discovery, USA) and the con- centration of each metalloprotease was determined from a standard 2.4 Analysis of proNGF and neuroserpin curve generated by the MSD Discovery Workbench software v. 4.0. (MesoScale Discovery, USA). Samples that fell below the detection Analysis of proNGF and neuroserpin in plasma and CSF was done threshold were not considered for analysis. Each sample was analyzed by Western blotting, following established procedures at the McGill in three independent experiments from separate aliquots by indepen- laboratory.15–17,26,40 Briefly, plasma samples (diluted in phosphate dent investigators. The average values of all assays were used for anal- buffered saline) and undiluted CSF were incubated in standard loading ysis. buffer under reducing conditions (5% β-mercaptoethanol), and loaded on 10% to 12% SDS-polyacrylamide gels and semi-dry transferred to nitrocellulose membranes (BioRad, USA) for 60 minutes at 220 mA. 2.6 Analysis of tPA Membranes were blocked for 1 hour in 5% bovine serum albumin (Sigma, USA) in tris-buffered saline-polysorbate 20 0.1% (TBS-T) and The plasma and CSF levels of tPA were measured with a Human Tis- incubated with anti-proNGF antibodies (Alomone Labs, Israel; at sue Type Plasminogen Activator ELISA (enzyme-linked immunosorbent 1:2000) or anti-neuroserpin antibodies (kindly provided by Dr. Daniel assay) kit (TPA; ab108914, Abcam, USA). tPA levels were measured in Lawrence, University of Michigan, USA; at 1:5000), overnight at 4◦ C undiluted plasma and in diluted CSF samples (1:2), following the pro- in blocking solution. Anti-mouse or anti-rabbit peroxidase-conjugated tocol detailed by the manufacturer. For the calibration curve used in secondary antibodies (Jackson Immunoresearch, USA) were applied the analysis of plasma samples, human tPA standards were diluted in for 1 hour at room temperature in blocking solution. Immunoblots tPA-depleted plasma, provided by the manufacturer. The assay range were developed with the Western Lightning Plus-ECL substrate for tPA quantification was 0.02 to 10 ng/mL. (PerkinElmer Inc, USA) on Hyblot CL films (Denville, Canada). Films were digitized and the relative levels of proNGF (27 kDa and 50 kDa bands) and neuroserpin (45 kDa band) were quantified with the 2.7 Statistical analysis Gel-Pro Analyzer (Media Cybernetics, Inc., USA) and the TotalLab CLIQS Software (TotalLab, UK). The densitometry of each sample Unless otherwise stated, data analyses were done with the soft- was normalized to that of an internal control, a plasma or CSF ware GraphPad Prism v7.00a (GraphPad, USA). Significance was set at sample that was loaded in all gels, and each value was expressed P < .05. Normality of data distribution was tested with the D’Agostino as relative fold-change with respect to the average of the control and Pearson omnibus K2 test. If data normality was violated, analysis group. ProNGF and neuroserpin determinations were done in three was done with non-parametric statistics. Outliers were removed at a independent experiments by independent investigators blinded to threshold of 3x the standard error of the mean. Differential expres- the clinical diagnosis of the cases. The values were averaged for sion of NGF metabolism-related proteins across clinical classifications analysis. Quantifiability across physiologically relevant concentra- was analyzed with a one-way analysis of variance (ANOVA) and Bon- tions/integrated optical density (IOD) values was established by ferroni post hoc tests, if normally distributed, or Kruskal-Wallis tests reconstitution experiments in which plasma/CSF was spiked with 2, 5, and Dunn’s post hoc correction if non-normal. The specificity of the 10, or 20 ng of human recombinant proNGF protein (Alomone Labs, biomarkers to DSAD was assessed by the area under a receiver oper- Israel), and subsequently analyzed by Western blotting as described ator characteristic (ROC) curve comparing the DSAD and aDS pop- above. As shown in Figure S1 in the Supplementary Files, responses ulations. Assessments of the effect of sex on biomarker expression were linear across the range of 2 to 10 ng and showed a minimal was performed with a two-way ANOVA or Friedman non-parametric (15%) increase in IOD response in the 20 ng sample in plasma (see test, in the case of non-normally distributed data, looking for main Figure S1A and S1B). In CSF, all responses were linear (see Figure effects of sex and sex-by-diagnosis interactions. Note that expression S1C and S1D). Spike-response patterns did not differ in plasma or CSF levels of each protein are not considered independent of one another samples from controls (HC) versus individuals with DS (Figure S1A and their relationships are described in Table S1 in the Supplemen- vs S1B; S1C vs S1D). All experimental values obtained by Western tary Files. The relationships between the CSF and plasma levels of each blotting fell within the linear range. The identity of each proNGF band NGF metabolism-related protein, as well as their associations with core was established and confirmed in previous reports by a variety of CSF biomarkers of AD, displayed evidence of heteroskedasticity and methods.15–17,26,40 were therefore assessed using the MM-estimation method of Crout PENTZ ET AL . 609 et al.,41 which is robust to outliers and heterogeneity of variance. This analysis was performed using R (R Project for Statistical Computing, https://www.R-project.org). 3 RESULTS Table 1 summarizes the demographic information of the study partici- pants. The aDS group was younger than DSAD and control individuals, although the age difference was not significant. There were no differ- ences in the proportion of females or proportion of APOE ε4 carriers between clinical groups. The DSAD group had a higher proportion of individuals with severe intellectual disability than the aDS group. 3.1 Increased proNGF levels in plasma and CSF from individuals with aDS and DSAD As there are no existing ELISAs that can differentiate different forms of proNGF, we assessed them using Western blotting. This allowed the detection of a 27 kDa proNGF form corresponding to unprocessed proNGF,42–44 and another one of higher molecular weight at 50 kDa, suggested to represent glycosylated proNGF42–45 and/or complexes of proNGF with macromolecules such as α2-macroglobulin.46 Analysis of the 27 kDa form of proNGF revealed a significant 40% increase in plasma in aDS as well as in DSAD individuals compared to non-trisomic controls (Figure 1A, P < .05). An 80% upregulation of the 27 kDa proNGF band was observed in paired CSF samples (from the same individuals) in both the aDS and DSAD groups compared to non- trisomic controls (Figure 1B, P < .01 and P < .001). CSF and plasma levels of proNGF 27 kDa significantly correlated (P < .01, r2 = 0.12, Table S1). F I G U R E 1 Upregulation of proNGF (nerve growth factor) in The 50 kDa form of proNGF was also upregulated in plasma from plasma and cerebrospinal fluid (CSF) from people with Down people with aDS and DSAD compared to controls (Figure 1C, P < .05), syndrome (DS). A, The 27 kDa form of proNGF, measured by Western similarly to the 27 kDa form. CSF analysis further revealed higher lev- blotting, is upregulated in plasma from Alzheimer’s disease els of 50 kDa proNGF in the aDS and DSAD groups compared to non- (AD)-asymptomatic people with Down syndrome (aDS) and in Down trisomic controls, and further elevated levels in the DSAD group com- syndrome with symptomatic AD (DSAD) compared to non-trisomic controls, one-way analysis of variance (ANOVA), F = 5.03, P = .01 and pared to the aDS group (Figure 1D, P < .0001 and P < .01). Plasma and Bonferroni post hoc correction; *P < .05. B, ProNGF (27 kDa) is also CSF levels of proNGF 50 kDa did not correlate (Table S1). upregulated in CSF from people with aDS and DSAD compared to non-trisomic controls, Kruskal Wallis test P = .0002 H = 16.83 and Dunn’s post hoc correction; **P < .01; ***P < .001. C, The 50 kDa form 3.2 Neuroserpin and tPA levels in plasma and of proNGF is upregulated in plasma from people with aDS and DSAD CSF from individuals with DS and DSAD compared to non-trisomic controls, one-way ANOVA, F = 5.62, P = .006 and Bonferroni post hoc correction; *P < .05. D, 50 kDa proNGF is upregulated in the CSF of people with aDS compared to The plasma levels of neuroserpin were similar between all groups non-trisomic controls, and further upregulated in people with DSAD, (Figure 2A, P > .05). In contrast, CSF neuroserpin levels were increased one-way ANOVA, F = 21.17, P < .0001 and Bonferroni post-hoc in subjects with DS (both aDS and DSAD), compared to non-trisomic correction; ***P < .001. Data are displayed in box and whisker plots, in controls (Figure 2B, P < .001, P < .05). which the median is represented by the horizontal line and the whiskers go from each quartile to the minimum or maximum value. Plasma levels of the neuroserpin target, tPA, were reduced in aDS HC, Healthy controls: n = 16, aDS: n = 14, DSAD: n = 22; IOD, and DSAD compared to non-trisomic controls (Figure 2C, P < .001 integrated optical density and P < .01). However, no such alteration was detectable in CSF from the same individuals (Figure 2D, P > .05). We observed no correlation between plasma and CSF levels of neuroserpin or tPA (Table S1). 610 PENTZ ET AL . F I G U R E 2 Dysregulation of neuroserpin and tissue plasminogen activator (tPA) in plasma and cerebrospinal fluid (CSF) from people F I G U R E 3 Dysregulation of matrix metalloproteinases (MMP) 9, with Down syndrome (DS). A, Neuroserpin levels were assessed by 3, and 1 in plasma and cerebrospinal fluid (CSF) from people with Western blotting. No significant change in plasma neuroserpin levels Down syndrome (DS). A, No change between clinical classifications in were observed between clinical groups, one-way analysis of variance plasma levels of MMP-9, Kruskal Wallis test, P > .05, Healthy controls (ANOVA), P > .05. B, Neuroserpin levels were found upregulated in (HC): n = 16, AD-asymptomatic Down syndrome (aDS): n = 14, Down CSF from AD-asymptomatic people with Down syndrome (aDS) and syndrome with symptomatic Alzheimer’s disease (DSAD): n = 22. B, from people with Down syndrome and symptomatic Alzheimer’s CSF MMP-9 levels were elevated in people DSAD compared to aDS disease (DSAD) compared to non-trisomic controls, Kruskal Wallis test and non-trisomic controls, one-way analysis of variance (ANOVA), P = .001 H = 13.63 and Dunn’s post hoc correction; *P < .05; **P < .01. F = 5.90, P = .007 and Bonferroni post hoc correction; *P < .05, C, tPA levels were reduced in plasma from people with aDS and DSAD, **P < .01 HC: n = 13, DS: n = 10, DSAD: n = 12. C, Both DSAD and aDS compared to non-trisomic controls, one-way ANOVA, F = 12.07, groups showed greater plasma levels of MMP-3 compared to P < .0001 and Bonferroni post hoc correction; *P < .05. D, No change non-trisomic controls, Kruskal Wallis test P = .003 H = 11.83 and between clinical classifications in CSF levels of tPA, one-way ANOVA, Dunn’s post hoc correction; *P < .05; **P < .01, HC: n = 16, DS: n = 14, P > .05. tPA levels were determined by enzyme-linked immunosorbent DSAD: n = 22. D, CSF MMP-3 was likewise elevated in people with assay. Data are displayed in box and whisker plots, in which the median aDS and with DSAD compared to non-trisomic controls, Kruskal is represented by the horizontal line and the whiskers go from each Wallis test P = .001 H = 13.47 and Dunn’s post hoc correction; quartile to the minimum or maximum value. HC, Healthy controls: *P < .05; **P < .01, HC: n = 16, DS: n = 13, DSAD: n = 21. E, While no n = 16, aDS: n = 14, DSAD: n = 22; IOD, integrated optical density significant post hoc differences in plasma MMP-1 expression were observed between clinical classifications, trends of P = .08 and P = .07 toward upregulations in the aDS and DSAD groups compared to non-trisomic controls were observed P = .03, H = 6.85. F, MMP-1 was 3.3 Analysis of metalloproteases in plasma and significantly upregulated in CSF from people with aDS compared to CSF from individuals with aDS and DSAD healthy controls and DSAD, Kruskal Wallis test P = .0035 H = 11.31 and Dunn’s post hoc correction; *P < .05; ***P < .001, HC: n = 15, DS: n = 14, DSAD: n = 20. Metalloprotease levels were determined with a The NGF-degrading proteinase MMP-9 was found unaltered in plasma sensitive multiplex array from MesoScale Discovery. Data are between the different groups (Figure 3A, P > .05). However, analysis of displayed in box and whisker plots, in which the median is represented CSF samples revealed increased MMP-9 levels in the DSAD group com- by the horizontal line and the whiskers go from each quartile to pared to both the aDS group and the non-trisomic controls (Figure 3B, the minimum or maximum value PENTZ ET AL . 611 P < .01 and P < .05). CSF and plasma levels of MMP-9 did not correlate (Table S1). MMP-3, which has been proposed to activate pro-MMP-947 and which we have also recently shown to degrade mature NGF but not its pro-form (Pentz et al.88 ), was equivalently increased in plasma from people with aDS and DSAD (Figure 3C, P < .05 and P < .01). MMP-3 was also elevated in both aDS and DSAD groups compared to controls in CSF samples (Figure 3D, P < .01). Plasma and CSF levels of MMP-3 significantly correlated (P < .001, r2 = 0.41, Table S1). MMP-1, which can be activated by MMP-3,48 was unchanged across clinical classifications, though there was a trend toward upregulations in plasma in both the aDS and DSAD groups (Figure 3E, aDS vs HC: P = .08; DSAD vs HC: P = .07). In paired CSF samples, MMP-1 was ele- vated in the aDS group compared to both non-trisomic controls and to people with DSAD (Figure 3F, P < .001, P < .05). Plasma and CSF levels of MMP-1 did not correlate (Table S1). 3.4 Specificity of NGF pathway markers to AD in DS The ability of each marker of the NGF metabolic pathway to specifically signal changes associated with symptomatic AD in DS was assessed with the ROC curve analyses. Two candidates showed good and com- parable performance: the 50 kDa form of proNGF measured in CSF (Figure 4A, AUC = 0.86) and CSF MMP-9 (Figure 4B, AUC = 0.87). These two markers showed better specificity for differentiating aDS and DSAD than two of three core biomarkers of AD that were com- pared for reference, including CSF 181-phospho-tau (p-tau, Figure 4C, AUC = 0.85) and total tau (t-tau, Figure 4C; AUC = 0.75). The CSF Aβ42/40 ratio performed slightly better than the proposed markers, with an AUC = 0.88 (Figure 4C). All other NGF metabolism-related markers had AUC values between 0.51 and 0.76 (Figure S2 in Supple- mentary Files). 3.5 Associations between NGF metabolism-related proteins and core biomarkers of AD Using robust regression (MM Estimation, following Croux et al.41 ), we next assessed the relationship between NGF metabolism-related pro- F I G U R E 4 Cerebrospinal fluid (CSF) matrix metalloproteinase teins (which are not considered to be independent) and the established (MMP)-9 and proNGF (precursor of nerve growth factor) discriminate effectively between Alzheimer’s disease (AD) symptomatic and core AD biomarkers measured in CSF: the Aβ42/40 ratio, p-tau, and asymptomatic people with Down syndrome (DS). A, CSF levels of the t-tau (see Tables 2 and 3). Adjusted r2 values are given. In addition, 50 kDa proNGF form effectively identify people with Down syndrome we examined associations with each Aβ species individually and the with symptomatic Alzheimer’s disease (DSAD) from within the total p-tau/Aβ42 ratio (see Tables 2 and 3). The two composites have both DS sample group, with an area under the receiver operator been proposed to outperform their component individual measures as characteristic (ROC) curve of 0.86. B, CSF MMP-9 levels effectively biomarkers of AD.49–52 identify people with DSAD from within the total DS sample group, with an area under the ROC curve of 0.87. C, ROC curves for the core The expression of NGF metabolism-related markers in plasma was CSF biomarkers of AD demonstrate that MMP-9 and proNGF (50 kDa) typically uncorrelated to the three core AD CSF biomarkers, save measured in CSF outperformed CSF pTau-181 (AUC = 0.85) and total neuroserpin, which correlated weakly to Aβ40 (P = .02, r2 = 0.07, tau (AUC = 0.75), while being outperformed by the Aβ42/40 ratio Table 2), Aβ42 (P = .01, r2 = 0.09), and to the p-tau/Aβ42 ratio (P = .01, (AUC = 0.88) 612 PENTZ ET AL . TA B L E 2 Associations among plasma levels of NGF metabolism-related proteins, age, and core biomarkers of AD (individual components and composites) measured in cerebrospinal fluid, given by r2 and P values as determined by the robust MM estimation method of Croux et al.41 (all cases, n = 52) Plasma marker Age Aβ40 Aβ42 p-tau-181 t-tau Aβ42/40 p-tau/Aβ42 ProNGF 27 kDa P = .240 P = .66 P = .60 P = .63 P = .42 P = .31 P = .50 r2 = 0.07 r2 = -0.02 r2 = -0.01 r2 = 0.02 r2 = 0.00 r2 = 0.01 r2 = -0.01 ProNGF 50 kDa P = .348 P = .79 P = .67 P = .11 P = .08 P = 0.15 P = .09 r2 = 0.04 r2 = -0.02 r2 = -0.02 r2 = 0.01 r2 = 0.03 r2 = 0.03 r2 = 0.02 Neuroserpin P = .125 P = .02 P = .01 P = .14 P = .30 P = 0.15 P = .01 r2 = 0.11 r2 = 0.07 r2 = 0.09 r2 = 0.02 r2 = 0.00 r2 = 0.03 r2 = 0.07 tPA P = .945 P = .93 P = .72 P = .24 P = .34 P = .85 P = .51 r2 = 0.00 r2 = -0.02 r2 = -0.02 r2 = 0.00 r2 = 0.00 r2 = -0.02 r2 = -0.01 MMP-9 P = .515 P = .46 P = .93 P = .09 P = .05 P = .46 P = .22 r2 = 0.02 r2 = -0.01 r2 = -0.02 r2 = 0.02 r2 = 0.03 r2 = -0.01 r2 = 0.00 MMP-3 P = .491 P = .41 P = .40 P = .16 P = .08 P = .09 P = .22 r2 = 0.02 r2 = -0.01 r2 = -0.01 r2 = 0.03 r2 = 0.05 r2 = 0.04 r2 = 0.02 MMP-1 P = .569 P = .76 P = .93 P = .66 P = .52 P = .88 P = .65 r2 = 0.02 r2 = -0.02 r2 = -0.02 r2 = -0.02 r2 = -0.02 r2 = -0.02 r2 = -0.02 Abbreviations: Aβ, amyloid beta; AD, Alzheimer’s disease; MMP, matrix metalloproteinase; NGF, nerve growth factor; p-tau, phosphorylated tau; tPA, tissue plasminogen activator; t-tau, total tau. r2 = 0.07). The results of MM estimates for all associations between 3.6 Effects of sex on the plasma and CSF plasma NGF–related proteins and core AD biomarkers are given in expression of NGF metabolism-related proteins Table 2. In CSF, the 27 kDa form of proNGF correlated weakly to the Given that some of our markers showed specificity to symptomatic AD Aβ42/Aβ40 ratio (P = .04, r2 = 0.07, Table 3), as well as p-tau (P = .008, in DS (ie, proNGF 50 kDa and MMP-9) or efficacy as measurements of r2 = 0.10) and t-tau levels (P = .03, r2 = 0.10). A weak correlation to the pathological processes associated with AD, we investigated whether p-tau/Aβ42 ratio was also observed (P = .008, r2 = 0.10). In contrast, sex influenced the expression of NGF pathway proteins, as this could the 50 kDa proNGF form correlated well to the CSF Aβ42/Aβ40 ratio have implications for setting biomarker thresholds. We observed an (P < .0001, r2 = 0.38) as well as p-tau (P = .01, r2 = 0.26), t-tau (P < .005, interaction between sex and clinical diagnosis in the expression of r2 = 0.31), and the p-tau/Aβ42 ratio (P = .02, r2 = 0.28). A weak correla- CSF neuroserpin, with males displaying higher levels of neuroserpin tion between 50 kDa proNGF in CSF and Aβ42 levels was also observed in the non-trisomic controls and DSAD groups, but with females (P = .04, r2 = 0.07). There were also significant correlations between displaying higher levels in the aDS group (Figure 5A, two-way ANOVA, CSF neuroserpin and the Aβ42/Aβ40 ratio (P = .01, r2 = 0.09), as well as Pinteraction < .05). MMP-9 was differentially expressed by sex in plasma, p-tau (P = .04, r2 = 0.05) t-tau (P = .04, r2 = 0.07), and the p-tau/Aβ42 with males expressing higher levels across all clinical conditions ratio (P = .01, r2 = 0.06). Conversely, no significant correlations were (Figure 5B, two-way ANOVA, Pmain effect sex < .01). Likewise, males observed between tPA and core CSF biomarkers of AD. The results of had consistently greater levels of MMP-3, both in plasma (Figure 5C, MM estimates for all associations between CSF NGF–related proteins two-way ANOVA, Pmain effect sex < .001) and in CSF (Figure 5D, two-way and core AD biomarkers are given in Table 3. ANOVA, Pmain effect sex < .05). No other main effects of sex or interac- CSF MMP-9 only correlated to measures of tauopathy: p-tau tions between sex and clinical diagnosis were observed for other NGF (P < .0001, r2 = 0.22) and t-tau (P = .005, r2 = 0.19), and better still pathway markers (Figure S3 in Supplementary Files). to the p-tau/ Aβ42 ratio (P < .0001, r2 = 0.26). CSF MMP-3, in con- trast, correlated well to the Aβ42/Aβ40 ratio (P = .002, r2 = 0.21), though yet stronger was the association between MMP-3 levels and 4 DISCUSSION CSF Aβ40 alone (P < .001, r2 = 0.44). CSF MMP-3 also correlation to t- tau (P = .003, r2 = 0.34) and to the p-tau/ Aβ42 ratio (P = .05, r2 = 0.19), People born with DS inexorably develop AD neuropathology through though a strong positive association between CSF MMP-3 and p-tau their lifetimes,1–3,53–57 with lifetime risk estimates now more than did not survive correction (P = .17, r2 = 0.50). Last, CSF MMP-1 corre- 90%.58 The primary cause of this vulnerability is attributed to the trip- lated to individual measures of CSF Aβ42 (P = .02, r2 = 0.33), but not lication of chromosome 21, which encodes the amyloid precursor pro- to Aβ42, to their ratio, or to any measures of tauopathy. The results of tein (APP) gene, from which Aβ peptides are produced,6,59–61 though MM estimates for all associations between CSF NGF–related proteins other triplicated genes in chromosome 21 are also known to con- and core AD biomarkers are given in Table 3. tribute to cognitive impairment and to influence the development of PENTZ ET AL . 613 TA B L E 3 Associations among CSF levels of NGF metabolism-related proteins, age, and core biomarkers of AD (individual components and composites) measured in CSF, given by r2 and p values for each association as determined by the robust MM estimation method of Croux et al.41 (all cases, n = 52) CSF marker Age Aβ40 Aβ42 p-tau-181 t-tau Aβ42/40 p-tau/Aβ42 ProNGF 27 kDa P = .705 P = .28 P = .60 P = .008 P = .03 P = .04 P = .008 r2 = 0.00 r2 = 0.00 r2 = -0.01 r2 = 0.10 r2 = 0.10 r2 = 0.07 r2 = 0.10 ProNGF 50 kDa P = .461 P = .33 P = .04 P = .01 P = .005 P < .001 P = .02 r2 = 0.01 r2 = -0.01 r2 = 0.07 r2 = 0.26 r2 = 0.31 r2 = 0.38 r2 = 0.28 Neuroserpin P = .249 P = .22 P = .42 P = .04 P = .04 P = .01 P = .01 r2 = 0.03 r2 = 0.00 r2 = -0.01 r2 = 0.05 r2 = 0.07 r2 = 0.09 r2 = 0.06 tPA P = .462 P = .94 P = .44 P = .37 P = .25 P = .36 P = .38 r2 = 0.01 r2 = -0.02 r2 = -0.01 r2 = -0.01 r2 = -0.01 r2 = 0.00 r2 = -0.01 MMP-9 P = .051 P = .89 P = .10 P < .001 P = .005 P = .06 P < .001 r2 = 0.10 r2 = -0.03 r2 = 0.04 r2 = 0.22 r2 = 0.19 r2 = 0.09 r2 = 0.26 MMP-3 P = .108 P < .001 P = .35 P = .17 P = .003 P = .002 P = .05 r2 = 0.05 r2 = 0.44 r2 = 0.04 r2 = 0.50 r2 = 0.34 r2 = 0.21 r2 = 0.19 MMP-1 P < .001 P = .08 P = .02 P = .44 P = .45 P = .62 P = .69 r2 = 0.38 r2 = 0.19 r2 = 0.33 r2 = -0.01 r2 = -0.02 r2 = -0.01 r2 = -0.02 Abbreviations: Aβ, amyloid beta; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; MMP, matrix metalloproteinase; NGF, nerve growth factor; p-tau, phos- phorylated tau; tPA, tissue plasminogen activator; t-tau, total tau. AD pathology in DS.62–65 The inexorable progression of AD pathology Both the 27 and 50 kDa forms of proNGF were increased in DS in this population, generally unmixed with other age-related diseases, plasma and CSF (irrespective of AD symptomatology) compared to makes it ideal for assessing the ability of biofluid proteins to reflect non-trisomic controls; however, the 50 kDa form was further elevated pathological changes occurring in the brain and thereby to support in the DSAD group compared to aDS. While the 27 kDa proNGF band these alterations. Biomarkers of preclinical AD-associated processes represents the unmodified neuronal form of the NGF precursor,72–74 could be especially valuable, as this is the stage at which therapeutic higher molecular weight forms have also been frequently reported, intervention is most likely to be viable.66 including a 41 kDa form secreted by cortical cells20 and a 50 kDa gly- AD, both in DS and in the sporadic population, is associated with the cosylated form.42,75,76 degeneration of basal forebrain cholinergic neurons.5,7–14 This phe- It is unsurprising that CSF markers outperformed plasma markers nomenon contributes to cognitive decline in the early phases of the as signals of neuropathology. First, the CSF compartment is in direct disease67,68 and is associated with disruptions of the NGF metabolic contact with the CNS. Second, brain-derived pools of molecules pathway,15–18 as basal forebrain cholinergic neurons depend on a con- unaltered in plasma, such as MMP-9 and neuroserpin, may simply tinuous target-derived supply of NGF for the maintenance of a healthy be too small compared to those derived from other sources, such as cholinergic phenotype.18,19,69–71 macrophages and neutrophils.77,78 Importantly, the plasma results Initial studies on the expression of NGF metabolism-related pro- confirm the dysregulation of NGF metabolism-related proteins that teins in frontal, temporal, and parietal cortex had shown that adults had been reported previously in a different and independent DS popu- with DS and AD-dementia express higher levels of proNGF and neu- lation from Sicily, Italy, which similarly demonstrated the upregulation roserpin protein, lower levels of tPA mRNA, and higher levels of of proNGF, MMP-9, and MMP-3 in plasma samples, with no changes MMP-9 activity and mRNA (likely reflecting higher MMP-9 protein to neuroserpin.16 Novel biomarkers are needed in DS, in both research expression),15 and indicating reduced maturation of proNGF to mNGF and clinical contexts, to track specific pathological events and thereby and increased degradation of mNGF. Furthermore, these changes also enable AD precision medicine.79,80 In this respect, we investigated occur in sporadic AD, both at symptomatic stages and prior to the onset whether NGF pathway biomarkers reflected processes specific to of overt dementia, where they correlate to cognitive decline and brain symptomatic individuals with DS that distinguished them from the rest cholinergic dysfunction.25,26 of the DS population. ROC analysis revealed that MMP-9 (AUC = 0.87) The present results in matched body fluids from people with DS and the 50 kDa form of proNGF measured in CSF (AUC = 0.86) outper- indicate that brain NGF pathway alterations are reflected in plasma formed classical CSF biomarkers of AD, including p-tau (AUC = 0.85) and, more faithfully, in CSF, as plasma did not reveal the upregula- and t-tau (0.75), but not the Aβ42/40 ratio (0.88). Thus, we propose tions in MMP-9 or neuroserpin seen in DS brains.15 The finding of that our markers are reflecting changes that are associated with the NGF metabolic changes in individuals asymptomatic for AD supports onset of symptomatic AD in DS. Furthermore, proNGF and MMP-9 an early dysregulation of NGF metabolism alongside AD pathology in measured in CSF could assist in the identification of AD-related NGF DS and the utility of the methods described here for monitoring these dysmetabolism and cholinergic dysfunction in the DS population. This changes in research and potentially clinical settings. claim could be further investigated in future studies using markers 614 PENTZ ET AL . F I G U R E 5 Sex influences the expression of certain nerve growth factor (NGF) metabolism-related proteins in plasma and cerebrospinal fluid (CSF). A, Sex/group interaction in the expression of CSF neuroserpin (two-way analysis of variance, interaction P < .05). B, Main effect of sex in the expression of plasma matrix metalloproteinase (MMP)-9 with consistently higher levels in males. C, Main effect of sex in the expression of plasma MMP-3 (P < .01) with consistently higher levels in males. D, Main effect of sex (P < .05) in the expression of CSF MMP-3 with consistently higher levels in males of cholinergic dysfunction accessible in plasma or CSF, or preferably, and treatment.84 The present study further suggests that sex-specific by basal forebrain volumetry with structural magnetic resonance thresholds may also aid the clinical implementation of biomarkers. imaging79,80 or the positron emission tomography imaging of cholin- We observed sex-related differences in MMP-9 (most significantly in ergic synapses.81,82 It is nonetheless worth noting that the direct plasma but also in CSF), which was one of the markers best able to iden- impact of NGF dysregulation on the cholinergic/cognitive phenotype tify individuals with AD dementia from DS at large, as well as MMP-3, has been demonstrated experimentally in rodents18,19 and that NGF which has been shown to inform longitudinal risk of AD-associated cog- dysmetabolism correlates to early cognitive decline and cholinergic nitive decline and has been reported to be increased in males in other dysfunction in the human AD brain.26 studies.16,85 Therefore, these results would indicate that any clinical While current biomarkers are able to effectively diagnose DSAD thresholds established for these markers should take the differential from the wider DS population, proNGF could also play a role in sub- expression of males and females into account. These differences may typing and staging AD given that AD pathology and cholinergic degen- also be relevant to the sex-related dimorphisms observed in the cholin- eration differ in extent among neuroanatomical subtypes of AD.83 The ergic system and its dysfunction in AD.86 ability of proNGF to predict responses to cholinesterase inhibitor ther- Whether the NGF biofluid signature reported in DS plasma and CSF apy should also be investigated in this regard. Importantly, it is likely is unique to AD in DS remains to be thoroughly examined. Neuropatho- that no single marker will prove to be an adequate diagnostic tool for logical studies have shown that proNGF (both forms), neuroserpin, AD in isolation; proNGF and other related markers may therefore have MMP-3, and MMP-9 (activity) are all increased, while tPA is unchanged utility as part of a larger diagnostic panel. in the frontal cortex from people with sporadic AD or MCI, as well as Significant sex-related dimorphisms have been observed in the inci- in cognitively normal people with elevated levels of brain amyloid.26 dence and nature of AD, with important implications for diagnosis These results accord well with the biomarker signature reported in the PENTZ ET AL . 615 present study, and support its generalizability. These results are also REFERENCES in accord with other accounts of increased proNGF in CSF from peo- 1. Lott IT. Down’s syndrome, aging, and Alzheimer’s disease: a clinical ple with AD,87 and with the altered expression of MMP-3, neuroserpin, review. 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