Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 CSF Biomarkers Monoaminergic impairment in Down syndrome with Alzheimer’s disease compared to early-onset Alzheimer’s disease Alain D. Dekkera,b, Yannick Vermeirena,b, Maria Carmona-Iraguic,d, Bessy Benejamd, Laura Videlad, Ellen Gelpie, Tony Aertsb, Debby Van Dama,b, Susana Fernandezd, Alberto Lleoc, Sebastian Videlad,f, Anne Siebenb, Jean-Jacques Martinb, Netherlands Brain Bankg, Rafael Blesac, Juan Forteac,d, Peter P. De Deyna,b,h,* a Department of Neurology and Alzheimer Research Center, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands b Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Wilrijk, Antwerp, Belgium c Department of Neurology, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Spain d Down Medical Center, Catalan Down Syndrome Foundation, Barcelona, Spain e Neurological Tissue Bank—Biobanc, Hospital Clinic Barcelona, Institut d’Investigacions Biomediques August Pi i Sunyer, Barcelona, Spain f Faculty of Health and Life Sciences, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain g Netherlands Institute for Neuroscience, Amsterdam, The Netherlands h Department of Neurology and Memory Clinic, Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken, Antwerp, Belgium Abstract Introduction: People with Down syndrome (DS) are at high risk for Alzheimer’s disease (AD). Defects in monoamine neurotransmitter systems are implicated in DS and AD but have not been comprehensively studied in DS. Methods: Noradrenaline, adrenaline, and their metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG); dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid; and serotonin and its metabolite 5-hydroxyindoleacetic acid were quantified in 15 brain regions of DS without AD (DS, n 5 4), DS with AD (DS1AD, n 5 17), early-onset AD (EOAD, n 5 11) patients, and healthy non-DS controls (n 5 10) in the general population. Moreover, monoaminergic concentrations were determined in cerebrospinal fluid (CSF)/plasma samples of DS (n 5 37/149), DS with prodromal AD (DS1pAD, n 5 13/36), and DS1AD (n 5 18/40). Results: In brain, noradrenergic and serotonergic compounds were overall reduced in DS1AD versus EOAD, while the dopaminergic system showed a bidirectional change. For DS versus non-DS controls, significantly decreased MHPG levels were noted in various brain regions, though to a lesser extent than for DS1AD versus EOAD. Apart from DOPAC, CSF/plasma concentrations were not altered between groups. Discussion: Monoamine neurotransmitters and metabolites were evidently impacted in DS, DS1AD, and EOAD. DS and DS1AD presented a remarkably similar monoaminergic profile, possibly related to early deposition of amyloid pathology in DS. To confirm whether monoaminergic alterations are indeed due to early amyloid b accumulation, future avenues include positron emission tomography studies of monoaminergic neurotransmission in relation to amyloid deposition, as well as relating monoaminergic concentrations to CSF/plasma levels of amyloid b and tau within individuals. Ó 2017 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/). Keywords: Alzheimer’s disease; Cerebrospinal fluid; Dementia; Dopamine; Down syndrome; Monoamines; MHPG; Neuro- transmitter; Noradrenaline; Plasma; Serotonin; Trisomy 21 The authors have declared no conflict of interest. E-mail address: p.p.de.deyn@umcg.nl *Corresponding author. Tel.: 131-50-361-4650; Fax: 131-50-361-1707. https://doi.org/10.1016/j.dadm.2017.11.001 2352-8729/ Ó 2017 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 100 A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 1. Introduction brain regions in early-onset AD patients (EOAD) compared with age- and gender-matched control subjects. In EOAD People with Down syndrome (DS), or trisomy 21, have an patients, lower levels of serotonergic compounds were found exceptionally high risk to develop Alzheimer’s disease in amygdala and hippocampus, complemented by lower NA (AD): 68%–80% of people are diagnosed with dementia levels in the prefrontal cortex and amygdala. No differences by the age of 65 years [1]. The additional copy of chromo- in MHPG levels could be observed [24]. some 21, encoding the amyloid precursor protein (APP), To the best of our knowledge, this study is the first to causes overproduction of amyloid b (Ab) peptides. Very comprehensively evaluate monoaminergic alterations in (1) early in life, intracellular Ab accumulation takes place in postmortem brain tissues and (2) (paired) CSF/plasma neurons, followed by extracellular Ab aggregation and samples from DS individuals with and without AD. Norad- subsequent deposition in characteristic Ab plaques [2–5]. renergic (NA; adrenaline; MHPG), dopaminergic (DA; 3,4- In DS brains, not only plaques but also neurofibrillary dihydroxyphenylacetic acid [DOPAC]; homovanillic acid tangles are omnipresent from the age of 40 years [6]. The [HVA]), and serotonergic (5-HT; 5-hydroxyindoleacetic onset of clinical dementia symptoms, however, is subject acid [5-HIAA]) compounds were quantified using reversed to a marked variation in time [7,8]. Because the dementia phase high-performance liquid chromatography (RP-HPLC). diagnosis in DS is complex, among others due to In one of the largest collections of DS brain tissue comorbidities, pre-existing intellectual disability, and (n 5 21), 15 regions of DS cases without and with a neuro- behavior [9], sensitive and specific biomarkers for AD in pathologically confirmed diagnosis of AD (DS and DS would be very valuable. In the general non-DS popula- DS1AD, respectively) were analyzed and compared with tion, the so-called “AD profile” (low Ab42, high total-tau, EOAD patients and healthy controls in the general popula- and high phosphorylated-tau) in cerebrospinal fluid (CSF) tion. Second, we report the monoaminergic results in has proven useful as a diagnostic aid [10]. However, the (paired) CSF/plasma samples obtained from the largest DS clinical utility in DS has not been demonstrated yet [11]. cohort to have undergone lumbar punctures, comparing DS Therefore, the study of alternative biomarkers for AD in without dementia (DS), DS with prodromal AD (DS1pAD), DS receives vast attention. and DS with clinically diagnosed AD (DS1AD). In this context, we previously analyzed monoamine neurotransmitters and metabolites in serum of 151 elderly DS individuals with AD (DS1AD) and without AD (DS), 2. Materials and methods but also in a nondemented DS group at blood sampling 2.1. Postmortem samples that developed dementia over time (converters). Remark- ably, serum levels of the primary noradrenergic metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were strongly 2.1.1. Study population decreased in DS1AD, but also in converted DS individuals. In total, postmortem samples from 21 elderly DS individ- Individuals with MHPG levels below median had a more uals were obtained from the Netherlands Brain Bank (NBB), than 10-fold increased risk of developing dementia, suggest- Netherlands Institute for Neuroscience (Amsterdam, The ing that decreased serum MHPG levels may be predictive for Netherlands), the Neurological Tissue Bank—Biobanc, conversion to AD [12]. Hospital Clinic Barcelona—Institut d’Investigacions Blood biomarkers, however, are subject to (confounding) Biomediques August Pi i Sunyer (IDIBAPS; Barcelona, peripheral effects. CSF biomarkers are generally regarded Spain), and the Institute Born-Bunge (IBB; Antwerp, better indicators of biochemical changes in the central Belgium). Specifically, brain samples from nine DS1AD in- nervous system because of their direct contact with the extra- dividuals were obtained from the NBB (open access: www. cellular space [13]. Very few studies have investigated brainbank.nl). All material has been collected from donors CSF biomarkers in (moreover small) DS cohorts [11], for or from whom written informed consent for a brain au- including two on monoamines [14,15]. Although a few topsy and the use of the material and clinical information postmortem studies were conducted several decades ago, a for research purposes had been obtained by the NBB. comprehensive profile of central monoaminergic changes Moreover, the IDIBAPS provided samples of two DS and in DS1AD is not established yet. Indeed, monoamines five DS1AD donors for whom written informed consent were quantified in a limited number of brain regions from was obtained from the next of kin. The study was approved a few DS cases with often long postmortem delays by the Hospital Clinic de Barcelona Ethics Committee and (PMDs). For instance, cell loss in the locus coeruleus in accordance with Spanish legislation. Finally, the IBB (LC), major source of noradrenaline (NA), and reduced provided samples of DS (n 5 2), DS1AD (n 5 3), EOAD pa- NA concentrations have been reported in elderly DS cases tients (n 5 11), and healthy controls without neurological [16–23], but an integrated study of regional changes in disease (n 5 10). Since DS1AD presents early in life, we NA, dopamine (DA), serotonin (5-HT), and their primary identified EOAD patients and controls ,75 years of age as metabolites is lacking. Vermeiren et al., for example, inves- comparison groups. Ethical approval was granted by the tigated monoaminergic profiles in a variety of postmortem medical ethics committee of the Hospital Network Antwerp A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 101 Table 1 Characteristics of postmortem study groups Characteristics DS (n 5 4) DS1AD (n 5 17) EOAD (n 5 11) Controls (n 5 10) P value a,b,c a b c Age at death in years (median; min.2max.) 39.5 (35.0–44.0) 62.0 (44.0–80.0) 67.2 (57.6–73.0) 65.5 (57.2–73.3) .004 Gender (N male and %) 2 (50) 5 (29.4) 8 (72.7) 6 (60) n.s. Psychoactive medication (yes/no/not reported) 3/0/1 10/3/4 3/8/0 2/8/0 .002 Postmortem delay in hours (median; min.–max.) 21.0 (11.5–36.0)a,b,c 7.3 (3.8–20.0)a,b,e 3.0 (2.8–7.0)b,d 5.4 (2.3–7.0)c,e ,.001 AD neuropathologic change Low High Intermediate (1)/High (10) Not (6)/Low (4) Available brain regions per study group Neocortex BA7: superior parietal lobule 2 13 11 10 BA9/10/46: (pre)frontal cortex 4 14 11 10 BA17: occipital pole (V1) 3 7 11 10 BA22: superior temporal gyrus 3 10 11 10 Limbic system Amygdala 2 10 10 10 Hippocampus 3 5 11 10 BA11/12: orbitofrontal cortex 4 6 11 10 Cingulate gyrus 3 8 11 10 Thalamus 3 11 11 10 Basal ganglia Caudate nucleus 3 16 11 10 Globus pallidus 2 8 11 10 Putamen 3 15 11 10 Substantia nigra 2 14 11 10 Metencephalon Locus coeruleus (in pons) – 10 10 10 Cerebellar cortex 2 9 10 10 Abbreviations: AD, Alzheimer’s disease; BA, Brodmann area; DS, Down syndrome without neuropathologic AD diagnosis; DS1AD, Down syndrome with neuropathologic AD diagnosis; EOAD, early-onset Alzheimer’s disease; n.s., not significant. NOTE. Gender and medication use were compared with Fisher’s exact test. Kruskal-Wallis tests were performed to compare ages and postmortem delays between the groups. Post hoc Mann-Whitney U tests were performed to identify significant group differences (P , .015): (a) DS vs. DS1AD; (b) DS vs. EOAD; (c) DS vs. controls; (d) DS1AD vs. EOAD; (e) DS1AD vs. controls. (ZNA, approval numbers 2805 and 2806). The study was IBB (DS: 20 and 36 hours; DS1AD: 15 hours, 20 hours, compliant with the World Medical Association Declaration and one unknown; and EOAD/controls: ,7 hours). Samples of Helsinki on Ethical Principles for Medical Research were dissected from the left hemispheres (undefined hemi- Involving Human Subjects. sphere for three IBB cases). Not all regions were available for all cases. Most samples from EOAD and controls have 2.1.2. Assessment of AD neuropathologic changes been published before [24]. For this study, Brodmann area Neuropathological analysis was conducted according to (BA)7, substantia nigra (SN), caudate nucleus, globus pal- the “ABC scoring” system [25]. Formalin-fixed paraffin- lidus, and putamen were additionally analyzed. embedded samples were sectioned in concordance with the minimally recommended brain regions (if available). If 2.2. CSF/plasma samples possible, additional sections of the cingulate gyrus, amygdala, pons at the level of the LC, and medulla oblongata were Samples of 241 DS adults were obtained from the Down included. Applied stains were hematoxylin-eosin, cresyl Alzheimer Barcelona Neuroimaging Initiative study, a pro- violet, Kl€uver-Barrera (myelin), and modified Bielschowsky spective biomarker study for AD in DS [26–28]. The silver staining. Moreover, antibodies against amyloid (4G8), person with DS and/or the legal representative provided phosphorylated-tau (AT8), ubiquitin, TDP-43, and p62 Lck li- written informed consent. The study was compliant with gands were used. All cases were diagnosed by experienced the World Medical Association Declaration of Helsinki on neuropathologists (E.G., A.S., and J.-J.M.) as not, low, inter- Ethical Principles for Medical Research Involving Human mediate, or high AD neuropathologic changes. Intermediate Subjects and approved by the ethics committee of the Sant and high signify the diagnosis of AD [25]. Pau hospital in Barcelona [27]. Neurologists and neuropsy- chologists established a consensus diagnosis of dementia, 2.1.3. Regional brain samples and dissection distinguishing between DS without dementia (DS), DS Table 1 shows the selection of frozen samples for RP- with prodromal AD (DS1pAD), and DS with diagnosed HPLC analyses. Brains were included in the three biobanks AD (DS1AD). Specifically, the DS group did not show ev- between 1990 and 2011 and stored at 280 C. Postmortem idence of cognitive decline. The DS1pAD group includes delays: NBB (,10 hours), IDIBAPS (,12 hours), and individuals who (1) presented cognitive/functional change 102 A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 but did not (yet) meet criteria for dementia or (2) showed sig- using Clarity Software (DataApex Ltd., 2008, Prague, nificant cognitive decline in longitudinal assessment. The Czech Republic). DS1AD group includes individuals with clear cognitive/ functional change meeting the dementia criteria (IWG-2 [29]). In the diagnostic procedure, medical comorbidities 2.4. Statistics and other possible causes of cognitive decline were assessed Histograms, normal quantile-quantile (Q-Q) plots, and (differential diagnostics). DS cases with cognitive decline Shapiro-Wilk tests (P , .05) demonstrated that the concentra- due to medical comorbidities or a psychiatric etiology tions in the brain and CSF/plasma were (largely) not normally were excluded (Fig. 1). Use of psychoactive medication distributed. Consequently, nonparametric Kruskal-Wallis tests around the moment of sampling was noted. Within the were applied to compare groups. If the P value was ,.05, post Down Alzheimer Barcelona Neuroimaging Initiative study, hoc Mann-Whitney U tests were conducted. In brain, the three participants are offered a lumbar puncture, which was found most relevant group comparisons were performed: DS versus to be feasible and safe [27]. For 68 individuals, paired CSF/ DS1AD, DS1AD versus EOAD, and DS versus controls. plasma samples were obtained. The other 157 participants The EOAD versus controls comparison has been largely provided plasma-only. CSF and plasma samples were drawn published before [24]. Regarding CSF/plasma samples, we on the same day. Lumbar punctures were performed between analyzed the total cohort (n 5 225), that is, all individuals 9 and 12 am, directly followed by plasma collection. Sam- regardless of medication use, as well as the medication-free ples were stored at 280 C. subpopulation because psychoactive medication may affect monoaminergic neurotransmission. Nonparametric Spear- man’s rank-order correlation tests established the relationship 2.3. Reversed-phase HPLC with age and between CSF and plasma concentrations. Cohort characteristics like gender and medication use were compared To quantify noradrenergic (NA; adrenaline; MHPG), using Pearson’s c2 tests or Fisher’s exact tests. To account for dopaminergic (DA; DOPAC; HVA), and serotonergic multiple comparisons, we applied the Benjamini-Hochberg (5-HT; 5-HIAA) compounds, a validated RP-HPLC setup procedure with a false discovery rate of 0.05 [34]. Original with ion pairing (octane-1-sulfonic acid sodium salt) and P values ,.015 were regarded significant. Finally, we evalu- amperometric electrochemical detection was used [30], previ- ated whether the results in the brain were possibly affected ously applied to CSF and blood samples [12] and brain by psychoactive medication and PMDs. Within each group, homogenates [24,31–33]. Concentrations were calculated we performed Mann-Whitney U tests to compare Fig. 1. Flow chart of CSF/plasma study groups. Abbreviations: AD, Alzheimer’s disease; CSF, cerebrospinal fluid; DS, Down syndrome without (clinical) dementia; DS1AD, DS with diagnosed AD dementia; DS1pAD, Down syndrome with prodromal AD. A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 103 Fig. 2. MHPG concentrations (ng/g tissue) for each study group in neocortical areas, the limbic system, locus coeruleus, and cerebellum. The boxes represent the IQR (25%–75%) with the black horizontal line indicating the median. The whiskers indicate values within 1.5 IQR. Mild outliers (1.5–3 IQR) are indicated with an open circle, whereas extreme outliers (.3 IQR) with an asterisk. One extreme value (EOAD, MHPG concentration in hippocampus of 3806 ng/g tissue) is not shown with respect to scaling. Evidently, MHPG levels were consistently lower in DS (vs. controls) and DS1AD (vs. EOAD). DS vs. DS1AD did not differ significantly. Individual comparison statistics are provided in Table 2. Abbreviations: BA, Brodmann area; DS, Down syndrome without neuropathologic AD diagnosis; DS1AD, Down syndrome with neuropathologic AD diagnosis; EOAD, early-onset Alzheimer’s disease; IQR, interquartile range; MHPG, 3-methoxy-4-hydroxyphenylglycol. monoaminergic concentrations between those taking psycho- nergic (Supplementary Table 2), and serotonergic active medication and a subgroup that did not and performed (Supplementary Table 3) systems. Spearman’s rank-order correlation tests to establish the associ- Since psychoactive medication may affect monoamin- ation between PMDs and monoaminergic concentrations. ergic concentrations, we assessed the donors’ clinical IBM SPSS Statistics, version 23.0, was used. documentation (Table 1). Comparing individuals who did and did not use psychoactive medication within each group yielded no significant monoaminergic differences in 3. Results DS1AD and control groups, whereas only a single signifi- Based on the measured concentrations, five accompa- cant difference was found in the EOAD group: NA levels nying ratios were calculated: (1) MHPG:NA (noradrenergic in the caudate nucleus were lower in individuals using medi- turnover), (2) DOPAC:DA (dopaminergic turnover) and cation (P 5 .014). In the DS group, the effect of medication (3) HVA:DA (dopaminergic turnover), (4) 5-HIAA:5-HT could not be established: three in four used medication, and (serotonergic turnover), and (5) HVA:5-HIAA (serotonergic for the last person, it was unknown. Nevertheless, the use of inhibition on dopaminergic neurotransmission). psychoactive medication did not appear to have evidently impacted monoaminergic concentrations in DS1AD, EOAD, and control groups. 3.1. Monoaminergic characterization of postmortem brain Given the very limited availability of postmortem DS tis- tissue sue, it was impossible to select for short PMDs, particularly Table 1 shows the general demographics, use of psycho- in the DS group. Apart from three cases, PMD was active medication, and PMDs for each of the four groups. ,12 hours in the DS1AD group. Because PMDs differed Table 2 provides the monoaminergic concentrations (median between groups (Table 1), we subsequently examined and quartiles) that differed significantly between the groups. whether PMD was associated with monoaminergic concen- Specifically, DS versus DS1AD, DS1AD versus EOAD, trations. Spearman’s rank-order correlation tests within each and DS versus controls were compared. EOAD and controls group revealed few significant associations with PMDs: were used as the reference group (compared in [24], thus HVA (cingulate gyrus, r 5 20.90, P 5 .002) in the DS1AD not further described here). The supplementary material group; MHPG (caudate nucleus, r 5 0.85, P 5 .001 and SN, provides all concentrations and the accompanying r 5 0.91, P , .001), DA (BA11/12, r 5 0.74, P 5 .01), HVA ratios for noradrenergic (Supplementary Table 1), dopami- (BA9/10/46, r 5 0.80, P 5 .003; BA22, r 5 0.88, P , .001; 104 A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 Table 2 Comparison of postmortem concentrations between the groups DS DS1AD EOAD Controls Brain region Compound N (n 5 4) (n 5 17) (n 5 11) (n 5 10) P value Neocortex BA7 MHPG 2/13/11/10 71.0 (63.3–) 72.3 (57.0–105.2)* 119.4 (106.0–189.5)* 259.7 (119.8–354.5) ,.001 DA 2/12/11/10 11.0 (6.5–) 18.7 (12.5–28.7) 10.7 (6.4–16.5) 6.9 (4.5–9.9) .004 5-HIAA 2/13/11/10 92.8 (70.4–) 40.2 (30.4–71.1) 55.3 (40.5–93.7) 106.4 (75.3–158.9) .003 BA9/10/46 MHPG 4/14/11/10 84.2 (77.7–86.9) 107.4 (63.4–133.8)** 471.2 (284.3–655.1)** 265.7 (132.4–629.6) ,.001 DA 4/14/11/10 127.5 (11.4–497.4) 373.9 (204.3–743.6)** 7.2 (2.5–9.3)** 7.5 (4.0–11.3) ,.001 5-HT 4/14/11/10 17.8 (13.2–30.6) 28.7 (15.3–49.8)* 11.1 (6.1–15.0)* 11.7 (7.4–16.7) .009 5-HIAA 4/14/11/10 81.1 (64.6–122.2) 62.2 (33.9–87.4)* 144.7 (110.5–186.6)* 164.1 (128.2–215.6) .001 BA17 MHPG 3/7/11/10 77.2 (61.3–)# 89.2 (73.6–114.6) 127.9 (101.6–182.0) 285.3 (244.0–530.1)# ,.001 5-HIAA 3/7/11/10 126.0 (93.6–) 92.1 (46.9–144.1) 95.6 (57.5–143.6) 200.8 (154.3–263.9) .008 BA22 NA 3/10/9/10 30.2 (18.3–) 17.3 (12.5–27.4) 10.0 (7.4–12.4) 18.7 (13.6–28.5) .014 MHPG 3/10/11/10 107.3 (80.4–)# 87.0 (60.0–125.4)** 520.7 (313.4–673.4)** 360.5 (260.4–630.7)# ,.001 DA 3/10/11/10 6.7 (6.5–) 11.3 (8.1–25.2)* 4.2 (3.2–6.8)* 9.1 (5.4–18.4) .041 5-HIAA 3/10/11/10 119.1 (98.7–) 84.0 (60.4–131.7)* 303.2 (119.6–450.2)* 171.5 (121.7–320.6) .004 Limbic system Amygdala NA 2/10/10/10 21.8 (19.5–) 25.3 (13.6–39.8)* 59.0 (46.9–78.5)* 84.5 (77.5–121.8) ,.001 MHPG 2/10/10/10 68.8 (58.2–) 70.9 (62.5–94.9)** 429.8 (180.0–964.3)** 304.8 (193.5–759.3) ,.001 HVA 2/10/10/10 265.8 (174.5–) 376.2 (258.9–617.2) 599.1 (398.7–866.2) 1132.5 (751.0–1421.4) .005 5-HT 2/10/10/10 59.3 (11.7–) 33.0 (18.6–49.6)* 121.1 (55.1–148.6)* 244.9 (221.7–297.2) ,.001 5-HIAA 2/10/10/10 197.9 (162.9–) 141.3 (102.8–227.1)** 522.4 (334.9–795.2)** 999.8 (754.5–1270.4) ,.001 Hippocampus Adrenaline 2/5/3/5 391.6 (236.5–) 42.3 (20.6–139.0) 6.4 (2.6–) 10.1 (6.4–14.1) .011 MHPG 3/5/11/10 75.9 (70.3–)# 97.2 (76.9–124.8)** 459.5 (193.2–1099.2)** 416.3 (232.9–713.5)# ,.001 5-HT 3/5/11/10 46.6 (28.0–) 13.5 (8.6–50.0) 44.4 (20.7–65.3) 87.8 (69.3–111.2) .003 5-HIAA 3/5/11/10 141.3 (110.6–) 47.7 (43.1–213.4)* 336.1 (257.8–476.2)* 383.9 (279.5–717.0) .004 BA11/12 MHPG 4/6/11/10 91.8 (70.6–105.7)# 103.1 (63.9–111.2)** 437.1 (352.6–545.6)** 361.7 (216.5–636.3)# ,.001 5-HIAA 4/6/11/10 98.2 (85.4–189.7) 56.7 (35.3–78.5)** 238.5 (183.5–344.3)** 229.9 (168.9–328.3) ,.001 Cingulate gyrus MHPG 3/8/11/10 128.0 (58.8–) 126.0 (104.6–153.6)** 567.8 (258.4–739.3)** 336.9 (158.1–587.5) ,.001 DA 3/8/11/10 224.2 (7.9–) 55.5 (40.6–211.2)** 10.5 (3.8–13.0)** 9.6 (3.0–17.4) ,.001 5-HIAA 3/8/11/10 66.0 (34.5–)# 102.6 (58.5–191.8)** 357.3 (281.2–379.3)** 387.2 (313.7–475.7)# ,.001 Thalamus MHPG 3/11/11/10 159.2 (131.3–)# 148.3 (110.2–177.8)** 793.0 (628.6–1442.6)** 441.9 (244.1–1345.4)# ,.001 5-HIAA 3/11/11/10 673.7 (508.8–) 689.3 (412.4–895.8)** 1584.5 (1237.1–1952.8)** 1525.8 (1168.1–1946.8) ,.001 Basal ganglia Caudate nucleus Adrenaline 2/11/7/6 533.3 (355.2–) 69.3 (42.7–151.0)* 268.5 (176.5–587.7)* 372.9 (185.7–743.0) .006 DA 3/16/11/10 4297.2 (1845.8–) 2395.2 (1178.5–2784.8)** 4721.2 (3403.8–6905.9)** 3965.1 (2987.2–4420.5) .003 HVA 3/16/11/10 2732.0 (2231.3–) 3145.6 (1522.7–3756.0)* 4681.2 (3714.5–5640.3)* 4372.3 (3564.4–6818.8) .014 5-HT 3/16/11/10 121.5 (33.1–) 55.8 (35.6–97.0)** 168.3 (139.5–230.2)** 240.0 (188.0–285.2) ,.001 5-HIAA 3/16/11/10 170.9 (76.6–) 217.6 (106.3–284.2)** 537.2 (362.3–727.8)** 579.6 (441.0–784.9) ,.001 Globus pallidus DOPAC 2/8/11/10 208.3 (28.0–) 131.7 (61.8–141.5)* 39.5 (19.5–85.8)* 20.1 (10.5–34.7) .004 5-HT 2/8/11/10 149.7 (89.7–) 97.4 (70.5–120.4)* 171.9 (117.6–207.7)* 161.8 (140.3–215.5) .022 5-HIAA 2/8/11/10 1009.2 (384.6–) 439.5 (329.9–978.8)* 984.5 (864.7–1407.3)* 1320.2 (1019.2–1614.4) .015 Putamen Adrenaline 2/8/11/10 390.4 (325.6) 135.0 (40.9–219.9)* 581.9 (187.2–1523.4)* 339.6 (100.7–797.8) .038 DOPAC 3/15/11/10 207.5 (165.1–) 611.6 (274.7–851.6) 421.9 (235.7–625.7) 202.2 (119.0–299.7) .008 5-HT 3/15/11/10 189.5 (43.9–) 77.8 (35.2–159.6)* 189.2 (153.9–219.8)* 219.7 (200.5–326.3) ,.001 5-HIAA 3/15/11/10 260.0 (231.7–) 372.7 (203.7–669.5)** 790.9 (638.7–1026.1)** 998.4 (781.9–1400.3) ,.001 Substantia nigra DA 2/14/11/10 164 (126.8–) 157.2 (89.0–292.4)** 572.7 (284.9–623.7)** 624.0 (295.7–936.8) ,.001 DOPAC 2/14/11/10 47.2 (47.1–) 58.0 (27.1–87.4)* 189.7 (80.3–211.0)* 47.1 (29.2–101.0) .005 HVA 2/14/11/10 3061.7 (2010.5–) 2421.8 (1995.7–3007.8)** 3799.4 (3461.0–4385.5)** 4331.0 (3241.6–5349.9) ,.001 5-HT 2/14/11/10 208.3 (167.6–) 364.1 (243.1–420.4) 487.2 (389.2–531.2) 461.0 (404.1–588.8) .009 Metencephalon Locus coeruleus NA 0/10/10/10 – 88.6 (52.7–11.4)** 255.1 (171.9–400.7)** 347.3 (248.6–501.8) ,.001 MHPG 0/10/10/10 – 201.8 (151.8–259.5)* 429.6 (304.6–530.7)* 572.4 (158.9–836.2) .032 DOPAC 0/10/10/10 – 12.3 (8.1–19.2)** 39.0 (23.6–71.3)** 55.5 (33.1–98.6) ,.001 HVA 0/10/10/10 – 730.2 (482.0–1003.5) 1009.7 (905.7–1443.4) 1351.0 (1195.0–1482.2) ,.001 (Continued ) A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 105 Table 2 Comparison of postmortem concentrations between the groups (Continued ) DS DS1AD EOAD Controls Brain region Compound N (n 5 4) (n 5 17) (n 5 11) (n 5 10) P value Cerebellum Adrenaline 0/7/4/6 – 36.4 (34.7–44.5)* 6.6 (3.6–12.7)* 21.4 (10.3–50.2) .017 MHPG 2/9/11/10 124.6 (100.3–) 101.1 (85.2–148.6)* 421.1 (232.5–708.2)* 518.1 (386.8–713.4) .001 DA 2/9/11/10 32.1 (8.4–) 15.5 (12.2–22.1)** 3.6 (1.5–8.6)** 3.6 (3.2–5.4) ,.001 DOPAC 2/9/11/10 35.1 (6.4–) 16.4 (11.3–26.8)* 8.2 (5.8–11.1)* 8.0 (6.0–8.3) .005 5-HT 2/9/11/10 66.5 (22.2–) 25.0 (14.5–34.9)** 4.6 (2.1–9.1)** 2.6 (2.3–6.8) ,.001 5-HIAA 2/9/11/10 30.8 (21.9–) 41.7 (33.8–56.5)** 206.0 (92.8–301.4)** 98.5 (73.7–207.2) ,.001 Abbreviations: AD, Alzheimer’s disease; BA, Brodmann area; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; DA, dopamine; DS, Down syndrome without neuropathologic AD diagnosis; DS1AD, Down syndrome with neuropathologic AD diagnosis; DOPAC, 3,4-dihydroxyphenylacetic acid; EOAD, early-onset Alzheimer’s disease; HVA, homovanillic acid; MHPG, 3-methoxy-4-hydroxyphenylglycol; NA, noradrenaline; n.s., not significant. NOTE. Monoamines and metabolites (ng/g tissue) are expressed as median (50%) with the interquartile range (25%–75%) between brackets. Kruskal-Wallis tests were used to compare the four groups. If P ,.05, post hoc Mann-Whitney U tests were performed. After all analyses had been conducted, we accounted for multiple comparisons by performing the Benjamini-Hochberg procedure. Here, we provide P values (Kruskal-Wallis) that remained significant after correction (P ,.015). Those in italics are no longer regarded significant but are nevertheless provided since the post hoc Mann-Whitney U tests remained significant in the Benjamini-Hochberg procedure. Post hoc comparisons were performed for DS vs. controls (#P , .015) and DS1AD vs. EOAD (*P , .015; **P ,.001). The accompanying ratios and the nonsignificant comparisons are provided in the supplementary material (Supplementary Tables 1-3). BA11/12, r 5 0.91, P , .001; hippocampus, r 5 0.86, cingulate gyrus, and cerebellum and lower in the basal ganglia P 5 .001; thalamus, r 5 0.75, P 5 .007; and cerebellum, (caudate nucleus and SN). Similarly, in DS1AD (vs. EOAD), r 5 0.78, P 5 .004) in the EOAD group; and 5-HT in the HVA was reduced in the caudate nucleus and SN. Conse- globus pallidus (r 5 20.85, P 5 .002) in the control group. quently, the HVA:DA ratio, indicative of dopaminergic turn- Although the PMD range was the largest in the DS1AD over, was consistently lower in DS1AD (vs. EOAD) in group, most significant correlations were observed in the cortical areas (BA7, BA9/10/46, and BA22), limbic regions EOAD group with overall lower PMDs (range: 2.8–7 hours). (BA11/12, cingulate gyrus, and thalamus), and the cerebellum. Although an effect of PMDs cannot be ruled out, our main In contrast, the HVA:DA ratio was increased in the SN. The findings do not appear to be evidently affected by PMDs. pattern for DOPAC was bidirectional as well: values were decreased in the SN and LC and increased in the globus pallidus 3.1.1. Noradrenergic system and cerebellum. The DOPAC:DA ratio was significantly lower Between groups, NA differed significantly in the BA22, in the BA9/10/46, BA22, cingulate gyrus, and LC for DS1AD amygdala, and LC. Specifically, NA levels were not altered versus EOAD. In short, the dopaminergic system was evidently between DS and DS1AD, but lower in DS1AD compared affected in DS1AD with higher DA levels (and thus lower with EOAD (amygdala and LC, Table 2). This pattern was HVA:DA and DOPAC:DA ratios) in cortical areas, limbic re- more pronounced for MHPG (Fig. 2). Compared with gions, and the cerebellum and lower DA and HVA levels in EOAD, DS1AD presented significantly lower MHPG levels the basal ganglia. in the LC, cortical (BA7, BA9/10/46, and BA22) and limbic projection areas (amygdala, hippocampus, BA11/12, cingu- 3.1.3. Serotonergic system late gyrus, and thalamus), and cerebellum. Consequently, the 5-HT and 5-HIAA did not differ significantly between DS MHPG:NA ratio was consistently lower in DS1AD (BA9/ and DS1AD. 5-HIAA levels in the cingulate gyrus and the 10/46, BA22, hippocampus, BA11/12, cingulate gyrus, thal- 5-HIAA:5-HT ratio in the (pre)frontal cortex were signifi- amus, and cerebellum), indicating a reduced noradrenergic cantly lower in DS than in controls. Compared with turnover. Moreover, MHPG was significantly lower in DS EOAD, 5-HT levels in DS1AD were significantly lower compared with controls (Fig. 2) for BA17, BA22, hippocam- in the amygdala and basal ganglia (caudate nucleus, globus pus, BA11/12, and thalamus. Neither NA nor MHPG levels pallidus, and putamen) and higher in the (pre)frontal cortex differed significantly in the basal ganglia, whereas adrena- and cerebellum. In comparison with EOAD, 5-HIAA was line levels were lower in the caudate nucleus and putamen consistently lower in DS1AD, namely in cortical areas and higher in the cerebellum in DS1AD (vs. EOAD). Taken (BA9/10/46, BA22), limbic system (amygdala, hippocam- together, the noradrenergic system—particularly MHPG— pus, BA11/12, cingulate gyrus, and thalamus), the basal was strongly impaired in DS (vs. controls) and DS1AD ganglia (caudate nucleus, globus pallidus, and putamen), (vs. EOAD). and the cerebellum. Similarly, the 5-HIAA:5-HT ratio was reduced in the BA9/10/46, BA22, BA11/12, and cerebellum 3.1.2. Dopaminergic system in DS1AD versus EOAD, thus indicating an overall No significant differences were observed for DS versus decreased serotonergic turnover in DS1AD. In summary, DS1AD and DS versus controls. Compared with EOAD, bidi- a serotonergic deficit became apparent in DS1AD, with a rectional dopaminergic changes became evident in DS1AD: pronounced overall reduction in 5-HIAA levels (and thus a DA levels were significantly higher in the BA9/10/46, BA22, reduced 5-HIAA:5-HT ratio) as compared with EOAD. 106 A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 Finally, the HVA:5-HIAA ratio, indicating serotonergic Abbreviations: APOE, apolipoprotein E gene; DS, Down syndrome without (clinical) dementia; DS1AD, Down syndrome with diagnosed AD dementia; DS1pAD, Down syndrome with prodromal AD; n.s., P value performed to identify significant group differences (P ,.015): (a) DS vs. DS1pAD; (b) DS vs. DS1AD; and (c) DS1pAD vs. DS1AD. Gender, APOE status, and medication use were compared using Pearson’s NOTE. Age at the moment of sampling is provided as median with range (minimum–maximum). The Kruskal-Wallis test was performed to compare ages between groups. Post hoc Mann Whitney U tests were ,.001 .014 .002 n.s. n.s. n.s. n.s. n.s. n.s. n.s. inhibition on dopaminergic neurotransmission, clearly differed between groups. Apart from a significantly higher HVA:5-HIAA ratio in the cingulate gyrus in DS (vs. con- trols), significance was, again, observed for the DS1AD Diagnosed AD dementia (DS1AD, n 5 40) versus EOAD comparison. The ratio was invariably higher in DS1AD for cortical regions (BA9/10/46 and BA22), 1 (ε2/3), 1 (ε2/4), 30 (ε3/3), 8 (ε3/4) limbic system (amygdala, hippocampus, BA11/12, and cingulate gyrus), and the cerebellum, suggestive of a reduced serotonergic inhibition on the dopaminergic system. b,c 3.2. Monoaminergic characterization of (paired) CSF/ 55.1 (42.1–69.2) plasma samples 25 (62.5) 14 (35.0) 24 (60.0) 9 (22.5) 10 (25.0) 13 (32.5) 8 (20.0) 5 (12.5) Samples of 225 DS individuals were included in analysis (Fig. 1). Paired samples were available for 68 individuals and plasma-only samples for 157 cases. Tables 3 and 4, respectively, show the study group characteristics and monoaminergic concentrations. The accompanying ratios are 1 (ε2/2), 6 (ε2/3), 21 (ε3/3), 8 (ε3/4) Prodromal AD (DS1pAD, n 5 36) provided in the supplementary material, Supplementary Table 4. Remarkably, CSF/plasma concentrations did not differ between the three groups apart from DOPAC levels in CSF (medication-free subpopulation) and plasma (total population and medication-free subpopulation). DOPAC levels were a,c 49.8 (37.7–59.4) consistently higher in DS1AD compared with DS (CSF medication-free, P 5 .001; plasma total, P , .001; and plasma 16 (44.4) 12 (33.3) 19 (52.8) 11 (30.6) 7 (19.4) 8 (22.2) 3 (8.3) 1 (2.8) medication-free, 0.002) but did not differ for the DS versus DS1pAD and DS1pAD versus DS1AD comparisons. Simi- larly, plasma HVA (total), plasma 5-HIAA (total), and CSF DOPAC:DA (medication-free) were higher in DS1AD versus DS. In contrast, CSF HVA:5-HIAA (total) was decreased in 1 (ε2/2), 15 (ε2/3), 2 (ε2/4), 106 (ε3/3), 23 (ε3/4), 2 (ε4/4) DS1AD. Moreover, DA (r 5 20.31, P 5 .012), DOPAC (r 5 10.71, P , .001), MHPG (r 5 10.70, P , .001), and adrenaline (r 5 10.49, P , .001) correlated significantly in CSF and plasma (paired samples, total population). Groups differed in age with DS1AD logically being the oldest. DOPAC (CSF, r 5 10.362, P 5 .002; plasma, r 5 10.386, P , .001), HVA (plasma, r 5 10.169, P 5 .011), and 5- HIAA (CSF, r 5 10.365, P 5 .002; plasma, r 5 10.345, No dementia (DS, n 5 149) P , .001) correlated significantly with age. After exclusion of individuals younger than 45 years, that is, resembling the a,b elderly DS cohort in our previously published serum study 36.9 (19.2–61.2) [12], comparison between DS (CSF/plasma, n 5 8; plasma- only, n 5 34), DS1pAD (CSF/plasma, n 5 11; plasma-only, 82 (55.0) 61 (40.9) 56 (37.6) 36 (24.2) 20 (13.4) 13 (8.7) 14 (9.4) 1 (0.7) n 5 31), and DS1AD (CSF/plasma, n 5 16; plasma-only, Characteristics of CSF/plasma study groups n 5 38) yielded no significant monoaminergic differences, again suggesting that DOPAC changes most likely relate to ag- ing rather than dementia status. c2 test or Fisher’s exact test. Any psychoactive drugs (%) Age (median; min.–max.) Gender (N male and %) 4. Discussion Levothyroxine (%) Antidepressants Antipsychotics Monoaminergic profiles were evaluated in 15 postmor- Antiepileptics Antidementia not significant. Characteristics Anxiolytics tem brain regions and (paired) CSF/plasma samples. In APOE status brain, pronounced noradrenergic, dopaminergic, and seroto- Table 3 nergic differences were found for DS1AD versus EOAD and to a lesser extent for DS versus controls (primarily A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 107 Table 4 Comparison of CSF/plasma concentrations between the groups Compound CSF or plasma Total or medication free N DS DS1pAD DS1AD P value NA CSF Total 37/13/18 2.8 (0.9–5.5) 1.4 (0.6–4.9) 1.8 (0.8–5.6) n.s. Medication free 21/7/10 2.8 (1.0–6.9) 1.4 (0.6–4.7) 1.4 (0.8–5.5) n.s. Plasma Total 145/35/37 0.4 (0.2–0.9) 0.6 (0.2–0.9) 0.6 (0.4–1.1) n.s. Medication free 90/16/15 0.5 (0.2–1.0) 0.4 (0.1–0.7) 0.4 (0.2–1.0) n.s. Adrenaline CSF Total 35/13/16 0.5 (0.3–0.9) 0.7 (0.3–1.3) 0.4 (0.3–1.2) n.s. Medication free 20/7/10 0.7 (0.3–1.0) 0.8 (0.2–2.1) 0.4 (0.4–1.2) n.s. Plasma Total 147/35/39 2.9 (1.9–4.2) 2.6 (1.9–4.0) 3.3 (1.4–4.3) n.s. Medication free 92/17/16 2.9 (2.0–4.0) 3.0 (2.0–4.2) 2.7 (1.2–3.9) n.s. MHPG CSF Total 37/13/18 30.2 (21.5–44.6) 24.0 (18.8–40.0) 22.4 (19.6–46.0) n.s. Medication free 21/7/10 31.1 (21.8–45.0) 24.0 (15.9–41.1) 28.5 (20.7–43.4) n.s. Plasma Total 149/36/40 76.2 (51.2–102.4) 70.9 (50.4–111.6) 75.6 (55.3–114.4) n.s. Medication free 93/17/16 77.3 (51.5–100.6) 70.4 (45.5–119.3) 63.0 (47.9–107.5) n.s. DA CSF Total 37/13/18 0.6 (0.4–0.9) 0.5 (0.2–0.7) 0.6 (0.3–1.2) n.s. Medication free 21/7/10 0.6 (0.4–0.9) 0.6 (0.3–1.0) 0.8 (0.4–1.3) n.s. Plasma Total 149/35/40 0.5 (0.3–1.0) 0.5 (0.3–1.1) 0.7 (0.3–1.3) n.s. Medication free 93/16/16 0.5 (0.3–1.0) 0.6 (0.4–1.1) 0.7 (0.4–1.9) n.s. DOPAC CSF Total 37/13/18 0.6 (0.4–1.3) 1.2 (0.6–2.7) 1.7 (0.7–3.3) .033 Medication free 21/7/10 0.5 (0.4–1.0)xx 0.9 (0.5–1.5) 2.8 (1.1–5.6)xx .003 Plasma Total 149/36/40 2.7 (1.9–4.1)xx 3.1 (2.6–4.3) 4.3 (3.0–6.2)xx ,.001 Medication free 93/17/16 2.5 (1.9–3.6)x 3.1 (2.7–3.6) 5.3 (2.9–6.2)x .004 HVA CSF Total 37/13/18 54.6 (40.5–67.7) 55.1 (43.3–74.8) 55.3 (33.3–67.3) n.s. Medication free 21/7/10 56.3 (41.5–64.1) 46.5 (35.2–79.5) 56.4 (33.1–67.2) n.s. Plasma Total 149/36/40 9.4 (7.5–11.6)x 10.3 (7.4–12.7) 10.6 (8.7–15.0)x .039 Medication free 93/17/16 9.5 (7.4–12.2) 10.5 (7.6–13.0) 9.9 (8.5–14.2) n.s. 5-HT CSF Total 11/4/6 0.1 (0.1–0.2) 0.1 (0.1–0.2) 0.1 (0.1–0.2) n.s. Medication free 6/2/2 0.1 (0.1–0.2) 0.1 (0.1–) 0.1 (0.0–) n.s. Plasma Total 149/36/40 9.5 (3.9–20.3) 11.1 (2.4–20.8) 9.7 (5.2–17.2) n.s. Medication free 93/17/16 12.4 (7.1–25.0) 17.3 (8.0–28.6) 10.6 (8.4–20.7) n.s. 5-HIAA CSF Total 37/13/18 24.9 (18.1–28.9) 26.5 (21.5–35.4) 28.9 (23.2–34.3) n.s. Medication free 21/7/10 26.6 (22.6–29.7) 26.5 (22.0–36.1) 33.2 (25.2–38.4) n.s. Plasma Total 149/36/40 4.5 (3.7–5.4)x 4.7 (4.2–5.7) 5.0 (4.1–6.6)x .020 Medication free 93/17/16 4.5 (3.6–5.4) 5.3 (4.2–6.5) 4.9 (3.6–6.7) n.s. Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; DA, dopamine; DS, Down syn- drome without (clinical) dementia; DS1pAD, Down syndrome with prodromal AD; DS1AD, DS with diagnosed AD dementia; DOPAC, 3-4- dihydroxyphenylacetic acid; HVA, homovanillic acid; MHPG, 3-methoxy-4-hydroxyphenylglycol; NA, noradrenaline; n.s., not significant. NOTE. Concentrations of monoamines and metabolites (ng/ml) are expressed as median (50%) with the interquartile range (25%–75%) between brackets. The number (N) of samples is provided as certain compounds were not detectable in all samples. Kruskal-Wallis tests were performed to compare the three groups. If P ,.05, post hoc Mann-Whitney U tests were performed. After all analyses had been conducted, we accounted for multiple comparisons by perform- ing the Benjamini-Hochberg procedure. The P values in italics are no longer regarded significant but are nevertheless provided since the post hoc Mann-Whitney U tests remained significant in the Benjamini-Hochberg procedure. Post hoc comparisons were performed for DS vs. DS1pAD, DS vs. DS1AD (xP ,.015 and xx P ,.001) and DS1pAD vs. DS1AD. The accompanying ratios are provided in the supplementary material (Supplementary Table 4). decreased MHPG levels), but not for DS versus DS1AD. DS1AD (even more than in EOAD), but also in DS, thus Similarly, CSF/plasma concentrations were virtually unal- already before the neuropathological criteria for AD were tered between the diagnostic DS groups. met. In AD, studies have demonstrated LC neuronal DA is produced in the SN and ventral tegmental area loss and reduced NA levels [21,35–39]. Noradrenergic (VTA). In AD, a variable SN neuronal loss and diminished abnormalities have been implicated in DS too [40]. Here, DA levels have been described [41,42]. Whereas previous we demonstrate that the noradrenergic system was more studies did not report evident dopaminergic alterations in severely impacted in DS1AD versus EOAD and to a lesser DS [16,18], we found significantly increased DA levels (and extent in DS versus non-DS controls. NA, MHPG, and the thus decreased HVA:DA and DOPAC:DA ratios) in cortical MHPG:NA ratio were significantly reduced in most brain areas, limbic regions and cerebellum, and a general areas, but not in the basal ganglia, which is in accordance decrease in DA and HVA levels in the basal ganglia. with the modest noradrenergic innervation of the basal Indeed, lower DA levels in the caudate nucleus have been ganglia [35]. These results are also in agreement with earlier reported in DS1AD versus EOAD and age-matched controls studies reporting AD-related loss of LC neurons [21–23,36] [20]. Ascending dopaminergic projections are subdivided into and reduced NA levels in various brain regions in DS1AD the nigrostriatal (from SN to striatum), mesolimbic (from compared with controls [16–20]. Our results demonstrate VTA to limbic system), and mesocortical (from VTA to cor- that MHPG concentrations were most severely impacted in tex) pathways [35]. Previously, a mild cell loss (though often 108 A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 not significant) in the SN, but also in the VTA, was found in than four such cases (limiting the generalizability of the find- DS1AD compared with controls or younger counterparts ings in this group) because inclusion of DS cases in brain [23,36,43,44]. Our results may suggest a more severe banks, those without pathology in particular, is very limited. impairment of the nigrostriatal pathway (reduced DA levels In fact, the 21 cases analyzed here were obtained by three in caudate and SN), whereas the mesolimbic and large brain banks in a timeframe of 25 years. Contemporary mesocortical pathways seem to be somewhat overactive, standardized (multicenter) brain banking efforts for DS are possibly as a compensatory mechanism. thus imperative [51,52], focusing, among others, on the Concerning the serotonergic system, neuronal loss in the collection of tissues with short(er) PMDs and good clinical dorsal raphe nuclei (5-HT production site) and reduced documentation. Although our main findings did not appear levels of 5-HT and 5-HIAA in various brain regions have to be evidently impacted by PMDs or psychoactive been reported in AD and DS [16–18,35–37,39,45]. medication use, such effects cannot be fully ruled out Compared with EOAD, we observed an even more severe because of the unavailability of DS tissue with low PMDs serotonergic impairment in DS1AD, presenting decreased and no psychoactive medication use. 5-HIAA levels in 11 brain regions, while 5-HT was reduced The apparent lack of monoaminergic changes between in the amygdala and basal ganglia but increased in the (pre) DS and DS1AD in the brain was also reflected in CSF/ frontal cortex and cerebellum. plasma. The CSF/plasma groups were distinguished based Interestingly, the DS and DS1AD groups showed remark- on a clinical dementia diagnosis, whereas from a neuropath- ably similar monoaminergic profiles, although both groups ological perspective, the (amyloid) pathology is likely to be had different AD neuropathologic changes (low vs. high). quite comparable. In future studies, it would be useful to Importantly, the four DS cases with low AD neuropathologic relate monoaminergic values in DS to (in vivo) pathologic change already presented high amyloid burden (“ABC staging, such as the CSF “AD profile” [11] or positron scoring system” [25]: A2,B1,C2; A3,B1,C1; A3,B0,C0, and emission tomography of Ab/tau [5]. Furthermore, mounting A3,B0,C0, respectively). The third copy of the APP gene in evidence indicates an important role of neuroinflammation DS causes very early Ab overproduction and accumulation. in the pathogenesis of AD (in DS) [53], and it would thus Deposition of Ab plaques occurs at ages as early as 12 years be valuable to look further than Ab and tau pathology and and precedes tau pathology by many years [4]. Previously, examine the role of neuroinflammatory processes in mono- noradrenergic and serotonergic depletion was found to be aminergic alterations as well. more severe in EOAD (mutations in APP or PSEN1/2, Surprisingly, the CSF/plasma results did not reflect promoting the amyloidogenic pathway) than in late-onset earlier results obtained in serum [12]. Whereas MHPG, for AD [38]. Inverse relations between Ab accumulation and, instance, was evidently decreased in DS1AD serum, respectively, NA, DA, and 5-HT signaling have been MHPG levels were virtually unaltered in CSF/plasma. This described [35,46]. This may suggest that the raises the question what causes this apparent discrepancy. monoaminergic system is particularly affected by (early) Our methodology has been validated [30], and the reported Ab pathology, being altered long before full-blown AD values have orders of magnitude comparable to earlier pathology is present. For a comprehensive summary about studies [14,15,54]. The—likely multifactorial—answer the pathophysiological link between monoaminergic alter- remains to be elucidated, including the effect of ations and AD pathology, see the review by Trillo et al. [35]. (alterations in) peripheral determinants, such as non-brain In the context of abnormal brain development, sources of catecholamines (e.g., the sympathetic nervous monoamines were quantified in the frontal cortex of fetal system is the main source of peripheral NA) and enzymes DS tissue (20 weeks) compared with controls. DA, 5-HT, involved in catecholaminergic turnover [55], as well as and 5-HIAA levels were significantly reduced in DS [47]. (pre)analytical variables. O’Bryant et al. (2015) addressed This suggests that monoamines are already impacted by tri- variables that can impact findings in blood, including somy 21 itself, which may be further impaired by progres- controllable variables (e.g., fasting status, tube type, centri- sive Ab pathology during life. Compared with fugation parameters, time from collection to freezing, and age-matched controls, smaller brain volumes were found freezing temperature) and uncontrollable variables (e.g., in DS, among others of (pre)frontal cortex, hippocampus, diet, activity level, comorbidities, and medication). In partic- brainstem and cerebellum [48–50]. Fewer neurons ular, serum versus plasma, type of needle, additive in the (cortical dysgenesis), altered neuronal distribution, and collection tubes, and presence of hemolysis may influence reduced synaptic density were described in DS as well the stability and detectability of biomarkers [56]. In CSF, [49]. Consequently, the compensatory reserve is likely to similar variables may impact biomarker levels [57,58]. be lower, which could result in a particularly early vulnera- Indeed, a few variables differ identifiably between our bility (functional impact) to additional neuropathology. To serum and plasma studies, such as fasting status, storage differentiate between the alterations caused by trisomy 21 temperature, and storage time. Retrospectively identifying and AD pathology, respectively, future monoaminergic the cause of the discrepancy is virtually impossible. New studies should include DS samples without early Ab plaque initiatives should, therefore, systematically study the effect load. In the present study, we were unable to collect more of these variables on monoaminergic concentrations. A.D. Dekker et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 99-111 109 In conclusion, this study is the first to comprehensively for their immunohistochemical work, and Laia Mu~noz and examine monoaminergic alterations in a unique collection Raul Nu~nez (Sant Pau) for the laboratory and sample of postmortem brain regions and (paired) CSF/plasma sam- handling. ples of DS individuals. Despite various limitations described previously, brain samples of DS1AD (vs. EOAD) revealed Supplementary data generalized impairments in the noradrenergic and seroto- Supplementary data related to this article can be found at nergic systems (overall decrease) and a bidirectional https://doi.org/10.1016/j.dadm.2017.11.001. dopaminergic change. For DS (vs. controls), significantly decreased MHPG levels were noted, though to a lesser extent than for DS1AD (vs. EOAD). DS and DS1AD groups showed remarkably similar monoaminergic profiles in the RESEARCH IN CONTEXT brain. CSF/plasma concentrations did not differ between the diagnostic DS groups either. The underlying cause for the discrepancy with earlier serum findings remains unclear 1 Systematic review: Alterations in monoamine neuro- and requires further study. To confirm whether the more transmitters and metabolites have been implicated in profound monoaminergic alterations in DS (vs. non-DS) Alzheimer’s disease (AD) and Down syndrome (DS). are indeed due to early Ab accumulation, (longitudinal) However, monoaminergic profiles have not been studies using positron emission tomography imaging of extensively studied in cerebrospinal fluid (CSF) and monoamines might provide a new avenue. For instance, neu- postmortem brain samples of DS with/without AD. roimaging of NA transporters in LC and key projection areas using [11C]methylreboxetine [59] in relation to amyloid 2 Interpretation: This is the first study to comprehen- deposition (e.g. [11C]Pittsburgh compound B) may be of sively characterize DS samples with regard to AD utmost importance in this respect. Moreover, to further diagnosis. In CSF/plasma, monoaminergic levels investigate disease progression, it would be valuable to were not related to the clinical status of dementia in relate monoaminergic concentrations to CSF/plasma levels DS. In brain, evident noradrenergic and serotonergic of Ab and tau within individuals, for instance to the CSF deficits were found in DS1AD versus early-onset AD profile (low Ab42, high total-tau, and high AD patients, and to a lesser extent in DS versus phosphorylated-tau) [11]. non-DS healthy controls. Our results reveal a rather similar monoaminergic profile in both DS and DS1AD, possibly caused by early trisomy 21–related Acknowledgments accumulation of amyloid b (Ab). This study was financially supported by the Gratama- 3 Future directions: Positron emission tomography Stichting/Stichting Groninger Universiteitsfonds (2015–04), studies of monoaminergic neurotransmission may the Research School for Behavioural and Cognitive Neurosci- reveal whether monoaminergic impairment in DS re- ences of the University of Groningen (RUG), Interuniversity lates to early Ab accumulation. Longitudinal studies Poles of Attraction of the Belgian Federal Science Policy in relation to Ab imaging would be of utmost impor- Office (IAP network P7/16), the Belgian Foundation for tance. Alzheimer Research (SAO-FRA 15002), and the Carlos III National Institute of Health of Spain (CM14/00029, PI13/ 01532, PI11/02425 and PI14/01126) jointly funded by Fondo Europeo de Desarrollo Regional, Uni on Europea, ‘Una man- References era de hacer Europa’, Fundaci o Bancaria La Caixa, and Marat o de TV3 (20141210). 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