University of Groningen Down & Alzheimer Dekker, Alain Daniel

University of Groningen

Down & Alzheimer

Dekker, Alain Daniel

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2017

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Dekker, A. D. (2017). Down & Alzheimer: Behavioural biomarkers of a forced marriage. Rijksuniversiteit

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Severe Monoaminergic Impairment in Down

Syndrome with Alzheimer’s Disease Compared to

Early-Onset Alzheimer’s Disease

Alain D. Dekker

a,b

– Yannick Vermeiren

a,b

– Maria Carmona-Iragui

c,d

Bessy Benejam

d

_{– Laura Videla}

d

_{– Ellen Gelpi}

e

_{– Tony Aerts}

b

_{– Debby Van Dam}

a,b

Susana Fernández

d

_{– Alberto Lleó}

c

_{– Sebastian Videla}

d,f

Anne Sieben

b

– Jean-Jacques Martin

b

– Netherlands Brain Bank

g

Rafael Blesa

c

– Juan Fortea

c,d

and Peter P. De Deyn

a,b,h

a _{University of Groningen and University Medical Center Groningen}

b _{Institute Born-Bunge, University of Antwerp}

c _{Hospital de la Santa Creu i Sant Pau, Barcelona}

d _{Catalan Down Syndrome Foundation, Barcelona}

e _{Neurological Tissue Bank – Biobanc, Hospital Clinic Barcelona}

f _{Universitat Pompeu Fabra, Barcelona}

g _{Netherlands Institute for Neuroscience, Amsterdam}

h _{Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken}

submitted for publication

6

Abstract

Background: 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: Noradrenergic, dopaminergic and

serotonergic compounds were quantified in 15 brain regions of DS without AD (DS, n=4),

DS with AD (DS+AD, n=17), early-onset AD patients (EOAD, n=11) and healthy non-DS

controls (n=10). Moreover, monoaminergic concentrations were determined in

CSF/plasma samples of DS (n=37/149), DS with prodromal AD (DS+pAD, n=13/36) and

DS+AD (n=18/40). Results: In brain, noradrenergic and serotonergic compounds were

overall reduced in DS+AD vs EOAD, while the dopaminergic system showed a bidirectional

change. Apart from DOPAC, CSF/plasma concentrations were not altered between groups.

Discussion: Monoamine neurotransmitters and metabolites were evidently impacted in

DS, DS+AD and EOAD. DS and DS+AD presented a remarkably similar monoaminergic

profile, possibly related to early deposition of amyloid pathology in DS.

6.1. Introduction

People with Down syndrome (DS), or trisomy 21, have an exceptionally high risk to

develop Alzheimer’s disease (AD): 68-80% is diagnosed with dementia by the age of 65

(Wiseman et al., 2015). The additional copy of chromosome 21, encoding the amyloid

precursor protein (APP), causes overproduction of amyloid-β (Aβ) peptides from birth

onwards, resulting in early aggregation and deposition of characteristic Aβ plaques

(Lemere et al., 1996). In DS brains, not only plaques, but also neurofibrillary tangles, are

omnipresent from the age of 40 (Mann, 1988). The onset of clinical dementia symptoms,

however, is subject to a marked variation in time (Krinsky-McHale et al., 2008; Zigman and

Lott, 2007). Since the dementia diagnosis in DS is complex, among others due to

co-morbidities, pre-existing intellectual disability and behavior (Dekker et al., 2015b),

sensitive and specific biomarkers for AD in DS would be very valuable. In the general,

non-DS population, the so-called ‘AD profile’ (low Aβ42, high total-tau, and high

phosphorylated-tau) in cerebrospinal fluid (CSF) has proven useful as diagnostic aid

(Blennow et al., 2015). However, the clinical utility in DS has not been demonstrated yet

(Dekker et al., 2017). Therefore, the study of alternative biomarkers for AD in DS receives

vast attention.

In this context, we previously analyzed monoamine neurotransmitters and

metabolites in serum of 151 elderly DS individuals with AD (DS+AD) and without AD (DS),

but also in a non-demented group at blood sampling that developed dementia over time

(converters). Remarkably, serum levels of the primary noradrenergic metabolite

3-methoxy-4-hydroxyphenylglycol (MHPG) were strongly decreased in DS+AD, but also in

converters. Individuals with MHPG levels below median had a more than tenfold

increased risk of developing dementia, suggesting that decreased serum MHPG levels may

be predictive for conversion to AD (Dekker et al., 2015a).

Blood biomarkers, however, are subject to (confounding) peripheral effects. CSF

biomarkers are generally regarded better indicators of biochemical changes in the central

nervous system due to their direct contact with the extracellular space (Hampel et al.,

2012). Very few studies have investigated CSF biomarkers in (moreover small) DS cohorts

(Dekker et al., 2017), including two on monoamines (Kay et al., 1987; Schapiro et al.,

1987). Although a few post-mortem studies were conducted several decades ago, a

comprehensive profile of central monoaminergic changes in DS+AD is not established yet.

Indeed, monoamines were quantified in a limited number of brain regions from a few DS

cases with often long post-mortem delays. For instance, cell loss in the locus coeruleus

(LC), major source of noradrenaline (NA), and reduced NA concentrations have been

reported in elderly DS cases (German et al., 1992; Godridge et al., 1987; Mann et al.,

1987b; Marcyniuk et al., 1988; Reynolds and Godridge, 1985; Risser et al., 1997; Yates et

al., 1983, 1981), but an integrated study of regional changes in NA, dopamine (DA),

serotonin (5-HT) and their primary metabolites is lacking.

To the best of our knowledge, this study is the first to comprehensively evaluate

monoaminergic alterations in (1) post-mortem brain tissues, and (2) (paired) CSF/plasma

samples from DS individuals with and without AD. Noradrenergic (NA; adrenaline; MHPG),

dopaminergic (DA; 3,4-dihydroxyphenylacetic acid, DOPAC; homovanillic acid, HVA) and

serotonergic (5-HT; 5-hydroxyindoleacetic acid, 5-HIAA) compounds were quantified using

reversed-phase HPLC (RP-HPLC). In one of the largest collections of DS brain tissue (n=21),

15 regions of DS cases without (DS) and with a neuropathologically confirmed diagnosis of

AD (DS+AD) were analyzed and compared to early-onset AD patients (EOAD) and healthy

controls in the general population. Secondly, we report the monoaminergic results in

(paired) CSF/plasma samples obtained from the largest DS cohort to have undergone

lumbar punctures, comparing DS without dementia (DS), DS with prodromal AD (DS+pAD),

and DS with clinically diagnosed AD (DS+AD).

6.2. Materials & Methods

Post-mortem samples

Study population

In total, post-mortem samples from 21 elderly DS individuals were obtained from The

Netherlands Brain Bank, Netherlands Institute for Neuroscience (NBB; Amsterdam, The

Netherlands), the Neurological Tissue Bank-Biobanc, Hospital Clinic Barcelona-Institut

d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS; Barcelona, Spain) and the

Institute Born-Bunge (IBB; Antwerp, Belgium). Specifically, brain samples from 9 DS+AD

individuals were obtained from NBB (open access: www.brainbank.nl). All material has

been collected from donors for or from whom written informed consent for a brain

autopsy and the use of the material and clinical information for research purposes had

been obtained by NBB. Moreover, IDIBAPS provided samples of 2 DS and 5 DS+AD donors

for whom written informed consent was obtained from the next of kin. The study was

approved by the local ethics committee and in accordance with Spanish legislation. Finally,

IBB provided samples of DS (n=2), DS+AD (n=3), EOAD patients (n=11) and healthy

controls without neurological disease (n=10). Since DS+AD presents early in life, we

identified EOAD patients and controls <75 years of age as comparison groups. Ethics

approval was granted by the medical ethics committee of the Hospital Network Antwerp

(ZNA, approval numbers 2805 and 2806). The study was compliant with the Declaration of

Helsinki.

Assessment of AD neuropathologic change

Neuropathological analysis was conducted according to the ‘ABC scoring’ system (Montine

et al., 2012). Formalin-fixed paraffin-embedded samples were sectioned in concordance

with the minimally recommended brain regions (if available). If possible, additional

sections of the cingulate gyrus, amygdala, pons at the level of the LC, and medulla

oblongata were included. Applied stains were hematoxylin-eosin, cresyl violet,

Klüver-Barrera (myelin) and modified Bielschowsky silver staining. Moreover, antibodies against

amyloid (4G8), phosphorylated-tau (AT8), ubiquitin, TDP-43 and p62 Lck ligands were

used. All cases were diagnosed by experienced neuropathologists (EG, AS and JJM) as Not,

Low, Intermediate or High AD neuropathologic change. Intermediate and High signify the

diagnosis of AD (Montine et al., 2012).

Regional brain samples and dissection

Table 6.1 shows the selection of frozen samples for RP-HPLC analyses. Brains were

included in the three biobanks between 1990 and 2011 and stored at -80°C. Post-mortem

delays: NBB (<10 hours), IDIBAPS (<12 hours) and IBB (DS: 20 and 36 hours; DS+AD: 15, 20,

and one unknown; EOAD/controls: <7 hours). Samples were dissected from the left

hemispheres (indecisive for three IBB cases). Not all regions were available for all cases.

Most samples from EOAD and controls have been published before (Vermeiren et al.,

2016). For this study, Brodmann area (BA)7, substantia nigra (SN), caudate nucleus, globus

pallidus and putamen were additionally analyzed.

CSF/plasma samples

Samples of 241 DS adults were obtained from the Down Alzheimer Barcelona

Neuroimaging Initiative (DABNI), a prospective biomarker study for AD in DS

(Carmona-Iragui et al., 2017, 2016; Fortea et al., 2016). The person with DS and/or the legal

representative provided written informed consent. The study was compliant with the

Declaration of Helsinki and locally approved (Carmona-Iragui et al., 2016). Neurologists

and neuropsychologists established a consensus diagnosis of dementia, distinguishing

between DS without dementia (DS), DS with prodromal AD (DS+pAD), DS with diagnosed

AD (DS+AD). DS cases with cognitive decline due to psychiatric etiology were excluded

(Figure 6.2). Use of psychoactive medication around the moment of sampling was noted.

Within the DABNI study, participants are offered a lumbar puncture, which was found to

be feasible and safe (Carmona-Iragui et al., 2016). For 68 individuals, paired CSF/plasma

samples were obtained. The other 157 participants provided plasma-only. Samples were

stored at -80°C.

RP-HPLC

To quantify noradrenergic (NA; adrenaline; MHPG), dopaminergic (DA; DOPAC; HVA) and

serotonergic (5-HT; 5-HIAA) compounds, a validated RP-HPLC set-up with ion-pairing

(octane-1-sulfonic acid sodium salt, OSA) and amperometric electrochemical detection

was used (Van Dam et al., 2014), previously applied to CSF and blood samples (Dekker et

al., 2015a) and brain homogenates (Vermeiren et al., 2016, 2015, 2014a, 2014b).

Concentrations were calculated using Clarity

TM

_{software (DataApex Ltd., 2008, Prague,}

Czech Republic).

Statistics

Histograms, Normal Quantile-Quantile (Q-Q) plots and Shapiro-Wilk tests (P<0.05)

demonstrated that the concentrations in brain and CSF/plasma were (largely) not

normally distributed. Consequently, non-parametric Kruskal-Wallis tests were applied to

compare groups. If the P-value was <0.05, post-hoc Mann-Whitney U tests were

conducted. In brain, we compared: DS vs DS+AD, DS+AD vs EOAD and DS vs controls.

Regarding CSF/plasma samples, we analyzed the total cohort (n=225), i.e. all individuals

regardless of medication use, as well as the medication-free subpopulation since

psychoactive medication may affect monoaminergic neurotransmission. Non-parametric

Spearman’s rank-order correlation tests established the relationship with age, and

between CSF and plasma concentrations. Cohort characteristics like gender and

medication use were compared using Pearson’s Chi-Square tests or Fisher’s Exact tests. To

account for multiple comparisons, we applied a Benjamini-Hochberg procedure with a

false discovery rate of 0.05. Original P-values below 0.015 were regarded significant. IBM

SPSS Statistics version 23.0 was used.

6.3. Results

Based on the measured concentrations, five accompanying ratios were calculated:

(1) MHPG:NA (noradrenergic turnover), (2) DOPAC:DA and (3) HVA:DA (both dopaminergic

turnover), (4) 5-HIAA:5-HT (serotonergic turnover), and (5) HVA:5-HIAA (serotonergic

inhibition on dopaminergic neurotransmission).

Monoaminergic characterization of post-mortem brain tissue

Table 6.1 shows the general demographics, Table 6.2 provides the monoaminergic

concentrations and ratios (median and quartiles) that differed significantly between the

four groups. Specifically, DS vs DS+AD, DS+AD vs EOAD and DS vs controls were compared.

EOAD and controls were used as reference group (compared in (Vermeiren et al., 2016),

thus not further described here). In a few cases, the Kruskall-Wallis test was no longer

significant, while individual post-hoc Mann-Whitney U tests remained significant. The

supplementary material provides all concentrations and ratios for noradrenergic (Table

S1), dopaminergic (S2) and serotonergic (S3) systems. The use of psychoactive medication

did not evidently impact the monoaminergic concentrations within each group.

Table 6.1: Characteristics of post-mortem study groups

DS

(n=4) DS+AD (n=17) (n=11) EOAD Controls (n=10) P-value

Age at death in years

(median; min.-max.) (35.0-44.0) 39.5 (44.0-80.0) 62.0 (57.6-73.0) 67.2 (57.2-73.3) 65.5 0.004

Gender (N male and %) 2 (50%) 5 (29.4%) 8 (72.7%) 6 (60%) n.s.

Psychoactive medication (yes/no/n.r.) 3/0/1 10/3/4 3/8/0 2/8/0 0.002

Post-mortem delay in hours

(median; min.-max.) (11.5-36.0) 21.0 (3.8-20.0) 7.3 (2.8-7.0) 3.0 (2.3-7.0) 5.4 <0.001

AD neuropathologic change _{Low High} Intermediate/

High Not/Low

Available brain regions per study group

Neocortex: BA7 2 13 11 10

BA9/10/46 4 14 11 10

BA17 3 7 11 10

BA22 3 10 11 10

Limbic system: Amygdala 2 10 10 10

Hippocampus 3 5 11 10

BA11/12 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

A Kruskal-Wallis test was performed to compare ages and post-mortem delay between the groups. Gender and medication use were compared with Fisher’s Exact test. Abbreviations: BA, Brodmann area; BA7, superior parietal lobule; BA9/10/46, (pre)frontal cortex; BA11/12, orbitofrontal cortex; BA17, occipital pole (V1); BA22, superior temporal gyrus; DS, Down syndrome without neuropathologic AD diagnosis; DS+AD, Down syndrome with neuropathologic AD diagnosis; EOAD, early-onset Alzheimer’s disease; n.r., not reported; n.s., not significant.

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Br ai n r eg ion Co m po un d/ ra tio N DS ( n= 4) DS+ AD (n =17 ) EO AD (n =1 1) Co ntr ol s (n =1 0) P-va lu e Neoc orte x BA 7 su pe rio r par ie tal lo bul e M HP G 2, 13, 11, 10 71. 0 ( 63. 3– ) 72. 3 ( 57. 0– 105. 2) * 11 9. 4 ( 10 6. 0– 18 9. 5) * 25 9. 7 ( 11 9. 8– 35 4. 5) <0. 00 1 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) 0. 00 4 HV A:D A 2, 12, 11, 10 10. 0 ( 6. 4– ) 6. 1 ( 3. 9– 8. 7) * 11. 5 ( 8. 3– 17. 8) * 17. 3 ( 10. 5– 29. 8) 0. 00 5 5-HI AA 2, 13, 11, 10 92. 8 ( 70. 4– ) 40. 2 ( 30. 4– 71. 1) 55. 3 ( 40. 5– 93. 7) 10 6. 4 ( 75. 3– 15 8. 9) 0. 00 3 HV A:5 -H IA A 2, 13, 11, 10 1. 1 ( 0. 8– ) 2. 6 ( 1. 6– 3. 6) 1. 8 ( 1. 1– 2. 6) 0. 9 ( 0. 7– 1. 3) 0. 00 1 BA 9/ 10/ 46 pr ef ro nt al co rte x M HP G 4, 14, 11, 10 84. 2 ( 77. 7– 86. 9) 10 7. 4 ( 63. 4– 13 3. 8) ** 47 1. 2 ( 28 4. 3– 65 5. 1) ** 26 5. 7 ( 13 2. 4– 62 9. 6) <0. 00 1 MH PG: N A 4, 14, 11, 10 8. 9 ( 3. 2– 15. 3) 6. 0 ( 2. 3– 12. 4) ** 77. 8 ( 11. 7– 203. 3) ** 9. 7 ( 4. 7– 32. 7) 0. 00 4 DA 4, 14, 11, 10 12 7. 5 ( 11. 4– 49 7. 4) 37 3. 9 ( 20 4. 3– 74 3. 6) ** 7. 2 ( 2. 5– 9. 3) ** 7. 5 ( 4. 0– 11. 3) <0. 00 1 DO PA C: DA 4, 14, 11, 10 0. 7 ( 0. 2– 2. 1) 0. 1 ( 0. 0– 0. 8) ** 1. 4 ( 1. 0– 3. 0) ** 1. 2 ( 0. 7– 2. 8) <0. 00 1 HV A:D A 4, 14, 11, 10 11. 7 ( 5. 1– 19. 2) 4. 7 ( 0. 5– 8. 2) ** 18. 7 (13. 2– 35. 8) ** 24. 8 ( 10. 7– 79. 3) <0. 00 1 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) 0. 00 9 5-HI AA 4, 14, 11, 10 81. 1 ( 64. 6– 122. 2) 62. 2 ( 33. 9– 87. 4) * 14 4. 7 ( 11 0. 5– 18 6. 6) * 16 4. 1 ( 12 8. 2– 21 5. 6) 0. 00 1 5-HI AA :5 -HT 4, 14, 11, 10 5. 9 ( 3. 2– 8. 3) # 4. 2 ( 2. 3– 9. 2) ** 15. 0 ( 13. 1– 32. 6) ** 16. 3 ( 10. 9– 19. 8) # <0. 00 1 HV A:5 -H IA A 4, 14, 11, 10 1. 3 ( 0. 9– 1. 7) 2. 2 ( 1. 4– 3. 2) ** 1. 0 ( 0. 7– 1. 1) ** 0. 8 ( 0. 7– 1. 1) 0. 00 1 BA 17 oc cip ita l p ole (V 1) M HP G 3, 7, 11 ,10 77. 2 ( 61. 3– ) # 89. 2 (73. 6– 114. 6) 12 7. 9 ( 10 1. 6– 18 2. 0) 28 5. 3 ( 24 4. 0– 53 0. 1) # <0. 00 1 5-HI AA 3, 7, 11 ,10 12 6. 0 ( 93. 6– ) 92. 1 ( 46. 9– 144. 1) 95. 6 ( 57. 5– 143. 6) 20 0. 8 ( 15 4. 3– 26 3. 9) 0. 00 8 HV A:5 -H IA A 3, 7, 11 ,10 0. 7 ( 0. 6– ) 1. 3 ( 0. 9– 2. 4) 0. 7 ( 0. 3– 0. 8) 0. 2 ( 0. 2– 0. 6) 0. 00 3 BA 22 su pe rio r te mp ora l gy rus 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) 0. 01 4 M HP G 3, 10, 11, 10 10 7. 3 ( 80. 4– ) # 87. 0 ( 60. 0– 125. 4) ** 52 0. 7 ( 31 3. 4– 67 3. 4) ** 36 0. 5 ( 26 0. 4– 63 0. 7) # <0. 00 1 MH PG: N A 3, 10 ,9 ,10 3. 5 ( 3. 1– ) 5. 1 (3. 8– 9. 3) ** 67. 1 ( 19. 2– 91. 1) ** 20. 0 ( 7. 8– 46. 9) <0. 00 1 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) (0. 04 1) DO PA C: DA 3, 10, 10, 10 1. 7 ( 0. 4– ) 0. 4 ( 0. 3– 1. 1) ** 2. 5 ( 1. 2– 3. 7) ** 1. 0 ( 0. 5– 1. 5) 0. 00 5 HV A:D A 3, 10, 11, 10 17. 5 (11. 2– ) 9. 8 ( 5. 8– 19. 2) * 32. 4 ( 19. 5– 44. 5) * 18. 3 ( 13. 9– 25. 7) 0. 01 4 5-HI AA 3, 10, 11, 10 11 9. 1 ( 98. 7– ) 84. 0 ( 60. 4– 131. 7) * 30 3. 2 ( 11 9. 6– 45 0. 2) * 17 1. 5 ( 12 1. 7– 32 0. 6) 0. 00 4 5-HI AA :5 -HT 3, 10, 11, 10 10. 2 ( 8. 3– ) 16. 9 ( 12. 5– 20. 5) * 67. 8 ( 28. 0– 147. 7) * 23. 5 (18. 0– 36. 7) 0. 00 2 HV A:5 -H IA A 3, 10, 11, 10 1. 1 ( 1. 0– ) 1. 7 ( 1. 2– 2. 2) * 0. 4 ( 0. 3– 1. 1) * 0. 8 ( 0. 6– 1. 0) 0. 00 5 Lim bic sys tem Am yg da la 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) <0. 00 1 M HP G 2, 10, 10, 10 68. 8 ( 58. 2– ) 70. 9 ( 62. 5– 94. 9) ** 42 9. 8 ( 18 0. 0– 96 4. 3) ** 30 4. 8 ( 19 3. 5– 75 9. 3) <0. 00 1 HV A 2, 10, 10, 10 26 5. 8 ( 17 4. 5– ) 37 6. 2 ( 25 8. 9– 61 7. 2) 59 9. 1 ( 39 8. 7– 86 6. 2) 11 32. 5 ( 751. 0– 142 1. 4) 0. 00 5 DO PA C: DA 2, 10, 10, 10 0. 9 ( 0. 3– ) 1. 0 ( 0. 5– 2. 2) 0. 4 ( 0. 3– 0. 9) 0. 2 ( 0. 2– 0. 3) 0. 00 8 5-HT 2, 10, 10, 10 59. 3 ( 11. 7– ) 33. 0 ( 18. 6– 49. 6) * 12 1. 1 ( 55. 1– 14 8. 6) * 24 4. 9 ( 22 1. 7– 29 7. 2) <0. 00 1 5-HI AA 2, 10, 10, 10 19 7. 9 ( 16 2. 9– ) 14 1. 3 ( 10 2. 8– 22 7. 1) ** 52 2. 4 ( 33 4. 9– 79 5. 2) ** 99 9. 8 ( 75 4. 5– 12 70. 4) <0. 00 1 HV A:5 -H IA A 2, 10, 10, 10 1. 3 ( 1. 1– ) 3. 0 ( 1. 5– 3. 7) * 1. 2 ( 0. 9– 1. 8) * 1. 0 ( 0. 8– 1. 3) 0. 01 4 Hi pp oc am pu s Adr ena line 2, 5, 3, 5 39 1. 6 ( 23 6. 5– ) 42. 3 ( 20. 6– 139. 0) 6. 4 ( 2. 6– ) 10. 1 ( 6. 4– 14. 1) 0. 01 1 M HP G 3, 5, 11 ,10 75. 9 ( 70. 3– ) # 97. 2 ( 76. 9– 124. 8) ** 45 9. 5 ( 19 3. 2– 10 99. 2) ** 41 6. 3 ( 23 2. 9– 71 3. 5) # <0. 00 1 MH PG: N A 3, 5, 10 ,10 3. 5 ( 3. 1– ) 3. 5 ( 2. 9– 4. 6) * 13. 1 ( 5. 0– 59. 9) * 8. 2 ( 2. 3– 31. 4) (0. 04 0) 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) 0. 00 3 5-HI AA 3, 5, 11 ,10 14 1. 3 ( 11 0. 6– ) 47. 7 ( 43. 1– 213. 4) * 33 6. 1 ( 25 7. 8– 47 6. 2) * 38 3. 9 (279 .5 –7 17. 0) 0. 00 4 HV A:5 -H IA A 3, 5, 11 ,10 1. 3 ( 0. 5– ) 2. 4 ( 1. 1– 3. 6) * 0. 6 ( 0. 4– 1. 2) * 0. 6 ( 0. 5– 0. 9) (0. 01 9)

Ta bl e 6. 2 (c ont inue d) Br ai n r eg ion Co m po un d/ ra tio N DS ( n= 4) DS+ AD (n =17 ) EO AD (n =1 1) Co ntr ol s (n =1 0) P-va lu e Lim bic sys tem BA 11/ 12 or bi to fr ont al co rte x M HP G 4, 6, 11 ,10 91. 8 ( 70. 6– 105. 7) # 10 3. 1 ( 63. 9– 11 1. 2) ** 43 7. 1 ( 35 2. 6– 54 5. 6) ** 36 1. 7 ( 21 6. 5– 63 6. 3) # <0. 00 1 MH PG: N A 4, 6, 11 ,10 5. 4 ( 2. 1– 14. 4) 5. 3 ( 3. 5– 10. 8) * 50. 6 ( 27. 7– 73. 3) * 18. 9 ( 11. 5– 37. 4) 0. 00 6 HV A:D A 4, 5, 11 ,10 26. 4 (8. 9– 41. 5) 11. 1 ( 4. 2– 14. 8) ** 38. 2 ( 25. 8– 53. 7) ** 33. 4 ( 26. 7– 47. 0) 0. 01 3 5-HI AA 4, 6, 11 ,10 98. 2 ( 85. 4– 189. 7) 56. 7 ( 35. 3– 78. 5) ** 23 8. 5 ( 18 3. 5– 34 4. 3) ** 22 9. 9 ( 16 8. 9– 32 8. 3) <0. 00 1 5-HI AA :5 -HT 4, 6, 11 ,10 6. 5 ( 5. 5– 7. 5) 7. 4 ( 6. 4– 8. 9) * 21. 0 ( 14. 4– 28. 1) * 13. 3 ( 8. 5– 22. 1) 0. 00 3 HV A:5 -H IA A 4, 6, 11 ,10 1. 2 ( 0. 8– 2. 3) 2. 6 ( 1. 7– 3. 1) ** 0. 8 ( 0. 5– 1. 0) ** 0. 7 ( 0. 6– 0. 8) 0. 00 1 Ci ng ul ate gy ru s M HP G 3, 8, 11 ,10 12 8. 0 ( 58. 8– ) 12 6. 0 ( 10 4. 6– 15 3. 6) ** 56 7. 8 ( 25 8. 4– 73 9. 3) ** 33 6. 9 ( 15 8. 1– 58 7. 5) <0. 00 1 MH PG: N A 3, 8, 10 ,10 3. 4 ( 2. 0– ) 3. 6 ( 2. 0– 5. 2) ** 30. 6 ( 12. 0– 54. 1) ** 9. 3 ( 3. 4– 21. 9) 0. 00 3 DA 3, 8, 11 ,10 22 4. 2 ( 7. 9– ) 55. 5 ( 40. 6– 211. 2) ** 10. 5 ( 3. 8– 13. 0) ** 9. 6 ( 3. 0– 17. 4) <0. 00 1 DO PA C: DA 3, 8, 11 ,10 0. 1 ( 0. 1– ) 0. 2 ( 0. 1– 0. 5) ** 1. 3 ( 1. 0– 2. 9) ** 1. 2 ( 0. 5– 1. 6) <0. 00 1 HV A:D A 3, 8, 11 ,10 0. 7 ( 0. 6– ) 1. 6 ( 1. 1– 5. 1) ** 23. 4 ( 15. 7– 44. 5) ** 24. 7 ( 17. 3– 98. 1) <0. 00 1 5-HI AA 3, 8, 11 ,10 66. 0 ( 34. 5– ) # 10 2. 6 ( 58. 5– 19 1. 8) ** 35 7. 3 ( 28 1. 2– 37 9. 3) ** 38 7. 2 ( 31 3. 7– 47 5. 7) # <0. 00 1 HV A:5 -H IA A 3, 8, 11 ,10 3. 1 ( 1. 9– ) # 1. 8 ( 0. 9– 2. 4) * 0. 7 (0. 5– 0. 9) * 0. 6 ( 0. 5– 0. 8) # <0. 00 1 Th al am us M HP G 3, 11, 11, 10 15 9. 2 ( 13 1. 3– ) # 14 8. 3 ( 11 0. 2– 17 7. 8) ** 79 3. 0 ( 62 8. 6– 14 42. 6) ** 44 1. 9 ( 24 4. 1– 13 45. 4) # <0. 00 1 MH PG: N A 3, 11, 10, 10 2. 7 ( 0. 4– ) 2. 1 ( 0. 7– 2. 9) * 5. 8 ( 4. 5– 12. 6) * 2. 9 ( 1. 0– 6. 1) 0. 01 0 HV A:D A 3, 11, 11, 10 17. 4 ( 1. 9– ) 8. 8 ( 3. 4– 17. 8) * 39. 7 ( 25. 9– 56. 9) * 30. 2 ( 24. 7– 50. 5) 0. 01 0 5-HI AA 3, 11, 11, 10 67 3. 7 ( 50 8. 8– ) 68 9. 3 ( 41 2. 4– 89 5. 8) ** 15 84. 5 ( 123 7. 1– 19 52. 8) ** 15 25. 8 ( 116 8. 1– 19 46. 8) <0. 00 1 5-HI AA :5 -HT 3, 11, 11, 10 3. 6 ( 3. 2– ) 5. 3 ( 4. 2– 8. 7) 9. 7 (7. 3– 13. 1) 8. 9 ( 5. 7– 9. 4) 0. 00 9 Bas al gangl ia Ca ud ate nu cl eu s Adr ena line 2, 11 ,7 ,6 53 3. 3 ( 35 5. 2– ) 69. 3 ( 42. 7– 151. 0) * 26 8. 5 ( 17 6. 5– 58 7. 7) * 37 2. 9 ( 18 5. 7– 74 3. 0) 0. 00 6 DA 3, 16, 11, 10 42 97. 2 ( 184 5. 8– ) 23 95. 2 ( 117 8. 5– 27 84. 8) ** 47 21. 2 ( 340 3. 8– 69 05. 9) ** 39 65. 1 ( 298 7. 2– 44 20. 5) 0. 00 3 HV A 3, 16, 11, 10 27 32. 0 ( 223 1. 3– ) 31 45. 6 ( 152 2. 7– 37 56. 0) * 46 81. 2 ( 371 4. 5– 56 40. 3) * 43 72. 3 ( 356 4. 4– 68 18. 8) 0. 01 4 5-HT 3, 16, 11, 10 12 1. 5 ( 33. 1– ) 55. 8 ( 35. 6– 97. 0) ** 16 8. 3 ( 13 9. 5– 23 0. 2) ** 24 0. 0 ( 18 8. 0– 28 5. 2) <0. 00 1 5-HI AA 3, 16, 11, 10 17 0. 9 ( 76. 6– ) 21 7. 6 ( 10 6. 3– 28 4. 2) ** 53 7. 2 ( 36 2. 3– 72 7. 8) ** 57 9. 6 ( 44 1. 0– 78 4. 9) <0. 00 1 Gl ob us pa lli du s DOP AC 2, 8, 11 ,10 20 8. 3 ( 28. 0– ) 13 1. 7 ( 61. 8– 14 1. 5) * 39. 5 ( 19. 5– 85. 8) * 20. 1 ( 10. 5– 34. 7) 0. 00 4 DO PA C: DA 2, 8, 11 ,10 0. 1 ( 0. 1– ) 0. 2 ( 0. 2– 0. 4) 0. 1 ( 0. 1– 0. 2) 0. 1 ( 0. 0– 0. 1) 0. 00 9 5-HT 2, 8, 11 ,10 14 9. 7 ( 89. 7– ) 97. 4 ( 70. 5– 120. 4) * 17 1. 9 ( 11 7. 6– 20 7. 7) * 16 1. 8 ( 14 0. 3– 21 5. 5) (0. 02 2) 5-HI AA 2, 8, 11 ,10 10 09. 2 ( 384. 6– ) 43 9. 5 ( 32 9. 9– 97 8. 8) * 98 4. 5 ( 86 4. 7– 14 07. 3) * 13 20. 2 ( 101 9. 2– 16 14. 4) (0. 01 5) HV A:5 -H IA A 2, 8, 11 ,10 10. 4 ( 3. 2– ) 6. 2 ( 4. 6– 12. 9) 4. 0 ( 2. 7– 4. 9) 3. 1 ( 2. 3– 3. 6) 0. 01 2 Pu ta m en Adr ena line 2, 8, 11 ,10 39 0. 4 ( 32 5. 6) 13 5. 0 ( 40. 9– 21 9. 9) * 58 1. 9 ( 18 7. 2– 15 23. 4) * 33 9. 6 ( 10 0. 7– 79 7. 8) (0. 03 8) DOP AC 3, 15, 11, 10 20 7. 5 ( 16 5. 1– ) 61 1. 6 ( 27 4. 7– 85 1. 6) 42 1. 9 ( 23 5. 7– 62 5. 7) 20 2. 2 ( 11 9. 0– 29 9. 7) 0. 00 8 DO PA C: DA 3, 15, 11, 10 0. 1 ( 0. 0– ) 0. 1 ( 0. 1– 0. 3) 0. 1 ( 0. 0– 0. 1) 0. 0 ( 0. 0– 0. 1) 0. 00 1 5-HT 3, 15, 11, 10 18 9. 5 ( 43. 9– ) 77. 8 ( 35. 2– 159. 6) * 18 9. 2 ( 15 3. 9– 21 9. 8) * 21 9. 7 ( 20 0. 5– 32 6. 3) <0. 00 1 5-HI AA 3, 15, 11, 10 26 0. 0 (231 .7 –) 37 2. 7 ( 20 3. 7– 66 9. 5) ** 79 0. 9 ( 63 8. 7– 10 26. 1) ** 99 8. 4 ( 78 1. 9– 14 00. 3) <0. 00 1 HV A:5 -H IA A 3, 15, 11, 10 21. 3 ( 6. 8– ) 15. 4 ( 10. 7– 19. 2) 9. 4 ( 7. 8– 14. 1) 6. 9 ( 5. 0– 10. 0) 0. 00 6 Su bs ta nti a ni gr a DA 2, 14, 11, 10 16 4 ( 126. 8– ) 15 7. 2 ( 89. 0– 29 2. 4) ** 57 2. 7 (284 .9 –6 23. 7) ** 62 4. 0 ( 29 5. 7– 93 6. 8) <0. 00 1 DOP AC 2, 14, 11, 10 47. 2 ( 47. 1– ) 58. 0 ( 27. 1– 87. 4) * 18 9. 7 ( 80. 3– 21 1. 0) * 47. 1 ( 29. 2– 101. 0) 0. 00 5 HV A 2, 14, 11, 10 30 61. 7 ( 201 0. 5– ) 24 21. 8 ( 199 5. 7– 30 07. 8) ** 37 99. 4 ( 346 1. 0– 43 85. 5) ** 43 31. 0 ( 324 1. 6– 53 49. 9) <0. 00 1 DO PA C: DA 2, 14, 11, 10 0. 3 ( 0. 2– ) 0. 4 ( 0. 2– 0. 5) 0. 2 ( 0. 2– 0. 5) 0. 1 ( 0. 1– 0. 1) 0. 00 1 HV A:D A 2, 14, 11, 10 18. 1 ( 15. 9– ) 17. 0 ( 9. 6– 23. 4) * 8. 3 ( 5. 3– 12. 9) * 8. 2 ( 4. 8– 9. 7) 0. 00 8 5-HT 2, 14, 11, 10 20 8. 3 ( 16 7. 6– ) 36 4. 1 ( 24 3. 1– 42 0. 4) 48 7. 2 ( 38 9. 2– 53 1. 2) 46 1. 0 (404 .1 –5 88. 8) 0. 00 9

Ta bl e 6.2 (c ont inue d) Br ai n r eg ion Co m po un d/ ra tio N DS ( n= 4) DS+ AD (n =17 ) EO AD (n =1 1) Co ntr ol s (n =1 0) P-va lu e Met enc ephal on Lo cu s co er ul eus NA 0, 10, 10, 10 – 88. 6 ( 52. 7– 11. 4) ** 25 5. 1 ( 17 1. 9– 40 0. 7) ** 34 7. 3 ( 24 8. 6– 50 1. 8) <0. 00 1 M HP G 0, 10, 10, 10 – 20 1. 8 ( 15 1. 8– 25 9. 5) * 42 9. 6 ( 30 4. 6– 53 0. 7) * 57 2. 4 ( 15 8. 9– 83 6. 2) (0. 03 2) DOP AC 0, 10, 10, 10 – 12. 3 ( 8. 1– 19. 2) ** 39. 0 ( 23. 6– 71. 3) ** 55. 5 ( 33. 1– 98. 6) <0. 00 1 HV A 0, 10, 10, 10 – 73 0. 2 ( 48 2. 0– 10 03. 5) 10 09. 7 ( 905. 7– 144 3. 4) 13 51. 0 (1 19 5. 0– 14 82. 2) <0. 00 1 DO PA C: DA 0, 10, 10, 10 – 0. 5 ( 0. 3– 0. 6) * 1. 1 ( 0. 9– 1. 6) * 1. 4 ( 0. 7– 2. 0) 0. 00 2 Cer ebel lum Adr ena line 0, 7, 4, 6 – 36. 4 ( 34. 7– 44. 5) * 6. 6 ( 3. 6– 12. 7) * 21. 4 ( 10. 3– 50. 2) (0. 01 7) M HP G 2, 9, 11 ,10 12 4. 6 ( 10 0. 3– ) 10 1. 1 ( 85. 2– 14 8. 6) * 42 1. 1 (232 .5 –7 08. 2) * 51 8. 1 ( 38 6. 8– 71 3. 4) 0. 00 1 MH PG: N A 2, 9, 11 ,10 2. 7 ( 2. 1– ) 2. 4 ( 1. 7– 3. 9) * 20. 2 ( 5. 5– 32. 3) * 13. 6 ( 7. 8– 18. 4) 0. 00 4 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) <0. 00 1 DOP AC 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) 0. 00 5 HV A:D A 2, 9, 11 ,10 6. 2 ( 2. 5– ) 6. 0 ( 5. 2– 6. 9) ** 28. 7 ( 12. 1– 44. 9) ** 28. 5 ( 18. 5– 32. 8) <0. 00 1 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) <0. 00 1 5-HI AA 2, 9, 11 ,10 30. 8 (21. 9– ) 41. 7 ( 33. 8– 56. 5) ** 20 6. 0 ( 92. 8– 30 1. 4) ** 98. 5 ( 73. 7– 207. 2) <0. 00 1 5-HI AA :5 -HT 2, 9, 11 ,10 0. 7 ( 0. 4– ) 1. 5 ( 1. 1– 3. 3) ** 41. 8 ( 15. 7– 97. 1) ** 32. 9 ( 22. 0– 44. 0) <0. 00 1 HV A:5 -H IA A 2, 9, 11 ,10 3. 7 ( 3. 6– ) 2. 1 ( 1. 6– 2. 9) ** 0. 6 ( 0. 4– 1. 2) ** 0. 8 ( 0. 4– 1. 1) <0. 00 1 Mono am ine s a nd m et abo lite s ( ng /g ti ss ue ) a nd the c or re spo ndi ng ra tio s a re e xpr es se d a s m edi an (5 0%) wi th the in te rqua rt ile ra ng e ( 25% -7 5% ) b et we en br ac ke ts . A K rus ka ll-W al lis t es t w as u se d to co m pa re t he fo ur g ro ups . S ig ni fic an t P -v al ue s ( <0 .0 15 ) a re pr ov ide d. T ho se in ita lic s be twe en b ra ck et s a re no lo ng er r eg ar de d s ig ni fic ant a ft er c or re ct io n ( P< 0. 01 5) , b ut po st -h oc Ma nn -W hi tne y U te sts re m ai ne d s ig ni fic an t: D S v s c on tr ol s ( #< 0. 015) a nd D S+ AD v s E OA D ( *< 0. 015; * *< 0. 00 1) . A bb re vi ati on s: B A, B ro dm an n a re a; 5 -H IA A, 5 -h yd ro xy in do le ace tic a ci d; 5 -H T, s er ot on in; D A, do pa m ine ; DS , D own s ynd ro m e wi tho ut ne ur opa tho lo gi c A D di ag no sis ; D S+ AD , D own s yn dr om e w ith ne ur opa tho lo gi c A D di ag no sis ; D O PA C, 3 -4 -di hy dr ox yp he ny la ce tic a ci d; E O AD , e ar ly -o ns et A lzh eim er ’s di se as e; H VA , ho m ov an ill ic a ci d; M HP G , 3 -m et ho xy -4 -h yd ro xy phe ny lg ly co l; NA , no ra dr ena line ; n. s. , no t s ig ni fic ant .

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MHPG con cent rat ion (ng /g) Study gr oups DS DS+ AD EO AD Con tr ol s 1 2 3 4 5 6 7 8 11 9 BA 9/ 10/ 46 BA 17 BA 22 BA 7 2 3 4 5 6 7 8 9 10 1 Am yg dala Hi pp oc am pu s BA 11/ 12 Cin gu lat e gy ru s Th alam us Lo cu s c oe ru le us Ce re be llu m 11

Neocortex Limbic system

1 2 3 4 5 6 7 8 11 9 10 1 2 3 4 5 6 7 8 11 9 10 1 2 3 4 5 6 7 8 11 9 10

Noradrenergic system

Between groups, NA differed significantly in BA22, amygdala and LC. Specifically, NA levels

were not altered between DS and DS+AD, but lower in DS+AD compared to EOAD

(amygdala and LC, Table 6.2). This pattern was more pronounced for MHPG (Figure 6.1).

Compared to EOAD, DS+AD presented significantly lower MHPG levels in LC, cortical (BA7,

BA9/10/46, BA22) and limbic projection areas (amygdala, hippocampus, BA11/12,

cingulate gyrus, thalamus) and cerebellum. Consequently, the MHPG:NA ratio was

consistently lower in DS+AD (BA9/10/46, BA22, hippocampus, BA11/12, cingulate gyrus,

thalamus and cerebellum), indicating a reduced noradrenergic turnover. Moreover, MHPG

was significantly lower in DS compared to controls (Figure 6.1) for BA17, BA22,

hippocampus, BA11/12 and thalamus, and close to significance for BA9/10/46 (P=0.024).

Neither NA, nor MHPG levels differed significantly in basal ganglia, while adrenaline levels

were lower in caudate nucleus and putamen, and higher in cerebellum in DS+AD (vs

EOAD). Taken together, the noradrenergic system – particularly MHPG – was strongly

impaired in DS (vs controls) and DS+AD (vs EOAD).

Dopaminergic system

No significant differences were observed for DS vs DS+AD and DS vs controls. Compared to

EOAD, bidirectional dopaminergic changes became evident in DS+AD: DA levels were

significantly higher in BA7 (P=0.016), BA9/10/46, BA22, cingulate gyrus and cerebellum,

and lower in the basal ganglia (caudate nucleus and SN). Similarly, in DS+AD (vs EOAD),

HVA was reduced in caudate nucleus and SN, and close to significance in LC (P=0.019).

Consequently, the HVA:DA ratio, indicative of dopaminergic turnover, was consistently

lower in DS+AD (vs EOAD) in cortical areas (BA7, BA9/10/46 and BA22), limbic regions

(BA11/12, hippocampus (P=0.019), cingulate gyrus and thalamus) and cerebellum. In

contrast, the HVA:DA ratio was increased in SN. The pattern for DOPAC was bidirectional

as well: values were decreased in SN and LC, and increased in globus pallidus and

cerebellum. The DOPAC:DA ratio was significantly lower in BA9/10/46, BA22, cingulate

gyrus and LC, and higher in globus pallidus (P=0.016) for DS+AD vs EOAD. In short, the

dopaminergic system was evidently affected in DS+AD with higher DA levels (and thus

lower HVA:DA and DOPAC:DA ratios) in cortical areas, limbic regions and cerebellum, and

lower DA and HVA levels in basal ganglia.

Serotonergic system

5-HT and 5-HIAA did not differ between DS and DS+AD, although 5-HT and 5-HIAA levels in

BA11/12 tended to be higher in DS (P=0.019 for both). 5-HIAA levels in cingulate gyrus and

the 5-HIAA/5-HT ratio in (pre)frontal cortex were significantly lower in DS than in controls.

Compared to EOAD, 5-HT levels in DS+AD were significantly lower in amygdala and basal

ganglia (caudate nucleus, globus pallidus, putamen, and close to significance (P=0.025) in

SN) and higher in the (pre)frontal cortex and cerebellum. In comparison with EOAD,

5-HIAA was consistently lower in DS+AD, namely in cortical areas (BA9/10/46, BA22),

limbic system (amygdala, hippocampus, BA11/12, cingulate gyrus and thalamus), basal

ganglia (caudate nucleus, globus pallidus and putamen) and cerebellum. Similarly, the

5-HIAA:5-HT ratio was reduced in BA9/10/46, BA22, BA11/12, cingulate gyrus (P=0.020),

thalamus (P=0.019) and cerebellum in DS+AD vs EOAD, thus indicating an overall

decreased serotonergic turnover in DS+AD. In summary, a serotonergic deficit became

apparent in DS+AD, with a pronounced overall reduction in 5-HIAA levels (and thus a

reduced 5-HIAA:5-HT ratio) as compared to EOAD.

Finally, the HVA:5-HIAA ratio, indicating serotonergic inhibition on dopaminergic

neurotransmission clearly differed between groups. Apart from a significantly higher

HVA:5-HIAA ratio in the cingulate gyrus in DS (vs controls), significance was, again,

observed for the DS+AD vs EOAD comparison. The ratio was invariably higher in DS+AD for

cortical regions (BA9/10/46, BA17 (P=0.015) and BA22), limbic system (amygdala,

hippocampus, BA11/12 and cingulate gyrus), globus pallidus (P=0.016), putamen (P=0.018)

and cerebellum, suggestive of a reduced serotonergic inhibition on the dopaminergic

system.

Monoaminergic characterization of (paired) CSF/plasma samples

Samples of 225 DS individuals were included in analysis (Figure 6.2). Paired samples were

available for 68 individuals, plasma-only samples for 157 cases.

Figure 6.2: Flow chart of CSF/plasma study groups

Tables 6.3 and 6.4, respectively, show the study group characteristics and monoaminergic

concentrations and ratios. Remarkably, CSF/plasma concentrations did not differ between

the three groups apart from DOPAC levels in CSF (medication-free) and plasma (total and

medication-free). DOPAC levels were consistently higher in DS+AD compared to DS (CSF

medication-free, P=0.001; plasma total, P<0.001; plasma medication-free, 0.002), but did

not differ for the DS vs DS+pAD and DS+pAD vs DS+AD comparisons. Similarly, plasma HVA

(total), plasma 5-HIAA (total) and CSF DOPAC:DA (medication-free) were higher in DS+AD

vs DS. In contrast, CSF HVA:5-HIAA (total) was decreased in DS+AD. Moreover, DA

(r=

–0.31, P=0.012), DOPAC (r=

+0.71, P<0.001), MHPG (r=

+0.70, P<0.001) and adrenaline

(r=

+0.49, P<0.001) correlated significantly in CSF and plasma (paired samples, total

population). Groups differed in age with DS+AD logically being the oldest. DOPAC (CSF,

r=

+0.362, P=0.002; plasma, r=

+0.386, P<0.001), HVA (plasma, r=

+0.169, P=0.011), 5-HIAA

(CSF, r=

+0.365, P=0.002; plasma, r=

+0.345, P<0.001) correlated significantly with age.

After exclusion of individuals younger than 45 years of age, i.e. resembling the elderly DS

cohort in our previously published serum study (Dekker et al., 2015a), comparison

between DS (CSF/plasma, n=8; plasma-only, n=34), DS+pAD (11;31) and DS+AD (16;38)

yielded no significant monoaminergic differences, again suggesting that DOPAC changes

most likely relate to aging rather than dementia status.

Table 6.3: Characteristics of CSF/plasma study groups

No dementia

(DS, n=149) (DS+pAD, n=36) Prodromal AD Diagnosed AD dementia (DS+AD, n=40) P-value

Age (median; min.-max.) 36.9 (19.2-61.2) 49.8 (37.7-59.4) 55.1 (42.1-69.2) <0.001

Gender (N male and %) 82 (55.0%) 16 (44.4%) 25 (62.5%) n.s.

ApoE status 2 (e2/4), 106 (e3/3), 1 (e2/2), 15 (e2/3),

23 (e3/4), 2 (e4/4)

1 (e2/2), 6 (e2/3), 0 (e2/4), 21 (e3/3), 8 (e3/4),

0 (e4/4)

0 (e2/2), 1 (e2/3), 1 (e2/4), 30 (e3/3), 8 (e3/4),

0 (e4/4) n.s.

Levothyroxine 61 (40.9%) 12 (33.3%) 14 (35.0%) n.s.

Any psychoactive drugs 56 (37.6%) 19 (52.8%) 24 (60.0%) n.s.

- antidepressants 36 (24.2%) 11 (30.6%) 9 (22.5%) n.s.

- anti-epileptics 13 (8.7%) 7 (19.4%) 10 (25.0%) 0.014

- antipsychotics 20 (13.4%) 8 (22.2%) 13 (32.5%) 0.017

- anxiolytics 14 (9.4%) 3 (8.3%) 8 (20.0%) n.s.

- anti-dementia 1 (0.7%) 1 (2.8%) 5 (12.5%) 0.002

Age at the moment of sampling is provided as median with range (minimum-maximum). A Kruskal-Wallis test was performed to compare ages between groups. Gender, ApoE status and medication use were compared using a Pearson’s Chi-Square test or Fisher’s Exact test. Abbreviations: ApoE, Apolipoprotein E; n.s., not significant.

6.4. Discussion

Monoaminergic profiles were evaluated in 15 post-mortem brain regions and (paired)

CSF/plasma samples. In brain, pronounced noradrenergic, dopaminergic and serotonergic

differences were found for DS+AD vs EOAD and DS vs controls, but not for DS vs DS+AD.

Similarly, CSF/plasma concentrations were virtually unaltered between the diagnostic DS

groups.

In AD, studies have demonstrated LC neuronal loss and reduced NA levels (Arai et

al., 1992; German et al., 1992; Lyness et al., 2003; Mann et al., 1985; Simic et al., 2017;

Trillo et al., 2013). Noradrenergic abnormalities have been implicated in DS too (Phillips et

al., 2016). Here, we demonstrate that the noradrenergic system was more severely

impacted in DS/DS+AD than in EOAD or non-DS controls. NA, MHPG and the MHPG:NA

ratio were significantly reduced in most brain areas, but not in the basal ganglia, which is

in accordance with the modest noradrenergic innervation of the basal ganglia (Trillo et al.,

2013). These results are also in agreement with earlier studies reporting AD-related loss of

LC neurons (German et al., 1992; Mann et al., 1987b, 1985; Marcyniuk et al., 1988) and

reduced NA levels in various brain regions in DS+AD compared to controls (Godridge et al.,

1987; Reynolds and Godridge, 1985; Risser et al., 1997; Yates et al., 1983, 1981). Our

results demonstrate that MHPG concentrations were most severely impacted in DS+AD

(even more than in EOAD), but also in DS, thus already before the neuropathological

criteria for AD were met.

DA is produced in the SN and ventral tegmental area (VTA). In AD, a variable SN

neuronal loss and diminished DA levels have been described (Storga et al., 1996; Zarow et

al., 2003). Whereas previous studies did not report evident dopaminergic alterations in DS

(Godridge et al., 1987; Risser et al., 1997), we found significantly increased DA levels (and

thus decreased HVA:DA and DOPAC:DA ratios) in cortical areas, limbic regions and

cerebellum, and a general decrease in DA and HVA levels in basal ganglia. Indeed, lower

DA levels in caudate nucleus have been reported in DS+AD vs EOAD and age-matched

controls (Yates et al., 1983). Ascending dopaminergic projections are subdivided into the

nigrostriatal (from SN to striatum), mesolimbic (from VTA to limbic system) and

mesocortical (from VTA to cortex) pathways (Trillo et al., 2013). Previously, mild cell loss

(though often not significant) in the SN, but also in the VTA, was found in DS+AD

compared to controls or younger counterparts (Gibb et al., 1989; Mann et al., 1987a,

1987b, 1985). Our results may suggest a more severe impairment of the nigrostriatal

pathway (reduced DA levels in caudate and SN), while the mesolimbic and mesocortical

pathways seem to be somewhat overactive, possibly as a compensatory mechanism.

Concerning the serotonergic system, neuronal loss in the dorsal raphe nuclei

(5-HT production site) and reduced levels of 5-HT and 5-HIAA in various brain regions have

been reported in AD and DS (Godridge et al., 1987; Lyness et al., 2003; Mann et al., 1985;

Reynolds and Godridge, 1985; Risser et al., 1997; Seidl et al., 1999; Simic et al., 2017; Trillo

et al., 2013). Compared to EOAD, we observed an even more severe serotonergic

impairment in DS+AD, presenting decreased 5-HIAA levels in 11 brain regions, while 5-HT

was reduced in amygdala and basal ganglia, but increased in the (pre)frontal cortex and

cerebellum.

Interestingly, the DS and DS+AD groups showed remarkably similar

monoaminergic profiles, although both groups had different AD neuropathologic changes

(low vs high). Importantly, the four DS cases with low AD neuropathologic change already

presented high amyloid burden (resp. A2,B1,C2; A3,B1,C1; A3,B0,C0 and A3,B0,C0). The

third copy of the APP gene in DS causes Aβ overproduction and accumulation from birth

onwards. Deposition of Aβ plaques occurs at ages as early as 12 years and precedes tau

pathology by many years (Lemere et al., 1996). Previously, noradrenergic and serotonergic

depletion was found to be more severe in EOAD (mutations in APP or PSEN1/2, promoting

the amyloidogenic pathway) than in late-onset AD (Arai et al., 1992). Inverse relations

between Aβ accumulation and respectively NA, DA and 5-HT signaling have been

described (Cirrito et al., 2011; Trillo et al., 2013). This may suggest that the

monoaminergic system is particularly affected by (early) Aβ pathology, being altered long

before full-blown AD-pathology is present.

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Co m po un d/ ra tio CS F/ pl as m a To ta l/ m ed ic ati on -fr ee N DS DS +p AD DS+ AD P-va lu e N A CS F to ta l 37, 13 ,18 2. 8 ( 0. 9– 5. 5 ) 1. 4 ( 0. 6– 4. 9) 1. 8 ( 0. 8– 5. 6) n. s. m ed ic at io n-fre e 21, 7, 10 2. 8 ( 1. 0– 6. 9) 1. 4 (0. 6– 4. 7) 1. 4 ( 0. 8– 5. 5) n. s. pl as m a to ta l 14 5, 35 ,37 0. 4 ( 0. 2– 0. 9) 0. 6 ( 0. 2– 0. 9) 0. 6 ( 0. 4– 1. 1) n. s. m ed ic at io n-fre e 90, 16 ,15 0. 5 ( 0. 2– 1. 0) 0. 4 ( 0. 1– 0. 7) 0. 4 ( 0. 2– 1. 0) n. s. Ad re na lin e CS F to ta l 35, 13 ,16 0. 5 ( 0. 3– 0. 9) 0. 7 ( 0. 3– 1. 3) 0. 4 ( 0. 3– 1. 2) n. s. m ed ic at io n-fre e 20, 7, 10 0. 7 ( 0. 3– 1. 0) 0. 8 ( 0. 2– 2. 1) 0. 4 ( 0. 4– 1. 2) n. s. pl as m a to ta l 14 7, 35 ,39 2. 9 ( 1. 9– 4. 2) 2. 6 ( 1. 9– 4. 0) 3. 3 ( 1. 4– 4. 3) n. s. m ed ic at io n-fre e 92, 17 ,16 2. 9 ( 2. 0– 4. 0) 3. 0 ( 2. 0– 4. 2) 2. 7 ( 1. 2– 3. 9) n. s. M HPG CS F to ta l 37, 13 ,18 30. 2 (21. 5– 44. 6) 24. 0 ( 18. 8– 40. 0) 22. 4 ( 19. 6– 46. 0) n. s. m ed ic at io n-fre e 21, 7, 10 31. 1 ( 21. 8– 45. 0) 24. 0 ( 15. 9– 41. 1) 28. 5 ( 20. 7– 43. 4) n. s. pl as m a to ta l 14 9, 36 ,40 76. 2 ( 51. 2– 102. 4) 70. 9 ( 50. 4– 111. 6) 75. 6 ( 55. 3– 114. 4) n. s. m ed ic at io n-fre e 93, 17 ,16 77. 3 (51. 5– 100. 6) 70. 4 ( 45. 5– 119. 3) 63. 0 ( 47. 9– 107. 5) n. s. M HP G: N A CS F to ta l 37, 13 ,18 11. 6 ( 6. 0– 31. 0) 29. 8 ( 6. 9– 34. 7) 18. 3 ( 6. 4– 28. 4) n. s. m ed ic at io n-fre e 21, 7, 10 10. 4 ( 5. 2– 31. 2) 29. 8 ( 5. 4– 35. 1) 22. 0 ( 8. 2– 32. 7) n. s. pl as m a to ta l 14 5, 35 ,37 18 5. 4 (9 6. 3– 39 2. 0) 16 5. 0 ( 10 4. 9– 26 0. 9) 11 1. 7 ( 72. 7– 20 3. 2) n. s. m ed ic at io n-fre e 90, 16 ,15 16 6. 3 ( 77. 7– 31 5. 6) 22 2. 3 ( 14 7. 8– 29 2. 9) 14 7. 7 ( 10 2. 2– 39 4. 9) n. s. DA CS F to ta l 37, 13 ,18 0. 6 ( 0. 4– 0. 9) 0. 5 ( 0. 2– 0. 7) 0. 6 ( 0. 3– 1. 2) n. s. m ed ic at io n-fre e 21, 7, 10 0. 6 (0. 4– 0. 9) 0. 6 ( 0. 3– 1. 0) 0. 8 ( 0. 4– 1. 3) n. s. pl as m a to ta l 14 9, 35 ,40 0. 5 ( 0. 3– 1. 0) 0. 5 ( 0. 3– 1. 1) 0. 7 ( 0. 3– 1. 3) n. s. m ed ic at io n-fre e 93, 16 ,16 0. 5 ( 0. 3– 1. 0) 0. 6 ( 0. 4– 1. 1) 0. 7 ( 0. 4– 1. 9) n. s. DOP AC CS F to ta l 37, 13 ,18 0. 6 ( 0. 4– 1. 3) 1. 2 ( 0. 6– 2. 7) 1. 7 (0. 7– 3. 3) (0. 03 3) m ed ic at io n-fre e 21, 7, 10 0. 5 ( 0. 4– 1. 0) §§ 0. 9 ( 0. 5– 1. 5) 2. 8 ( 1. 1– 5. 6) §§ 0. 00 3 pl as m a to ta l 14 9, 36 ,40 2. 7 ( 1. 9– 4. 1) §§ 3. 1 ( 2. 6– 4. 3) 4. 3 ( 3. 0– 6. 2) §§ <0. 00 1 m ed ic at io n-fre e 93, 17 ,16 2. 5 ( 1. 9– 3. 6) § 3. 1 ( 2. 7– 3. 6) 5. 3 ( 2. 9– 6. 2) § 0. 00 4 HVA CS F to ta l 37, 13 ,18 54. 6 ( 40. 5– 67. 7) 55. 1 ( 43. 3– 74. 8) 55. 3 ( 33. 3– 67. 3) n. s. m ed ic at io n-fre e 21, 7, 10 56. 3 ( 41. 5– 64. 1) 46. 5 ( 35. 2– 79. 5) 56. 4 ( 33. 1– 67. 2) n. s. pl as m a to ta l 14 9, 36 ,40 9. 4 ( 7. 5– 11. 6) § 10. 3 ( 7. 4– 12. 7) 10. 6 ( 8. 7– 15. 0) § (0. 03 9) m ed ic at io n-fre e 93, 17 ,16 9. 5 ( 7. 4– 12. 2) 10. 5 ( 7. 6– 13. 0) 9. 9 ( 8. 5– 14. 2) n. s. DO PA C: DA CS F to ta l 37, 13 ,18 0. 9 ( 0. 5– 4. 4) 2. 3 ( 0. 8– 17. 2) 2. 9 ( 0. 7– 7. 0) n. s. m ed ic at io n-fre e 21, 7, 10 0. 8 ( 0. 5– 2. 6) § 1. 2 ( 0. 7– 2. 9) 3. 2 ( 2. 4– 7. 4) § (0. 03 1) pl as m a to ta l 14 9, 35 ,40 5. 1 ( 2. 5– 11. 1) 6. 5 ( 3. 2– 10. 6) 7. 7 ( 3. 3– 13. 1) n. s. m ed ic at io n-fre e 93, 16 ,16 4. 3 ( 2. 5– 8. 7) 5. 9 ( 2. 9– 8. 3) 6. 0 ( 2. 8– 8. 8) n. s. HVA :D A CS F to ta l 37, 13 ,18 88. 9 ( 52. 2– 158. 1) 10 8. 6 ( 63. 6– 24 4. 6) 73. 8 ( 48. 1– 121. 9) n. s. m ed ic at io n-fre e 21, 7, 10 87. 0 (50. 7– 146. 9) 65. 6 ( 59. 5– 174. 9) 62. 4 ( 40. 9– 109. 0) n. s. pl as m a to ta l 14 9, 35 ,40 18. 4 ( 9. 4– 31. 5) 19. 9 ( 8. 9– 43. 4) 16. 4 ( 8. 5– 32. 9) n. s. m ed ic at io n-fre e 93, 16 ,16 17. 6 ( 9. 6– 31. 2) 16. 9 ( 7. 0– 33. 5) 16. 4 ( 5. 8– 25. 4) n. s.

Ta bl e 6. 4 (c ont inue d) Co m po un d/ ra tio CS F/ pl as m a To ta l/ m ed ic ati on -fr ee N DS DS +p AD DS+ AD P-va lu e 5-HT CS F to ta l 11 ,4 ,6 0. 1 ( 0. 1– 0. 2) 0. 1 ( 0. 1– 0. 2) 0. 1 ( 0. 1– 0. 2) n. s. m ed ic at io n-fre e 6, 2, 2 0. 1 ( 0. 1– 0. 2) 0. 1 ( 0. 1– ) 0. 1 ( 0. 0– ) n. s. pl as m a to ta l 14 9, 36 ,40 9. 5 ( 3. 9– 20. 3) 11. 1 ( 2. 4– 20. 8) 9. 7 ( 5. 2– 17. 2) n. s. m ed ic at io n-fre e 93, 17 ,16 12. 4 ( 7. 1– 25. 0) 17. 3 ( 8. 0– 28. 6) 10. 6 ( 8. 4– 20. 7) n. s. 5-HIA A CS F to ta l 37, 13 ,18 24. 9 ( 18. 1– 28. 9) 26. 5 ( 21. 5– 35. 4) 28. 9 ( 23. 2– 34. 3) n. s. m ed ic at io n-fre e 21, 7, 10 26. 6 ( 22. 6– 29. 7) 26. 5 (22. 0– 36. 1) 33. 2 ( 25. 2– 38. 4) n. s. pl as m a to ta l 14 9, 36 ,40 4. 5 ( 3. 7– 5. 4) § 4. 7 ( 4. 2– 5. 7) 5. 0 ( 4. 1– 6. 6) § (0. 02 0) m ed ic at io n-fre e 93, 17 ,16 4. 5 ( 3. 6– 5. 4) 5. 3 ( 4. 2– 6. 5) 4. 9 ( 3. 6– 6. 7) n. s. 5-HIA A: 5-HT CS F to ta l 11 ,4 ,6 19 8. 3 ( 14 7. 8– 34 0. 9) 16 6. 7 (143 .6 –1 96. 1) 16 1. 6 ( 13 6. 7– 55 5. 7) n. s. m ed ic at io n-fre e 6, 2, 2 23 6. 3 ( 16 5. 5– 34 8. 5) 18 2. 0 ( 16 0. 3– ) 77 2. 2 ( 15 2. 6– ) n. s. pl as m a to ta l 14 9, 36 ,40 0. 5 ( 0. 2– 1. 3) 0. 5 ( 0. 2– 1. 8) 0. 6 ( 0. 3– 1. 3) n. s. m ed ic at io n-fre e 93, 17 ,16 0. 3 ( 0. 2– 0. 6) 0. 4 ( 0. 2– 0. 7) 0. 4 (0. 2– 0. 7) n. s. HVA :5 -H IA A CS F to ta l 37, 13 ,18 2. 2 ( 1. 9– 2. 7) § 2. 0 ( 1. 7– 2. 3) 1. 9 ( 1. 2– 2. 2) § (0. 02 9) m ed ic at io n-fre e 21, 7, 10 2. 0 ( 1. 7– 2. 3) 1. 8 ( 1. 5– 2. 2) 1. 8 ( 1. 2– 2. 1) n. s. pl as m a to ta l 14 9, 36 ,40 2. 1 ( 1. 7– 2. 7) 2. 0 ( 1. 5– 2. 6) 2. 2 ( 1. 7– 2. 7) n. s. m ed ic at io n-fre e 93, 17 ,16 2. 2 ( 1. 8– 2. 8) 1. 8 ( 1. 4– 2. 8) 2. 3 ( 1. 8– 2. 7) n. s. Co nc ent ra tio ns o f m ono am ine s a nd m et ab ol ite s ( ng /m l), a s we ll a s t he c or re spo ndi ng ra tio s a re e xp re ss ed a s m edi an ( 50 %) wi th th e i nt er qu ar til e r an ge (25 % -7 5%) be twe en br ac ke ts . T he n um be r ( N) of s am pl es is pr ov ide d a s c er ta in c om po un ds we re no t de te ct abl e i n a ll s am pl es . A K rus ka l-W al lis te st wa s pe rf or m ed t o c om pa re the thr ee g ro ups . S ig ni fic an t P -v al ue s ( <0 .0 15 ) a re pr ov ide d. T ho se in ita lic s be twe en br ac ke ts a re no lo ng er r eg ar de d s ig ni fic an t a ft er c or re ct io n ( P< 0. 01 5) , b ut po st -ho c M ann -W hi tn ey U te sts r em ai ne d s ig ni fic ant . P os t-ho c c om pa ris ons we re p er fo rm ed f or D S v s DS+ pA D, DS v s DS+ AD ( § < 0. 01 5 a nd § §< 0. 00 1) a nd DS+ pA D v s DS +A D. A bb re vi at io ns : 5 -H IA A, 5 -hy dr ox yi ndo le ac et ic a ci d; 5 -H T, se ro to ni n; C SF , c er eb ro spi na l f lu id; D A, do pa m ine ; D S, D ow n s yn dr om e wi tho ut ( cl in ic al ) de m en tia ; D S+ pA D , D own s yn dr om e wi th pr odr om al A D; D S+ AD , D S w ith di ag no se d A D de m en tia ; D O PA C, 3 -4 -d ih yd ro xy ph en yl ace tic a ci d; H VA , h om ov an ill ic a ci d; M HP G , 3-m et ho xy -4 -hy dr ox yp he ny lg ly co l; NA , no ra dr ena line ; n. s. , no t s ig ni fic ant .

In the context of abnormal brain development, monoamines were quantified in

frontal cortex of fetal DS tissue (20 weeks) compared to controls. DA, 5-HT and 5-HIAA

levels were significantly reduced in DS (Whittle et al., 2007). This suggests that

monoamines are already impacted by trisomy 21 itself, which may be further impaired by

progressive Aβ pathology during life. Compared to age-matched controls, smaller brain

volumes were found in DS, among others of (pre)frontal cortex, hippocampus, brainstem

and cerebellum (Beacher et al., 2010; Teipel and Hampel, 2006; Wisniewski, 1990). Fewer

neurons (cortical dysgenesis), altered neuronal distribution and reduced synaptic density

was described in DS as well (Wisniewski, 1990). Consequently, the compensatory reserve

is likely to be lower, which could result in a particularly early vulnerability (functional

impact) to additional neuropathology. To differentiate between the alterations caused by

trisomy 21 and AD pathology, respectively, future monoaminergic studies should include

DS samples without early Aβ plaque load. However, inclusion of DS cases in brain banks,

those without pathology in particular, is very limited. In fact, the 21 cases analyzed here

were obtained by three brain banks in a timeframe of 25 years. Standardized (multicenter)

brain banking efforts for DS are thus imperative (Hartley et al., 2015).

The apparent lack of monoaminergic changes between DS and DS+AD in brain

was also reflected in CSF/plasma. The CSF/plasma groups were distinguished based on a

clinical dementia diagnosis, while from a neuropathologic perspective the (amyloid)

pathology is likely to be quite comparable. In future studies it would be useful to relate

monoaminergic values in DS to (in vivo) pathologic staging, such as the CSF ‘AD profile’

(Dekker et al., 2017) or positron emission tomography (PET) of Aβ/tau. Surprisingly, the

CSF/plasma results did not reflect earlier results in serum (Dekker et al., 2015a). Whereas

MHPG, for instance, was evidently decreased in DS+AD serum, MHPG levels were virtually

unaltered in CSF/plasma. This raises the question what causes this apparent discrepancy.

Our methodology has been validated (Van Dam et al., 2014) and the reported values have

orders of magnitude comparable to earlier studies (Coppus et al., 2007; Kay et al., 1987;

Schapiro et al., 1987). The – likely multifactorial – answer remains to be elucidated,

including the effect of (pre)analytical variables. O’Bryant and colleagues (2015) addressed

variables that can impact findings in blood, including controllable variables (e.g. fasting

status, tube type, centrifugation parameters, time from collection to freezing and freezing

temperature) and uncontrollable variables (e.g. diet, activity level, co-morbidities and

medication). In particular, serum vs plasma, type of needle, additive in the collection tubes

and presence of hemolysis may influence the stability and detectability of biomarkers

(O’Bryant et al., 2015). In CSF, similar variables may impact biomarker levels (Le Bastard et

al., 2015; Mattsson et al., 2011). Indeed, a few variables differ identifiably between our

serum and plasma studies, such as fasting status and storage temperature and time.

Retrospectively identifying the cause of the discrepancy is virtually impossible. New

initiatives should, therefore, exhaustively study the effect of these variables on

monoaminergic concentrations.

In conclusion, DS/DS+AD brain samples revealed generalized impairments in the

noradrenergic and serotonergic systems (overall decrease) and a bidirectional

dopaminergic change. CSF/plasma concentrations did not differ between groups. The

underlying cause for the discrepancy with earlier serum findings remains unclear and

requires further study. To confirm whether the more profound monoaminergic alterations

in DS (vs non-DS) are indeed due to early Aβ accumulation, (longitudinal) studies using PET

imaging of monoamines might provide a new avenue. For instance, neuroimaging of NA

transporters in LC and key projection areas using [

11

C]methylreboxetine (Pietrzak et al.,

2013) in relation to amyloid deposition (e.g. [

11

_{C]Pittsburgh compound B) may be of}

utmost importance in this respect.

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