university of groningen down & alzheimer dekker, alain daniel

University of Groningen

Down & AlzheimerDekker, Alain Daniel

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2017

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Dekker, A. D. (2017). Down & Alzheimer: Behavioural biomarkers of a forced marriage. RijksuniversiteitGroningen.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 02-10-2021

https://research.rug.nl/en/publications/down--alzheimer(7e35a356-4e2a-4c45-ba94-253505167cc7).html

Severe 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 Fernándezd – Alberto Lleóc – Sebastian Videlad,f Anne Siebenb – Jean-Jacques Martinb – Netherlands Brain Bankg

Rafael Blesac – Juan Forteac,d and Peter P. De Deyna,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.

176

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

177

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

178

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 ClarityTM 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

179

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)

EOAD (n=11)

Controls (n=10) P-value

Age at death in years (median; min.-max.)

39.5 (35.0-44.0)

62.0 (44.0-80.0)

67.2 (57.6-73.0)

65.5 (57.2-73.3) 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.)

21.0 (11.5-36.0)

7.3 (3.8-20.0)

3.0 (2.8-7.0)

5.4 (2.3-7.0) <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.

180

Tabl

e 6.

2: C

ompa

rison

of p

ost-

mor

tem

con

cent

ratio

ns a

nd ra

tios b

etw

een

the

grou

ps

Brai

n re

gion

Co

mpo

und/

ratio

N

D

S (n

=4)

DS+

AD (n

=17)

EO

AD (n

=11)

Co

ntro

ls (n

=10)

P-

valu

e Neocortex

BA7

su

perio

r pa

rieta

l lob

ule

MHP

G

2,13

,11,

10

71.0

(63.

3–)

72.3

(57.

0–10

5.2)

*

119.

4 (1

06.0

–189

.5) *

25

9.7

(119

.8–3

54.5

) <0

.001

DA

2,

12,1

1,10

11

.0 (6

.5–)

18

.7 (1

2.5–

28.7

) 10

.7 (6

.4–1

6.5)

6.

9 (4

.5–9

.9)

0.00

4 HV

A:DA

2,

12,1

1,10

10

.0 (6

.4–)

6.

1 (3

.9–8

.7) *

11

.5 (8

.3–1

7.8)

*

17.3

(10.

5–29

.8)

0.00

5 5-

HIAA

2,

13,1

1,10

92

.8 (7

0.4–

) 40

.2 (3

0.4–

71.1

) 55

.3 (4

0.5–

93.7

) 10

6.4

(75.

3–15

8.9)

0.

003

HVA:

5-HI

AA

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.

001

BA9/

10/4

6

pref

ront

al

cort

ex

MHP

G

4,14

,11,

10

84.2

(77.

7–86

.9)

107.

4 (6

3.4–

133.

8) *

* 47

1.2

(284

.3–6

55.1

) **

265.

7 (1

32.4

–629

.6)

<0.0

01

MHP

G:N

A 4,

14,1

1,10

8.

9 (3

.2–1

5.3)

6.

0 (2

.3–1

2.4)

**

77.8

(11.

7–20

3.3)

**

9.7

(4.7

–32.

7)

0.00

4 DA

4,

14,1

1,10

12

7.5

(11.

4–49

7.4)

37

3.9

(204

.3–7

43.6

) **

7.2

(2.5

–9.3

) **

7.5

(4.0

–11.

3)

<0.0

01

DOPA

C:DA

4,

14,1

1,10

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

.001

HV

A:DA

4,

14,1

1,10

11

.7 (5

.1–1

9.2)

4.

7 (0

.5–8

.2) *

* 18

.7 (1

3.2–

35.8

) **

24.8

(10.

7–79

.3)

<0.0

01

5-HT

4,

14,1

1,10

17

.8 (1

3.2–

30.6

) 28

.7 (1

5.3–

49.8

) *

11.1

(6.1

–15.

0) *

11

.7 (7

.4–1

6.7)

0.

009

5-HI

AA

4,14

,11,

10

81.1

(64.

6–12

2.2)

62

.2 (3

3.9–

87.4

) *

144.

7 (1

10.5

–186

.6) *

16

4.1

(128

.2–2

15.6

) 0.

001

5-HI

AA:5

-HT

4,14

,11,

10

5.9

(3.2

–8.3

) # 4.

2 (2

.3–9

.2) *

* 15

.0 (1

3.1–

32.6

) **

16.3

(10.

9–19

.8) #

<0.0

01

HVA:

5-HI

AA

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

occi

pita

l pol

e (V

1)

MHP

G

3,7,

11,1

0 77

.2 (6

1.3–

) # 89

.2 (7

3.6–

114.

6)

127.

9 (1

01.6

–182

.0)

285.

3 (2

44.0

–530

.1) #

<0.0

01

5-HI

AA

3,7,

11,1

0 12

6.0

(93.

6–)

92.1

(46.

9–14

4.1)

95

.6 (5

7.5–

143.

6)

200.

8 (1

54.3

–263

.9)

0.00

8 HV

A:5-

HIAA

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.

003

BA22

su

perio

r te

mpo

ral

gyru

s

NA

3,10

,9,1

0 30

.2 (1

8.3–

) 17

.3 (1

2.5–

27.4

) 10

.0 (7

.4–1

2.4)

18

.7 (1

3.6–

28.5

) 0.

014

MHP

G

3,10

,11,

10

107.

3 (8

0.4–

) # 87

.0 (6

0.0–

125.

4) *

* 52

0.7

(313

.4–6

73.4

) **

360.

5 (2

60.4

–630

.7) #

<0.0

01

MHP

G: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

.8–4

6.9)

<0

.001

DA

3,

10,1

1,10

6.

7 (6

.5–)

11

.3 (8

.1–2

5.2)

*

4.2

(3.2

–6.8

) *

9.1

(5.4

–18.

4)

(0.0

41)

DOPA

C:DA

3,

10,1

0,10

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:DA

3,

10,1

1,10

17

.5 (1

1.2–

) 9.

8 (5

.8–1

9.2)

*

32.4

(19.

5–44

.5) *

18

.3 (1

3.9–

25.7

) 0.

014

5-HI

AA

3,10

,11,

10

119.

1 (9

8.7–

) 84

.0 (6

0.4–

131.

7) *

30

3.2

(119

.6–4

50.2

) *

171.

5 (1

21.7

–320

.6)

0.00

4 5-

HIAA

:5-H

T 3,

10,1

1,10

10

.2 (8

.3–)

16

.9 (1

2.5–

20.5

) *

67.8

(28.

0–14

7.7)

*

23.5

(18.

0–36

.7)

0.00

2 HV

A:5-

HIAA

3,

10,1

1,10

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

Limbic system

Amyg

dala

NA

2,10

,10,

10

21.8

(19.

5–)

25.3

(13.

6–39

.8) *

59

.0 (4

6.9–

78.5

) *

84.5

(77.

5–12

1.8)

<0

.001

M

HPG

2,

10,1

0,10

68

.8 (5

8.2–

) 70

.9 (6

2.5–

94.9

) **

429.

8 (1

80.0

–964

.3) *

* 30

4.8

(193

.5–7

59.3

) <0

.001

HV

A 2,

10,1

0,10

26

5.8

(174

.5–)

37

6.2

(258

.9–6

17.2

) 59

9.1

(398

.7–8

66.2

) 11

32.5

(751

.0–1

421.

4)

0.00

5 DO

PAC:

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.

008

5-HT

2,

10,1

0,10

59

.3 (1

1.7–

) 33

.0 (1

8.6–

49.6

) *

121.

1 (5

5.1–

148.

6) *

24

4.9

(221

.7–2

97.2

) <0

.001

5-

HIAA

2,

10,1

0,10

19

7.9

(162

.9–)

14

1.3

(102

.8–2

27.1

) **

522.

4 (3

34.9

–795

.2) *

* 99

9.8

(754

.5–1

270.

4)

<0.0

01

HVA:

5-HI

AA

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.

014

Hip

poca

mpu

s

Adre

nalin

e 2,

5,3,

5 39

1.6

(236

.5–)

42

.3 (2

0.6–

139.

0)

6.4

(2.6

–)

10.1

(6.4

–14.

1)

0.01

1 M

HPG

3,

5,11

,10

75.9

(70.

3–) #

97.2

(76.

9–12

4.8)

**

459.

5 (1

93.2

–109

9.2)

**

416.

3 (2

32.9

–713

.5) #

<0.0

01

MHP

G: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

.3–3

1.4)

(0

.040

) 5-

HT

3,5,

11,1

0 46

.6 (2

8.0–

) 13

.5 (8

.6–5

0.0)

44

.4 (2

0.7–

65.3

) 87

.8 (6

9.3–

111.

2)

0.00

3 5-

HIAA

3,

5,11

,10

141.

3 (1

10.6

–)

47.7

(43.

1–21

3.4)

*

336.

1 (2

57.8

–476

.2) *

38

3.9

(279

.5–7

17.0

) 0.

004

HVA:

5-HI

AA

3,5,

11,1

0 1.

3 (0

.5–)

2.

4 (1

.1–3

.6) *

0.

6 (0

.4–1

.2) *

0.

6 (0

.5–0

.9)

(0.0

19)

181

Tabl

e 6.

2 (c

ontin

ued)

Brai

n re

gion

Co

mpo

und/

ratio

N

D

S (n

=4)

DS+

AD (n

=17)

EO

AD (n

=11)

Co

ntro

ls (n

=10)

P-

valu

e Limbic system

BA11

/12

orbi

tofr

onta

l co

rtex

MHP

G

4,6,

11,1

0 91

.8 (7

0.6–

105.

7) #

103.

1 (6

3.9–

111.

2) *

* 43

7.1

(352

.6–5

45.6

) **

361.

7 (2

16.5

–636

.3) #

<0.0

01

MHP

G:N

A 4,

6,11

,10

5.4

(2.1

–14.

4)

5.3

(3.5

–10.

8) *

50

.6 (2

7.7–

73.3

) *

18.9

(11.

5–37

.4)

0.00

6 HV

A:DA

4,

5,11

,10

26.4

(8.9

–41.

5)

11.1

(4.2

–14.

8) *

* 38

.2 (2

5.8–

53.7

) **

33.4

(26.

7–47

.0)

0.01

3 5-

HIAA

4,

6,11

,10

98.2

(85.

4–18

9.7)

56

.7 (3

5.3–

78.5

) **

238.

5 (1

83.5

–344

.3) *

* 22

9.9

(168

.9–3

28.3

) <0

.001

5-

HIAA

:5-H

T 4,

6,11

,10

6.5

(5.5

–7.5

) 7.

4 (6

.4–8

.9) *

21

.0 (1

4.4–

28.1

) *

13.3

(8.5

–22.

1)

0.00

3 HV

A:5-

HIAA

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

Cing

ulat

e gy

rus

MHP

G

3,8,

11,1

0 12

8.0

(58.

8–)

126.

0 (1

04.6

–153

.6) *

* 56

7.8

(258

.4–7

39.3

) **

336.

9 (1

58.1

–587

.5)

<0.0

01

MHP

G: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

.4–2

1.9)

0.

003

DA

3,8,

11,1

0 22

4.2

(7.9

–)

55.5

(40.

6–21

1.2)

**

10.5

(3.8

–13.

0) *

* 9.

6 (3

.0–1

7.4)

<0

.001

DO

PAC:

DA

3,8,

11,1

0 0.

1 (0

.1–)

0.

2 (0

.1–0

.5) *

* 1.

3 (1

.0–2

.9) *

* 1.

2 (0

.5–1

.6)

<0.0

01

HVA:

DA

3,8,

11,1

0 0.

7 (0

.6–)

1.

6 (1

.1–5

.1) *

* 23

.4 (1

5.7–

44.5

) **

24.7

(17.

3–98

.1)

<0.0

01

5-HI

AA

3,8,

11,1

0 66

.0 (3

4.5–

) # 10

2.6

(58.

5–19

1.8)

**

357.

3 (2

81.2

–379

.3) *

* 38

7.2

(313

.7–4

75.7

) # <0

.001

HV

A:5-

HIAA

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

.001

Thal

amus

MHP

G

3,11

,11,

10

159.

2 (1

31.3

–) #

148.

3 (1

10.2

–177

.8) *

* 79

3.0

(628

.6–1

442.

6) *

* 44

1.9

(244

.1–1

345.

4) #

<0.0

01

MHP

G:N

A 3,

11,1

0,10

2.

7 (0

.4–)

2.

1 (0

.7–2

.9) *

5.

8 (4

.5–1

2.6)

*

2.9

(1.0

–6.1

) 0.

010

HVA:

DA

3,11

,11,

10

17.4

(1.9

–)

8.8

(3.4

–17.

8) *

39

.7 (2

5.9–

56.9

) *

30.2

(24.

7–50

.5)

0.01

0 5-

HIAA

3,

11,1

1,10

67

3.7

(508

.8–)

68

9.3

(412

.4–8

95.8

) **

1584

.5 (1

237.

1–19

52.8

) **

1525

.8 (1

168.

1–19

46.8

) <0

.001

5-

HIAA

:5-H

T 3,

11,1

1,10

3.

6 (3

.2–)

5.

3 (4

.2–8

.7)

9.7

(7.3

–13.

1)

8.9

(5.7

–9.4

) 0.

009

Basal ganglia

Caud

ate

nucl

eus

Adre

nalin

e 2,

11,7

,6

533.

3 (3

55.2

–)

69.3

(42.

7–15

1.0)

*

268.

5 (1

76.5

–587

.7) *

37

2.9

(185

.7–7

43.0

) 0.

006

DA

3,16

,11,

10

4297

.2 (1

845.

8–)

2395

.2 (1

178.

5–27

84.8

) **

4721

.2 (3

403.

8–69

05.9

) **

3965

.1 (2

987.

2–44

20.5

) 0.

003

HVA

3,16

,11,

10

2732

.0 (2

231.

3–)

3145

.6 (1

522.

7–37

56.0

) *

4681

.2 (3

714.

5–56

40.3

) *

4372

.3 (3

564.

4–68

18.8

) 0.

014

5-HT

3,

16,1

1,10

12

1.5

(33.

1–)

55.8

(35.

6–97

.0) *

* 16

8.3

(139

.5–2

30.2

) **

240.

0 (1

88.0

–285

.2)

<0.0

01

5-HI

AA

3,16

,11,

10

170.

9 (7

6.6–

) 21

7.6

(106

.3–2

84.2

) **

537.

2 (3

62.3

–727

.8) *

* 57

9.6

(441

.0–7

84.9

) <0

.001

Glo

bus

palli

dus

DOPA

C 2,

8,11

,10

208.

3 (2

8.0–

) 13

1.7

(61.

8–14

1.5)

*

39.5

(19.

5–85

.8) *

20

.1 (1

0.5–

34.7

) 0.

004

DOPA

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.

009

5-HT

2,

8,11

,10

149.

7 (8

9.7–

) 97

.4 (7

0.5–

120.

4) *

17

1.9

(117

.6–2

07.7

) *

161.

8 (1

40.3

–215

.5)

(0.0

22)

5-HI

AA

2,8,

11,1

0 10

09.2

(384

.6–)

43

9.5

(329

.9–9

78.8

) *

984.

5 (8

64.7

–140

7.3)

*

1320

.2 (1

019.

2–16

14.4

) (0

.015

) HV

A:5-

HIAA

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

Puta

men

Adre

nalin

e 2,

8,11

,10

390.

4 (3

25.6

) 13

5.0

(40.

9–21

9.9)

*

581.

9 (1

87.2

–152

3.4)

*

339.

6 (1

00.7

–797

.8)

(0.0

38)

DOPA

C 3,

15,1

1,10

20

7.5

(165

.1–)

61

1.6

(274

.7–8

51.6

) 42

1.9

(235

.7–6

25.7

) 20

2.2

(119

.0–2

99.7

) 0.

008

DOPA

C:DA

3,

15,1

1,10

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

189.

5 (4

3.9–

) 77

.8 (3

5.2–

159.

6) *

18

9.2

(153

.9–2

19.8

) *

219.

7 (2

00.5

–326

.3)

<0.0

01

5-HI

AA

3,15

,11,

10

260.

0 (2

31.7

–)

372.

7 (2

03.7

–669

.5) *

* 79

0.9

(638

.7–1

026.

1) *

* 99

8.4

(781

.9–1

400.

3)

<0.0

01

HVA:

5-HI

AA

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

Subs

tant

ia

nigr

a

DA

2,14

,11,

10

164

(126

.8–)

15

7.2

(89.

0–29

2.4)

**

572.

7 (2

84.9

–623

.7) *

* 62

4.0

(295

.7–9

36.8

) <0

.001

DO

PAC

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.

2–10

1.0)

0.

005

HVA

2,14

,11,

10

3061

.7 (2

010.

5–)

2421

.8 (1

995.

7–30

07.8

) **

3799

.4 (3

461.

0–43

85.5

) **

4331

.0 (3

241.

6–53

49.9

) <0

.001

DO

PAC:

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.

001

HVA:

DA

2,14

,11,

10

18.1

(15.

9–)

17.0

(9.6

–23.

4) *

8.

3 (5

.3–1

2.9)

*

8.2

(4.8

–9.7

) 0.

008

5-HT

2,

14,1

1,10

20

8.3

(167

.6–)

36

4.1

(243

.1–4

20.4

) 48

7.2

(389

.2–5

31.2

) 46

1.0

(404

.1–5

88.8

) 0.

009

182

Tabl

e 6.

2 (c

ontin

ued)

Brai

n re

gion

Co

mpo

und/

ratio

N

D

S (n

=4)

DS+

AD (n

=17)

EO

AD (n

=11)

Co

ntro

ls (n

=10)

P-

valu

e Metencephalon

Locu

s co

erul

eus

NA

0,10

,10,

10

–

88.6

(52.

7–11

.4) *

* 25

5.1

(171

.9–4

00.7

) **

347.

3 (2

48.6

–501

.8)

<0.0

01

MHP

G

0,10

,10,

10

– 20

1.8

(151

.8–2

59.5

) *

429.

6 (3

04.6

–530

.7) *

57

2.4

(158

.9–8

36.2

) (0

.032

) DO

PAC

0,10

,10,

10

– 12

.3 (8

.1–1

9.2)

**

39.0

(23.

6–71

.3) *

* 55

.5 (3

3.1–

98.6

) <0

.001

HV

A 0,

10,1

0,10

–

730.

2 (4

82.0

–100

3.5)

10

09.7

(905

.7–1

443.

4)

1351

.0 (1

195.

0–14

82.2

) <0

.001

DOPA

C:DA

0,

10,1

0,10

–

0.5

(0.3

–0.6

) *

1.1

(0.9

–1.6

) *

1.4

(0.7

–2.0

) 0.

002

Cere

bellu

m

Adre

nalin

e 0,

7,4,

6 –

36.4

(34.

7–44

.5) *

6.

6 (3

.6–1

2.7)

*

21.4

(10.

3–50

.2)

(0.0

17)

MHP

G

2,9,

11,1

0 12

4.6

(100

.3–)

10

1.1

(85.

2–14

8.6)

*

421.

1 (2

32.5

–708

.2) *

51

8.1

(386

.8–7

13.4

) 0.

001

MHP

G: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

.8–1

8.4)

0.

004

DA

2,9,

11,1

0 32

.1 (8

.4–)

15

.5 (1

2.2–

22.1

) **

3.6

(1.5

–8.6

) **

3.6

(3.2

–5.4

) <0

.001

DO

PAC

2,9,

11,1

0 35

.1 (6

.4–)

16

.4 (1

1.3–

26.8

) *

8.2

(5.8

–11.

1) *

8.

0 (6

.0–8

.3)

0.00

5 HV

A:DA

2,

9,11

,10

6.2

(2.5

–)

6.0

(5.2

–6.9

) **

28.7

(12.

1–44

.9) *

* 28

.5 (1

8.5–

32.8

) <0

.001

5-

HT

2,9,

11,1

0 66

.5 (2

2.2–

) 25

.0 (1

4.5–

34.9

) **

4.6

(2.1

–9.1

) **

2.6

(2.3

–6.8

) <0

.001

5-

HIAA

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.

7–20

7.2)

<0

.001

5-

HIAA

:5-H

T 2,

9,11

,10

0.7

(0.4

–)

1.5

(1.1

–3.3

) **

41.8

(15.

7–97

.1) *

* 32

.9 (2

2.0–

44.0

) <0

.001

HV

A:5-

HIAA

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

.001

M

onoa

min

es a

nd m

etab

olite

s (n

g/g

tissu

e) a

nd th

e co

rres

pond

ing

ratio

s ar

e ex

pres

sed

as m

edia

n (5

0%) w

ith th

e in

terq

uart

ile ra

nge

(25%

-75%

) bet

wee

n br

acke

ts. A

Kru

skal

l-Wal

lis te

st w

as u

sed

to

com

pare

the

four

gro

ups.

Sig

nific

ant

P-va

lues

(<0.

015)

are

pro

vide

d. T

hose

in it

alic

s be

twee

n br

acke

ts a

re n

o lo

nger

reg

arde

d sig

nific

ant

afte

r co

rrec

tion

(P<0

.015

), bu

t po

st-h

oc M

ann-

Whi

tney

U

test

s re

mai

ned

signi

fican

t: DS

vs

cont

rols

(#<0

.015

) and

DS+

AD v

s EO

AD (*

<0.0

15; *

*<0.

001)

. Abb

revi

atio

ns: B

A, B

rodm

ann

area

; 5-H

IAA,

5-h

ydro

xyin

dole

acet

ic a

cid;

5-H

T, s

erot

onin

; DA,

dop

amin

e;

DS, D

own

synd

rom

e w

ithou

t ne

urop

atho

logi

c AD

dia

gnos

is; D

S+AD

, Dow

n sy

ndro

me

with

neu

ropa

thol

ogic

AD

diag

nosis

; DO

PAC,

3-4

-dih

ydro

xyph

enyl

acet

ic a

cid;

EO

AD, e

arly

-ons

et A

lzhei

mer

’s

dise

ase;

HVA

, hom

ovan

illic

aci

d; M

HPG

, 3-m

etho

xy-4

-hyd

roxy

phen

ylgl

ycol

; NA,

nor

adre

nalin

e; n

.s.,

not s

igni

fican

t.

183

Fi

gure

6.1

: M

HPG

con

cent

ratio

ns (

ng/g

tiss

ue)

for

each

stu

dy g

roup

in

neoc

ortic

al a

reas

, lim

bic

syst

em,

locu

s co

erul

eus

and

cere

bellu

m.

The

boxe

s re

pres

ent

the

inte

rqua

rtile

rang

e (IQ

R, 2

5-75

%) w

ith th

e bl

ack

horiz

onta

l lin

e in

dica

ting

the

med

ian.

The

whi

sker

s in

dica

te v

alue

s with

in 1

.5 IQ

R. M

ild o

utlie

rs (1

.5-3

IQR)

are

indi

cate

d w

ith

an o

pen

circ

le, e

xtre

me

outli

ers

(>3

IQR)

with

an

aste

risks

. One

ext

rem

e va

lue

(EO

AD, M

HPG

conc

entr

atio

n in

hip

poca

mpu

s of

380

6 ng

/g ti

ssue

) is

not s

how

n w

ith re

spec

t to

scal

ing.

Evi

dent

ly, M

HPG

leve

ls w

ere

cons

isten

tly lo

wer

in D

S (v

s co

ntro

ls) a

nd D

S+AD

(vs

EOAD

). DS

vs

DS+A

D di

d no

t diff

er s

igni

fican

tly. I

ndiv

idua

l com

paris

on s

tatis

tics

are

prov

ided

in T

able

6.2

. Abb

revi

atio

ns: B

A, B

rodm

ann

area

; DS,

Dow

n sy

ndro

me

with

out

neur

opat

holo

gic

AD d

iagn

osis;

DS+

AD, D

own

synd

rom

e w

ith n

euro

path

olog

ic A

D di

agno

sis; E

OAD

, ear

ly-o

nset

Alzh

eim

er’s

dise

ase.

MHPG concentration(ng/g)

Stud

ygr

oups

DSDS

+AD

EOAD

Cont

rols

12

34

56

78

119

BA9/

10/4

6

BA17

BA22

BA7

2 3 4 5 6 7 8 9 101

Amyg

dala

Hipp

ocam

pus

BA11

/12

Cing

ulat

egy

rus

Thal

amus

Locu

s coe

rule

us

Cere

bellu

m11

Neocortex Limbic system

12

34

56

78

119

101

23

45

67

811

910

12

34

56

78

119

10

184

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),

185

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

186

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)

Prodromal AD (DS+pAD, n=36)

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 1 (e2/2), 15 (e2/3),

2 (e2/4), 106 (e3/3), 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.,

187

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.

188

Tabl

e 6.

4: C

ompa

rison

of C

SF/p

lasm

a co

ncen

trat

ions

and

ratio

s bet

wee

n th

e gr

oups

Co

mpo

und/

ratio

CS

F/pl

asm

a To

tal/

med

icat

ion-

free

N

D

S D

S+pA

D

DS+

AD

P-va

lue

NA

CSF

tota

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

edic

atio

n-fr

ee

21,7

,10

2.8

(1.0

–6.9

) 1.

4 (0

.6–4

.7)

1.4

(0.8

–5.5

) n.

s.

plas

ma

tota

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

edic

atio

n-fr

ee

90,1

6,15

0.

5 (0

.2–1

.0)

0.4

(0.1

–0.7

) 0.

4 (0

.2–1

.0)

n.s.

Adre

nalin

e

CSF

tota

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

edic

atio

n-fr

ee

20,7

,10

0.7

(0.3

–1.0

) 0.

8 (0

.2–2

.1)

0.4

(0.4

–1.2

) n.

s.

plas

ma

tota

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

edic

atio

n-fr

ee

92,1

7,16

2.

9 (2

.0–4

.0)

3.0

(2.0

–4.2

) 2.

7 (1

.2–3

.9)

n.s.

MH

PG

CSF

tota

l 37

,13,

18

30.2

(21.

5–44

.6)

24.0

(18.

8–40

.0)

22.4

(19.

6–46

.0)

n.s.

med

icat

ion-

free

21

,7,1

0 31

.1 (2

1.8–

45.0

) 24

.0 (1

5.9–

41.1

) 28

.5 (2

0.7–

43.4

) n.

s.

plas

ma

tota

l 14

9,36

,40

76.2

(51.

2–10

2.4)

70

.9 (5

0.4–

111.

6)

75.6

(55.

3–11

4.4)

n.

s.

m

edic

atio

n-fr

ee

93,1

7,16

77

.3 (5

1.5–

100.

6)

70.4

(45.

5–11

9.3)

63

.0 (4

7.9–

107.

5)

n.s.

MH

PG:N

A

CSF

tota

l 37

,13,

18

11.6

(6.0

–31.

0)

29.8

(6.9

–34.

7)

18.3

(6.4

–28.

4)

n.s.

med

icat

ion-

free

21

,7,1

0 10

.4 (5

.2–3

1.2)

29

.8 (5

.4–3

5.1)

22

.0 (8

.2–3

2.7)

n.

s.

plas

ma

tota

l 14

5,35

,37

185.

4 (9

6.3–

392.

0)

165.

0 (1

04.9

–260

.9)

111.

7 (7

2.7–

203.

2)

n.s.

med

icat

ion-

free

90

,16,

15

166.

3 (7

7.7–

315.

6)

222.

3 (1

47.8

–292

.9)

147.

7 (1

02.2

–394

.9)

n.s.

DA

CSF

tota

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

edic

atio

n-fr

ee

21,7

,10

0.6

(0.4

–0.9

) 0.

6 (0

.3–1

.0)

0.8

(0.4

–1.3

) n.

s.

plas

ma

tota

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

edic

atio

n-fr

ee

93,1

6,16

0.

5 (0

.3–1

.0)

0.6

(0.4

–1.1

) 0.

7 (0

.4–1

.9)

n.s.

DO

PAC

CSF

tota

l 37

,13,

18

0.6

(0.4

–1.3

) 1.

2 (0

.6–2

.7)

1.7

(0.7

–3.3

) (0

.033

)

med

icat

ion-

free

21

,7,1

0 0.

5 (0

.4–1

.0) §§

0.

9 (0

.5–1

.5)

2.8

(1.1

–5.6

) §§

0.00

3 pl

asm

a to

tal

149,

36,4

0 2.

7 (1

.9–4

.1) §§

3.

1 (2

.6–4

.3)

4.3

(3.0

–6.2

) §§

<0.0

01

m

edic

atio

n-fr

ee

93,1

7,16

2.

5 (1

.9–3

.6) §

3.1

(2.7

–3.6

) 5.

3 (2

.9–6

.2) §

0.00

4

HVA

CSF

tota

l 37

,13,

18

54.6

(40.

5–67

.7)

55.1

(43.

3–74

.8)

55.3

(33.

3–67

.3)

n.s.

med

icat

ion-

free

21

,7,1

0 56

.3 (4

1.5–

64.1

) 46

.5 (3

5.2–

79.5

) 56

.4 (3

3.1–

67.2

) n.

s.

plas

ma

tota

l 14

9,36

,40

9.4

(7.5

–11.

6)§

10.3

(7.4

–12.

7)

10.6

(8.7

–15.

0)§

(0.0

39)

m

edic

atio

n-fr

ee

93,1

7,16

9.

5 (7

.4–1

2.2)

10

.5 (7

.6–1

3.0)

9.

9 (8

.5–1

4.2)

n.

s.

DO

PAC:

DA

CSF

tota

l 37

,13,

18

0.9

(0.5

–4.4

) 2.

3 (0

.8–1

7.2)

2.

9 (0

.7–7

.0)

n.s.

med

icat

ion-

free

21

,7,1

0 0.

8 (0

.5–2

.6) §

1.2

(0.7

–2.9

) 3.

2 (2

.4–7

.4) §

(0.0

31)

plas

ma

tota

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.

med

icat

ion-

free

93

,16,

16

4.3

(2.5

–8.7

) 5.

9 (2

.9–8

.3)

6.0

(2.8

–8.8

) n.

s.

HVA

:DA

CSF

tota

l 37

,13,

18

88.9

(52.

2–15

8.1)

10

8.6

(63.

6–24

4.6)

73

.8 (4

8.1–

121.

9)

n.s.

med

icat

ion-

free

21

,7,1

0 87

.0 (5

0.7–

146.

9)

65.6

(59.

5–17

4.9)

62

.4 (4

0.9–

109.

0)

n.s.

pl

asm

a to

tal

149,

35,4

0 18

.4 (9

.4–3

1.5)

19

.9 (8

.9–4

3.4)

16

.4 (8

.5–3

2.9)

n.

s.

m

edic

atio

n-fr

ee

93,1

6,16

17

.6 (9

.6–3

1.2)

16

.9 (7

.0–3

3.5)

16

.4 (5

.8–2

5.4)

n.

s.

189

Tabl

e 6.

4 (c

ontin

ued)

Co

mpo

und/

ratio

CS

F/pl

asm

a To

tal/

med

icat

ion-

free

N

D

S D

S+pA

D

DS+

AD

P-va

lue

5-H

T

CSF

tota

l 11

,4,6

0.

1 (0

.1–0

.2)

0.1

(0.1

–0.2

) 0.

1 (0

.1–0

.2)

n.s.

med

icat

ion-

free

6,

2,2

0.1

(0.1

–0.2

) 0.

1 (0

.1–)

0.

1 (0

.0–)

n.

s.

plas

ma

tota

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.

med

icat

ion-

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-H

IAA

CSF

tota

l 37

,13,

18

24.9

(18.

1–28

.9)

26.5

(21.

5–35

.4)

28.9

(23.

2–34

.3)

n.s.

med

icat

ion-

free

21

,7,1

0 26

.6 (2

2.6–

29.7

) 26

.5 (2

2.0–

36.1

) 33

.2 (2

5.2–

38.4

) n.

s.

plas

ma

tota

l 14

9,36

,40

4.5

(3.7

–5.4

) § 4.

7 (4

.2–5

.7)

5.0

(4.1

–6.6

) § (0

.020

)

med

icat

ion-

free

93

,17,

16

4.5

(3.6

–5.4

) 5.

3 (4

.2–6

.5)

4.9

(3.6

–6.7

) n.

s.

5-H

IAA:

5-H

T

CSF

tota

l 11

,4,6

19

8.3

(147

.8–3

40.9

) 16

6.7

(143

.6–1

96.1

) 16

1.6

(136

.7–5

55.7

) n.

s.

m

edic

atio

n-fr

ee

6,2,

2 23

6.3

(165

.5–3

48.5

) 18

2.0

(160

.3–)

77

2.2

(152

.6–)

n.

s.

plas

ma

tota

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

edic

atio

n-fr

ee

93,1

7,16

0.

3 (0

.2–0

.6)

0.4

(0.2

–0.7

) 0.

4 (0

.2–0

.7)

n.s.

HVA

:5-H

IAA

CSF

tota

l 37

,13,

18

2.2

(1.9

–2.7

) § 2.

0 (1

.7–2

.3)

1.9

(1.2

–2.2

) § (0

.029

)

med

icat

ion-

free

21

,7,1

0 2.

0 (1

.7–2

.3)

1.8

(1.5

–2.2

) 1.

8 (1

.2–2

.1)

n.s.

pl

asm

a to

tal

149,

36,4

0 2.

1 (1

.7–2

.7)

2.0

(1.5

–2.6

) 2.

2 (1

.7–2

.7)

n.s.

med

icat

ion-

free

93

,17,

16

2.2

(1.8

–2.8

) 1.

8 (1

.4–2

.8)

2.3

(1.8

–2.7

) n.

s.

Conc

entr

atio

ns o

f mon

oam

ines

and

met

abol

ites

(ng/

ml),

as

wel

l as

the

corr

espo

ndin

g ra

tios

are

expr

esse

d as

med

ian

(50%

) with

the

inte

rqua

rtile

rang

e (2

5%-7

5%) b

etw

een

brac

kets

. The

num

ber (

N)

of s

ampl

es is

pro

vide

d as

cer

tain

com

poun

ds w

ere

not d

etec

tabl

e in

all

sam

ples

. A K

rusk

al-W

allis

test

was

per

form

ed to

com

pare

the

thre

e gr

oups

. Sig

nific

ant P

-val

ues

(<0.

015)

are

pro

vide

d. T

hose

in

italic

s be

twee

n br

acke

ts a

re n

o lo

nger

reg

arde

d si

gnifi

cant

aft

er c

orre

ctio

n (P

<0.0

15),

but

post

-hoc

Man

n-W

hitn

ey U

tes

ts r

emai

ned

signi

fican

t. Po

st-h

oc c

ompa

rison

s w

ere

perf

orm

ed f

or D

S vs

DS

+pAD

, DS

vs D

S+AD

(§ <

0.01

5 an

d §§

<0.0

01) a

nd D

S+pA

D vs

DS+

AD. A

bbre

viat

ions

: 5-H

IAA,

5-h

ydro

xyin

dole

acet

ic a

cid;

5-H

T, se

roto

nin;

CSF

, cer

ebro

spin

al fl

uid;

DA,

dop

amin

e; D

S, D

own

synd

rom

e w

ithou

t (c

linic

al)

dem

entia

; DS

+pAD

, Do

wn

synd

rom

e w

ith p

rodr

omal

AD;

DS+

AD,

DS w

ith d

iagn

osed

AD

dem

entia

; DO

PAC,

3-4

-dih

ydro

xyph

enyl

acet

ic a

cid;

HVA

, ho

mov

anill

ic a

cid;

MHP

G,

3-m

etho

xy-4

-hyd

roxy

phen

ylgl

ycol

; NA,

nor

adre

nalin

e; n

.s.,

not s

igni

fican

t.

190

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

191

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 [11C]methylreboxetine (Pietrzak et al., 2013) in relation to amyloid deposition (e.g. [11C]Pittsburgh compound B) may be of utmost importance in this respect.

References

Arai, H., Ichimiya, Y., Kosaka, K., Moroji, T., Iizuka, R., 1992. Neurotransmitter changes in early- and late-onset Alzheimer-type dementia. Prog. Neuropsychopharmacol. Biol. Psychiatry 16, 883–90.

Beacher, F., Daly, E., Simmons, A., Prasher, V.P., Morris, R., Robinson, C., Lovestone, S., Murphy, K., Murphy, D.G.M., 2010. Brain anatomy and ageing in non-demented adults with Down’s syndrome: an in vivo MRI study. Psychol. Med. 40, 611–9.

Blennow, K., Dubois, B., Fagan, A.M., Lewczuk, P., de Leon, M.J., Hampel, H., 2015. Clinical utility of cerebrospinal fluid biomarkers in the diagnosis of early Alzheimer’s disease. Alzheimer’s Dement. 11, 58–69.

Carmona-Iragui, M., Balasa, M., Benejam, B., Alcolea, D.A., Fernandez, S., Videla, L., Sala, I., Sánchez-Saudinós, M.B., Morenas-Rodriguez, E., Ribosa-Nogué, R., Illán-Gala, I., Gonzalez-Ortiz, S., Clarimón, J., Schmitt, F.A., Powell, D.K., Bosch, B., Lladó, A., Rafii, M., Head, E., Molinuevo, J.L., Blesa, R., Videla, S., Lleó, A., Sánchez-Valle, R., Fortea, J., 2017. Cerebral amyloid angiopathy in Down syndrome and sporadic and autosomal-dominant Alzheimer’s disease. Alzheimer’s Dement. 1–10.

Carmona-Iragui, M., Santos, T., Videla, S., Fernandez, S., Benejam, B., Videla, L., Alcolea, D.A., Blennow, K., Blesa, R., Lleó, A., Fortea, J., 2016. Feasibility of Lumbar Puncture in the Study of Cerebrospinal Fluid Biomarkers for Alzheimer’s Disease in Subjects with Down Syndrome. J. Alzheimer’s Dis.

Cirrito, J.R., Disabato, B.M., Restivo, J.L., Verges, D.K., Goebel, W.D., Sathyan, A., Hayreh, D., D’Angelo, G., Benzinger, T., Yoon, H., Kim, J., Morris, J.C., Mintun, M.A., Sheline, Y.I., 2011. Serotonin signaling is associated with lower amyloid-β levels and plaques in transgenic mice and humans. Proc. Natl. Acad. Sci. U. S. A. 108, 14968–73.

Coppus, A.M.W., Fekkes, D., Verhoeven, W.M., Tuinier, S., Egger, J.I., van Duijn, C.M., 2007. Plasma amino acids and neopterin in healthy persons with Down’s syndrome. J. Neural Transm. 114, 1041–1045.

Dekker, A.D., Coppus, A.M.W., Vermeiren, Y., Aerts, T., van Duijn, C.M., Kremer, B.P., Naudé, P.J.W., Van Dam, D., De Deyn, P.P., 2015a. Serum MHPG strongly predicts conversion to Alzheimer’s disease in behaviorally characterized subjects with Down syndrome. J. Alzheimer’s Dis. 43, 871–891.

Dekker, A.D., Fortea, J., Blesa, R., De Deyn, P.P., 2017. Cerebrospinal fluid biomarkers for Alzheimer’s disease in Down syndrome. Alzheimer’s Dement. Diagnosis, Assess. Dis. Monit. 8, 1–10.

Dekker, A.D., Strydom, A., Coppus, A.M.W., Nizetic, D., Vermeiren, Y., Naudé, P.J.W., Van Dam, D., Potier, M.-C., Fortea, J., De Deyn, P.P., 2015b. Behavioural and psychological symptoms of dementia in Down syndrome: Early indicators of clinical Alzheimer’s disease? Cortex 73, 36–61.

Fortea, J., Carmona-Iragui, M., Fernandez, S., Benejam, B., Videla, L., Alcolea, D.A., Vilaplana, E., Clarimón, J., Videla, S., Blesa, R., Lleo, A., 2016. Down Alzheimer Barcelona Neuroimaging Initiative (DABNI): A Prospective Longitudinal Biomarker Cohort to Study Alzheimer’s Disease in Down Syndrome. Alzheimer’s Dement. J. Alzheimer’s Assoc. 12, P380–P381.

German, D.C., Manaye, K.F., White, C.L., Woodward, D.J., McIntire, D.D., Smith, W.K., Kalaria, R.N., Mann, D.M., 1992. Disease-specific patterns of locus coeruleus cell loss. Ann. Neurol. 32, 667–76.

Gibb, W.R., Mountjoy, C.Q., Mann, D.M., Lees, A.J., 1989. The substantia nigra and ventral tegmental area in Alzheimer’s disease and Down’s syndrome. J. Neurol. Neurosurg. Psychiatry 52, 193–200.

Godridge, H., Reynolds, G.P., Czudek, C., Calcutt, N.A., Benton, M., 1987. Alzheimer-like neurotransmitter deficits in adult Down’s syndrome brain tissue. J. Neurol. Neurosurg. Psychiatry 50, 775–778.

Hampel, H., Lista, S., Khachaturian, Z.S., 2012. Development of biomarkers to chart all Alzheimer’s disease stages: the royal road to cutting the therapeutic Gordian Knot. Alzheimer’s Dement. 8, 312–36.

Hartley, D., Blumenthal, T., Carrillo, M.C., DiPaolo, G., Esralew, L., Gardiner, K.J., Granholm, A.C., Iqbal, K., Krams, M., Lemere, C. a, Lott, I.T., Mobley, W.C., Ness, S., Nixon, R., Potter, H., Reeves, R.H., Sabbagh, M., Silverman, W.P., Tycko, B., Whitten, M., Wisniewski, T.M., 2015. Down syndrome and Alzheimer’s disease: Common pathways, common goals. Alzheimer’s Dement. 11, 700–709.

Kay, A.D., Schapiro, M.B., Riker, A.K., Haxby, J. V, Rapoport, S.I., Cutler, N.R., 1987. Cerebrospinal fluid monoaminergic metabolites are elevated in adults with Down’s syndrome. Ann. Neurol. 21, 408–411.

Krinsky-McHale, S.J., Devenny, D.A., Gu, H., Jenkins, E.C., Kittler, P., Murty, V. V, Schupf, N., Scotto, L., Tycko, B., Urv, T.K., Ye, L., Zigman, W.B., Silverman, W.P., 2008. Successful aging in a 70-year-old man with down syndrome: a case study. Intellect. Dev. Disabil. 46, 215–28.

Le Bastard, N., De Deyn, P.P., Engelborghs, S., 2015. Importance and impact of preanalytical variables on Alzheimer disease biomarker concentrations in cerebrospinal fluid. Clin. Chem. 61, 734–43.

Lemere, C.A., Blusztajn, J.K., Yamaguchi, H., Wisniewski, T.M., Saido, T.C., Selkoe, D.J., 1996. Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation. Neurobiol. Dis. 3, 16–32.

Lyness, S.A., Zarow, C., Chui, H.C., 2003. Neuron loss in key cholinergic and aminergic nuclei in Alzheimer disease: a meta-analysis. Neurobiol. Aging 24, 1–23.

Mann, D.M.A., 1988. Alzheimer’s disease and Down’s syndrome. Histopathology 13, 125–37.

192

Mann, D.M.A., Yates, P.O., Marcyniuk, B., 1987a. Dopaminergic neurotransmitter systems in Alzheimer’s disease and in Down’s syndrome at middle age. J. Neurol. Neurosurg. Psychiatry 50, 341–344.

Mann, D.M.A., Yates, P.O., Marcyniuk, B., Ravindra, C.R., 1987b. Loss of neurones from cortical and subcortical areas in Down’s syndrome patients at middle age. Quantitative comparisons with younger Down’s patients and patients with Alzheimer’s disease. J. Neurol. Sci. 80, 79–89.

Mann, D.M.A., Yates, P.O., Marcyniuk, B., Ravindra, C.R., 1985. Pathological evidence for neurotransmitter deficits in Down’s syndrome of middle age. J. Ment. Defic. Res. 29 (Pt 2), 125–135.

Marcyniuk, B., Mann, D.M.A., Yates, P.O., Ravindra, C.R., 1988. Topography of nerve cell loss from the locus caeruleus in middle aged persons with Down’s syndrome. J. Neurol. Sci. 83, 15–24.

Mattsson, N., Andreasson, U., Persson, S., Arai, H., Batish, S.D., Bernardini, S., Bocchio-Chiavetto, L., Blankenstein, M.A., Carrillo, M.C., Chalbot, S., Coart, E., Chiasserini, D., Cutler, N., Dahlfors, G., Duller, S., Fagan, A.M., Forlenza, O., Frisoni, G.B., Galasko, D., Galimberti, D., Hampel, H., Handberg, A., Heneka, M.T., Herskovits, A.Z., Herukka, S.-K., Holtzman, D.M., Humpel, C., Hyman, B.T., Iqbal, K., Jucker, M., Kaeser, S.A., Kaiser, E., Kapaki, E., Kidd, D., Klivenyi, P., Knudsen, C.S., Kummer, M.P., Lui, J., Lladó, A., Lewczuk, P., Li, Q.-X., Martins, R., Masters, C., McAuliffe, J., Mercken, M., Moghekar, A., Molinuevo, J.L., Montine, T.J., Nowatzke, W., O’Brien, R., Otto, M., Paraskevas, G.P., Parnetti, L., Petersen, R.C., Prvulovic, D., de Reus, H.P.M., Rissman, R.A., Scarpini, E., Stefani, A., Soininen, H., Schröder, J., Shaw, L.M., Skinningsrud, A., Skrogstad, B., Spreer, A., Talib, L., Teunissen, C.E., Trojanowski, J.Q., Tumani, H., Umek, R.M., Van Broeck, B., Vanderstichele, H., Vecsei, L., Verbeek, M.M., Windisch, M., Zhang, J., Zetterberg, H., Blennow, K., 2011. The Alzheimer’s Association external quality control program for cerebrospinal fluid biomarkers. Alzheimer’s Dement. 7, 386–395.e6.

Montine, T.J., Phelps, C.H., Beach, T.G., Bigio, E.H., Cairns, N.J., Dickson, D.W., Duyckaerts, C., Frosch, M.P., Masliah, E., Mirra, S.S., Nelson, P.T., Schneider, J.A., Thal, D.R., Trojanowski, J.Q., Vinters, H. V, Hyman, B.T., 2012. National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: a practical approach. Acta Neuropathol. 123, 1–11.

O’Bryant, S.E., Gupta, V., Henriksen, K., Edwards, M., Jeromin, A., Lista, S., Bazenet, C., Soares, H., Lovestone, S., Hampel, H., Montine, T., Blennow, K., Foroud, T., Carrillo, M.C., Graff-Radford, N., Laske, C., Breteler, M., Shaw, L.M., Trojanowski, J.Q., Schupf, N., Rissman, R.A., Fagan, A.M., Oberoi, P., Umek, R., Weiner, M.W., Grammas, P., Posner, H., Martins, R., 2015. Guidelines for the standardization of preanalytic variables for blood-based biomarker studies in Alzheimer’s disease research. Alzheimer’s Dement. 11, 549–60.

Phillips, C., Fahimi, A., Das, D., Mojabi, F.S., Ponnusamy, R., Salehi, A., 2016. Noradrenergic System in Down Syndrome and Alzheimer’s Disease: A Target for Therapy. Curr. Alzheimer Res. 68–83.

Pietrzak, R.H., Gallezot, J.-D., Ding, Y.-S., Henry, S., Potenza, M.N., Southwick, S.M., Krystal, J.H., Carson, R.E., Neumeister, A., 2013. Association of posttraumatic stress disorder with reduced in vivo norepinephrine transporter availability in the locus coeruleus. JAMA psychiatry 70, 1199–205.

Reynolds, G.P., Godridge, H., 1985. Alzheimer-like brain monoamine deficits in adults with Down’s syndrome. Lancet 2, 1368–1369.

Risser, D., Lubec, G., Cairns, N., Herrera-Marschitz, M., 1997. Excitatory amino acids and monoamines in parahippocampal gyrus and frontal cortical pole of adults with Down syndrome. Life Sci. 60, 1231–1237.

Schapiro, M.B., Kay, A.D., May, C., Ryker, A.K., Haxby, J. V, Kaufman, S., Milstien, S., Rapoport, S.I., 1987. Cerebrospinal fluid monoamines in Down’s syndrome adults at different ages. J. Ment. Defic. Res. 31 (Pt 3), 259–269.

Seidl, R., Kaehler, S.T., Prast, H., Singewald, N., Cairns, N., Gratzer, M., Lubec, G., 1999. Serotonin (5-HT) in brains of adult patients with Down syndrome. J. neural Transm. 57, 221–232.

Simic, G., Babić Leko, M., Wray, S., Harrington, C.R., Delalle, I., Jovanov-Milošević, N., Bazadona, D., Buee, L., de Silva, R., Di Giovanni, G., Wischik, C.M., Hof, P.R., 2017. Monoaminergic neuropathology in Alzheimer’s disease. Prog. Neurobiol. 151, 101–138.

Storga, D., Vrecko, K., Birkmayer, J.G., Reibnegger, G., 1996. Monoaminergic neurotransmitters, their precursors and metabolites in brains of Alzheimer patients. Neurosci. Lett. 203, 29–32.

Teipel, S.J., Hampel, H., 2006. Neuroanatomy of Down syndrome in vivo: A model of preclinical Alzheimer’s disease. Behav. Genet. 36, 405–415.

Trillo, L., Das, D., Hsieh, W., Medina, B., Moghadam, S., Lin, B., Dang, V., Sanchez, M.M., De Miguel, Z., Ashford, J.W., Salehi, A., 2013. Ascending monoaminergic systems alterations in Alzheimer’s disease. translating basic science into clinical care. Neurosci. Biobehav. Rev. 37, 1363–1379.

Van Dam, D., Vermeiren, Y., Aerts, T., De Deyn, P.P., 2014. Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical detection for the simultaneous and fast determination of eight biogenic amines and metabolites in human brain tissue. J. Chromatogr. 1353, 28–39.

Vermeiren, Y., Janssens, J., Aerts, T., Martin, J.-J., Sieben, A., Van Dam, D., De Deyn, P.P., 2016. Brain serotonergic and noradrenergic deficiencies in behavioral variant frontotemporal dementia compared to early-onset Alzheimer’s disease. J. Alzheimer’s Dis. 53, 1079–1096.

Vermeiren, Y., Van Dam, D., Aerts, T., Engelborghs, S., De Deyn, P.P., 2014a. Monoaminergic neurotransmitter alterations in postmortem brain regions of depressed and aggressive patients with Alzheimer’s disease. Neurobiol. Aging 35, 2691–2700.

Vermeiren, Y., Van Dam, D., Aerts, T., Engelborghs, S., De Deyn, P.P., 2014b. Brain region-specific monoaminergic correlates of neuropsychiatric symptoms in Alzheimer’s disease. J. Alzheimer’s Dis. 41, 819–33.

Vermeiren, Y., Van Dam, D., Aerts, T., Engelborghs, S., Martin, J.-J., De Deyn, P.P., 2015. The monoaminergic footprint of depression and psychosis in dementia with Lewy bodies compared to Alzheimer’s disease. Alzheimers. Res. Ther. 7, 1–18.

Whittle, N., Sartori, S.B., Dierssen, M., Lubec, G., Singewald, N., 2007. Fetal Down syndrome brains exhibit aberrant levels of

193

neurotransmitters critical for normal brain development. Pediatrics 120, e1465-71. Wiseman, F.K., Al-Janabi, T., Hardy, J., Karmiloff-Smith, A., Nizetic, D., Tybulewicz, V.L.J., Fisher, E.M., Strydom, A., 2015. A genetic

cause of Alzheimer disease: mechanistic insights from Down syndrome. Nat. Rev. Neurosci. 16, 564–74. Wisniewski, K.E., 1990. Down syndrome children often have brain with maturation delay, retardation of growth, and cortical

dysgenesis. Am. J. Med. Genet. Suppl. 7, 274–81. Yates, C.M., Ritchie, I.M., Simpson, J., Maloney, A.F., Gordon, A., 1981. Noradrenaline in Alzheimer-type dementia and Down

syndrome. Lancet 2, 39–40. Yates, C.M., Simpson, J., Gordon, A., Maloney, A.F.J., Allison, Y., Ritchie, I.M., Urquhart, A., 1983. Catecholamines and cholinergic

enzymes in pre-senile and senile Alzheimer-type dementia and Down’s syndrome. Brain Res. 280, 119–126. Zarow, C., Lyness, S.A., Mortimer, J.A., Chui, H.C., 2003. Neuronal loss is greater in the locus coeruleus than nucleus basalis and

substantia nigra in Alzheimer and Parkinson diseases. Arch. Neurol. 60, 337–341. Zigman, W.B., Lott, I.T., 2007. Alzheimer’s disease in Down syndrome: neurobiology and risk. Ment. Retard. Dev. Disabil. Res. Rev.

13, 237–246.

194

Supp

lem

enta

ry m

ater

ial,

Tabl

e S1

: (N

or)a

dren

ergi

c sy

stem

- co

mpa

rison

of p

ost-

mor

tem

con

cent

ratio

ns a

nd ra

tios b

etw

een

the

grou

ps

Brai

n re

gion

Co

mpo

und/

ratio

N

D

S (n

=4)

DS+

AD (n

=17)

EO

AD (n

=11)

Co

ntro

ls (n

=10)

P-

valu

e Neocortex

BA7

su

perio

r pa

rieta

l lob

ule

NA

2,13

,11,

10

33.8

(14.

5–)

17.3

(15.

4–31

.9)

22.9

(12.

2–45

.6)

28.0

(23.

0–46

.7)

n.s.

Ad

rena

line

0,4,

0,4

– 27

.2 (1

1.3–

201.

8)

– 20

.4 (6

.6–2

8.0)

n.

s.

MHP

G

2,13

,11,

10

71.0

(63.

3–)

72.3

(57.

0–10

5.2)

*

119.

4 (1

06.0

–189

.5) *

25

9.7

(119

.8–3

54.5

) <0

.001

M

HPG

:NA

2,13

,11,

10

3.3

(1.2

–)

3.7

(2.1

–7.5

) 4.

6 (3

.4–9

.5)

8.3

(2.9

–14.

1)

n.s.

BA9/

10/4

6

pref

ront

al

cort

ex

NA

4,14

,11,

10

19.0

(7.7

–29.

9)

19.3

(8.7

–35.

4)

5.5

(3.7

–21.

4)

23.2

(18.

7–32

.8)

(0.0

48)

Adre

nalin

e 2,

8,5,

6 11

6.0

(15.

5–)

16.3

(10.

5–16

5.0)

8.

2 (6

.0–1

1.8)

12

.1 (8

.0–3

6.8)

n.

s.

MHP

G

4,14

,11,

10

84.2

(77.

7–86

.9)

107.

4 (6

3.4–

133.

8) *

* 47

1.2

(284

.3–6

55.1

) **

265.

7 (1

32.4

–629

.6)

<0.0

01

MHP

G:N

A 4,

14,1

1,10

8.

9 (3

.2–1

5.3)

6.

0 (2

.3–1

2.4)

**

77.8

(11.

7–20

3.3)

**

9.7

(4.7

–32.

7)

0.00

4

BA17

oc

cipi

tal p

ole

(V1)

NA

3,7,

11,1

0 23

.2 (1

6.8–

) 13

.4 (6

.5–3

8.8)

26

.1 (2

0.3–

74.2

) 32

.8 (2

2.7–

48.0

) n.

s.

Adre

nalin

e 1,

2,2,

4 24

5.7

146.

8 (2

3.2–

) 2.

9 (2

.1–)

32

.2 (2

0.8–

46.3

) n.

s.

MHP

G

3,7,

11,1

0 77

.2 (6

1.3–

) # 89

.2 (7

3.6–

114.

6)

127.

9 (1

01.6

–182

.0)

285.

3 (2

44.0

–530

.1) #

<0.0

01

MHP

G:N

A 3,

7,11

,10

3.6

(1.9

–)

7.3

(3.5

–11.

3)

4.9

(1.6

–10.

4)

11.1

(4.5

–20.

6)

n.s.

BA22

su

perio

r te

mpo

ral g

yrus

NA

3,10

,9,1

0 30

.2 (1

8.3–

) 17

.3 (1

2.5–

27.4

) 10

.0 (7

.4–1

2.4)

18

.7 (1

3.6–

28.5

) 0.

014

Adre

nalin

e 1,

4,2,

5 21

9.4

32.8

(10.

7–20

2.9)

14

.1 (7

.8–)

15

.5 (9

.6–1

8.6)

n.

s.

MHP

G

3,10

,11,

10

107.

3 (8

0.4–

) # 87

.0 (6

0.0–

125.

4) *

* 52

0.7

(313

.4–6

73.4

) **

360.

5 (2

60.4

–630

.7) #

<0.0

01

MHP

G: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

.8–4

6.9)

<0

.001

Limbic system

Amyg

dala

NA

2,10

,10,

10

21.8

(19.

5–)

25.3

(13.

6–39

.8) *

59

.0 (4

6.9–

78.5

) *

84.5

(77.

5–12

1.8)

<0

.001

Ad

rena

line

1,10

,3,5

25

6.0

56

.6 (3

5.9–

80.9

) 17

.1 (1

4.3–

) 20

.4 (1

0.8–

42.5

) (0

.031

) M

HPG

2,

10,1

0,10

68

.8 (5

8.2–

) 70

.9 (6

2.5–

94.9

) **

429.

8 (1

80.0

–964

.3) *

* 30

4.8

(193

.5–7

59.3

) <0

.001

M

HPG

:NA

2,10

,10,

10

3.2

(2.4

–)

3.0

(1.8

–6.2

) 6.

4 (3

.4–2

2.7)

3.

0 (1

.4–9

.9)

n.s.

Hip

poca

mpu

s

NA

3,5,

10,1

0 21

.9 (2

1.2–

) 32

.6 (1

8.3–

42.5

) 25

.8 (1

9.0–

44.7

) 43

.0 (2

5.8–

81.9

) n.

s.

Adre

nalin

e 2,

5,3,

5 39

1.6

(236

.5–)

42

.3 (2

0.6–

139.

0)

6.4

(2.6

–)

10.1

(6.4

–14.

1)

0.01

1 M

HPG

3,

5,11

,10

75.9

(70.

3–) #

97.2

(76.

9–12

4.8)

**

459.

5 (1

93.2

–109

9.2)

**

416.

3 (2

32.9

–713

.5) #

<0.0

01

MHP

G: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

.3–3

1.4)

(0

.040

)

BA11

/12

orbi

tofr

onta

l co

rtex

NA

4,6,

11,1

0 23

.2 (6

.3–4

5.4)

15

.6 (9

.7–4

2.9)

9.

6 (6

.4–2

7.4)

20

.5 (1

3.9–

28.4

) n.

s.

Adre

nalin

e 2,

3,5,

6 11

8.4

(14.

8–)

12.1

(6.7

–)

4.2

(2.4

–9.8

) 9.

3 (6

.6–1

3.3)

n.

s.

MHP

G

4,6,

11,1

0 91

.8 (7

0.6–

105.

7) #

103.

1 (6

3.9–

111.

2) *

* 43

7.1

(352

.6–5

45.6

) **

361.

7 (2

16.5

–636

.3) #

<0.0

01

MHP

G:N

A 4,

6,11

,10

5.4

(2.1

–14.

4)

5.3

(3.5

–10.

8) *

50

.6 (2

7.7–

73.3

) *

18.9

(11.

5–37

.4)

0.00

6

Cing

ulat

e gy

rus

NA

3,8,

10,1

0 50

.9 (5

.9–)

31

.2 (2

7.7–

45.0

) 16

.6 (1

0.9–

27.7

) 33

.5 (2

6.4–

43.4

) n.

s.

Adre

nalin

e 0,

4,4,

4 –

68.3

(31.

7–78

.1)

12.8

(7.1

–34.

8)

18.0

(7.8

–51.

2)

n.s.

M

HPG

3,

8,11

,10

128.

0 (5

8.8–

) 12

6.0

(104

.6–1

53.6

) **

567.

8 (2

58.4

–739

.3) *

* 33

6.9

(158

.1–5

87.5

) <0

.001

M

HPG

:NA

3,8,

10,1

0 3.

4 (2

.0–)

3.

6 (2

.0–5

.2) *

* 30

.6 (1

2.0–

54.1

) **

9.3

(3.4

–21.

9)

0.00

3

Thal

amus

NA

3,11

,10,

10

48.8

(38.

3–)

79.5

(50.

6–18

3.1)

12

4.3

(110

.6–1

64.6

) 19

2.8

(145

.5–2

21.3

) n.

s.

Adre

nalin

e 1,

5,5,

5 26

9.6

7.1

(6.2

–10.

6)

7.0

(4.9

–12.

5)

9.5

(7.4

–12.

6)

n.s.

M

HPG

3,

11,1

1,10

15

9.2

(131

.3–)

# 14

8.3

(110

.2–1

77.8

) **

793.

0 (6

28.6

–144

2.6)

**

441.

9 (2

44.1

–134

5.4)

# <0

.001

M

HPG

:NA

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

195

Tabl

e S1

(con

tinue

d)

Br

ain

regi

on

Com

poun

d/ra

tio

N

DS

(n=4

) D

S+AD

(n=1

7)

EOAD

(n=1

1)

Cont

rols

(n=1

0)

P-va

lue

Basal ganglia

Caud

ate

nucl

eus

NA

3,16

,11,

10

58.9

(24.

2–)

31.7

(21.

7–47

.8)

21.9

(12.

4–83

.9)

31.0

(18.

2–45

.5)

n.s.

Ad

rena

line

2,11

,7,6

53

3.3

(355

.2–)

69

.3 (4

2.7–

151.

0) *

26

8.5

(176

.5–5

87.7

) *

372.

9 (1

85.7

–743

.0)

0.00

6 M

HPG

3,

16,1

1,10

64

.8 (6

1.9–

) 77

.7 (5

6.5–

97.3

) 11

6.7

(82.

2–14

9.8)

91

.8 (6

4.8–

171.

5)

n.s.

M

HPG

:NA

3,16

,11,

10

1.7

(1.1

–)

2.8

(1.4

–5.0

) 4.

0 (1

.3–1

0.2)

3.

2 (2

.1–5

.9)

n.s.

Glo

bus p

allid

us

NA

2,8,

11,1

0 26

.6 (2

2.4–

) 31

.8 (2

7.5–

93.3

) 66

.0 (4

5.3–

103.

8)

40.0

(28.

7–14

7.1)

n.

s.

Adre

nalin

e 1,

3,10

,9

268.

1 34

.0 (3

2.8–

) 18

7.5

(18.

4–10

53.8

) 22

2.3

(24.

3–64

0.7)

n.

s.

MHP

G

2,8,

11,1

0 84

.6 (7

8.3–

) 10

5.8

(92.

2–13

5.1)

76

.2 (5

7.1–

111.

4)

46.9

(35.

9–10

8.4)

n.

s.

MHP

G:N

A 2,

8,11

,10

3.2

(2.9

–)

2.7

(1.5

–4.2

) 1.

3 (0

.8–2

.5)

1.0

(0.4

–2.1

) n.

s.

Puta

men

NA

3,15

,11,

10

38.6

(32.

4–)

38.3

(26.

3–49

.8)

35.8

(24.

3–61

.2)

47.0

(36.

9–80

.7)

n.s.

Ad

rena

line

2,8,

11,1

0 39

0.4

(325

.6)

135.

0 (4

0.9–

219.

9) *

58

1.9

(187

.2–1

523.

4) *

33

9.6

(100

.7–7

97.8

) (0

.038

) M

HPG

3,

15,1

1,10

15

3.1

(97.

2–)

114.

7 (8

1.6–

156.

4)

94.8

(72.

3–13

2.1)

86

.7 (4

1.4–

139.

4)

n.s.

M

HPG

:NA

3,15

,11,

10

2.7

(2.5

–)

3.4

(2.0

–4.3

) 2.

6 (1

.5–4

.5)

1.4

(0.8

–2.8

) n.

s.

Subs

tant

ia

nigr

a

NA

2,14

,11,

10

28.7

(25.

1–)

96.8

(56.

0–15

7.1)

10

1.8

(67.

6–14

7.6)

10

5.0

(73.

4–16

8.8)

n.

s.

Adre

nalin

e 0,

2,6,

4 –

14.4

(6.3

–)

470.

8 (1

6.7–

1058

.4)

17.9

(8.8

–95.

9)

n.s.

M

HPG

2,

14,1

1,10

67

.6 (5

9.5–

) 10

4.2

(87.

5–13

8.7)

10

9.0

(74.

7–19

5.8)

15

0.5

(87.

7–20

7.5)

n.

s.

MHP

G:N

A 2,

14,1

1,10

2.

4 (2

.3–)

1.

5 (0

.6–2

.0)

1.2

(0.7

–1.9

) 1.

4 (0

.6–2

.4)

n.s.

Metencephalon

Locu

s co

erul

eus

NA

0,10

,10,

10

–

88.6

(52.

7–11

.4) *

* 25

5.1

(171

.9–4

00.7

) **

347.

3 (2

48.6

–501

.8)

<0.0

01

Adre

nalin

e 0,

0,6,

5 –

– 10

.5 (7

.0–1

8.7)

16

.5 (1

2.6–

38.8

) n.

s.

MHP

G

0,10

,10,

10

– 20

1.8

(151

.8–2

59.5

) *

429.

6 (3

04.6

–530

.7) *

57

2.4

(158

.9–8

36.2

) (0

.032

) M

HPG

:NA

0,10

,10,

10

– 2.

5 (1

.8–3

.6)

1.4

(1.2

–2.2

) 1.

1 (0

.5–2

.4)

(0.0

43)

Cere

bellu

m

NA

2,9,

11,1

0 50

.4 (3

1.5–

) 39

.4 (3

2.2–

69.8

) 22

.5 (1

4.4–

30.6

) 43

.3 (2

4.7–

65.0

) n.

s.

Adre

nalin

e 0,

7,4,

6 –

36.4

(34.

7–44

.5) *

6.

6 (3

.6–1

2.7)

*

21.4

(10.

3–50

.2)

(0.0

17)

MHP

G

2,9,

11,1

0 12

4.6

(100

.3–)

10

1.1

(85.

2–14

8.6)

*

421.

1 (2

32.5

–708

.2) *

51

8.1

(386

.8–7

13.4

) 0.

001

MHP

G: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

.8–1

8.4)

0.

004

Mon

oam

ines

and

met

abol

ites

(ng/

g tis

sue)

and

the

cor

resp

ondi

ng r

atio

s ar

e ro

unde

d of

f to

one

deci

mal

and

exp

ress

ed a

s m

edia

n (5

0%) w

ith t

he in

terq

uart

ile r

ange

(25%

-75%

) bet

wee

n br

acke

ts.

A Kr

uska

ll-W

allis

test

was

use

d to

com

pare

the

four

gro

ups.

Sig

nific

ant P

-val

ues

(<0.

015)

are

pro

vide

d. T

hose

in it

alic

s be

twee

n br

acke

ts a

re n

o lo

nger

rega

rded

sig

nific

ant a

fter

cor

rect

ion

(P<0

.015

). Po

st-h

oc M

ann-

Whi

tney

U te

sts:

DS

vs D

S+AD

, DS

vs c

ontr

ols

(#<0

.015

) and

DS+

AD v

s EO

AD (*

<0.0

15; *

*<0.

001)

. Abb

revi

atio

ns: B

A, B

rodm

ann

area

; DS,

Dow

n sy

ndro

me

with

out n

euro

path

olog

ic A

D

diag

nosis

; DS+

AD, D

own

synd

rom

e w

ith n

euro

path

olog

ic A

D di

agno

sis; E

OAD

, ear

ly-o

nset

Alz

heim

er’s

dise

ase;

MHP

G, 3

-met

hoxy

-4-h

ydro

xyph

enyl

glyc

ol; N

A, n

orad

rena

line;

n.s

., no

t sig

nific

ant.

196

Supp

lem

enta

ry m

ater

ial,

Tabl

e S2

: Dop

amin

ergi

c sy

stem

- co

mpa

rison

of p

ost-

mor

tem

con

cent

ratio

ns a

nd ra

tios b

etw

een

the

grou

ps

Brai

n re

gion

Co

mpo

und/

ratio

N

D

S (n

=4)

DS+

AD (n

=17)

EO

AD (n

=11)

Co

ntro

ls (n

=10)

P-

valu

e Neocortex

BA7

su

perio

r pa

rieta

l lob

ule

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.

004

DOPA

C 2,

13,1

1,10

26

.2 (2

4.1–

) 14

.6 (8

.7–2

4.2)

8.

3 (5

.9–1

6.0)

4.

3 (3

.4–1

0.4)

(0

.015

) HV

A 2,

13,1

1,10

94

.2 (8

8.0–

) 11

3.8

(95.

1–11

9.8)

13

5.4

(105

.2–1

41.8

) 11

7.8

(79.

8–14

9.1)

n.

s.

DOPA

C:DA

2,

12,1

1,10

2.

8 (1

.8–)

0.

7 (0

.5–0

.9)

0.7

(0.5

–1.5

) 0.

8 (0

.3–2

.4)

n.s.

HV

A:DA

2,

12,1

1,10

10

.0 (6

.4–)

6.

1 (3

.9–8

.7) *

11

.5 (8

.3–1

7.8)

*

17.3

(10.

5–29

.8)

0.00

5

BA9/

10/4

6

pref

ront

al

cort

ex

DA

4,14

,11,

10

127.

5 (1

1.4–

497.

4)

373.

9 (2

04.3

–743

.6) *

* 7.

2 (2

.5–9

.3) *

* 7.

5 (4

.0–1

1.3)

<0

.001

DO

PAC

4,14

,11,

10

7.0

(2.4

–24.

8)

14.2

(8.9

–22.

7)

8.5

(7.2

–12.

5)

6.6

(4.8

–12.

1)

n.s.

HV

A 4,

14,1

1,10

10

6.8

(86.

0–12

7.6)

13

6.2

(89.

7–17

8.9)

12

0.6

(98.

9–14

3.7)

14

9.4

(85.

2–19

9.4)

n.

s.

DOPA

C:DA

4,

14,1

1,10

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

.001

HV

A:DA

4,

14,1

1,10

11

.7 (5

.1–1

9.2)

4.

7 (0

.5–8

.2) *

* 18

.7 (1

3.2–

35.8

) **

24.8

(10.

7–79

.3)

<0.0

01

BA17

oc

cipi

tal p

ole

(V1)

DA

3,7,

11,1

0 5.

4 (4

.5–)

6.

1 (4

.5–1

9.4)

3.

0 (1

.6–1

2.3)

2.

2 (0

.9–4

.4)

n.s.

DO

PAC

3,7,

11,1

0 12

.6 (4

.7–)

6.

1 (3

.0–6

.8)

5.1

(2.6

–7.8

) 6.

9 (4

.4–1

2.2)

n.

s.

HVA

3,7,

11,1

0 92

.4 (7

0.7–

) 99

.7 (8

4.5–

136.

1)

72.9

(26.

8–10

3.5)

61

.0 (3

8.7–

95.8

) n.

s.

DOPA

C:DA

3,

7,11

,10

2.8

(0.3

–)

0.6

(0.3

–6.1

) 1.

2 (0

.3–1

.8)

2.9

(1.2

–24.

9)

n.s.

HV

A:DA

3,

7,11

,10

13.0

(10.

7–)

16.9

(7.2

–30.

4)

9.0

(2.8

–32.

9)

51.0

(13.

2–76

.5)

n.s.

BA22

su

perio

r te

mpo

ral g

yrus

DA

3,10

,11,

10

6.7

(6.5

–)

11.3

(8.1

–25.

2) *

4.

2 (3

.2–6

.8) *

9.

1 (5

.4–1

8.4)

(0

.041

) DO

PAC

3,10

,10,

10

11.2

(5.2

–)

5.0

(3.3

–17.

1)

10.9

(9.4

–14.

2)

7.7

(6.2

–11.

0)

n.s.

HV

A 3,

10,1

1,10

16

0.0

(113

.6–)

14

3.4

(102

.4–1

82.8

) 13

7.0

(115

.8–1

76.4

) 14

9.6

(109

.9–2

16.6

) n.

s.

DOPA

C:DA

3,

10,1

0,10

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:DA

3,

10,1

1,10

17

.5 (1

1.2–

) 9.

8 (5

.8–1

9.2)

*

32.4

(19.

5–44

.5) *

18

.3 (1

3.9–

25.7

) 0.

014

Limbic system

Amyg

dala

DA

2,10

,10,

10

20.3

(19.

4–)

21.2

(10.

1–52

.9)

70.8

(31.

7–99

.2)

56.5

(40.

2–10

4.0)

(0

.048

) DO

PAC

2,10

,10,

10

19.2

(6.4

–)

20.3

(9.3

–32.

8)

21.0

(16.

8–36

.9)

18.3

(9.5

–24.

8)

n.s.

HV

A 2,

10,1

0,10

26

5.8

(174

.5–)

37

6.2

(258

.9–6

17.2

) 59

9.1

(398

.7–8

66.2

) 11

32.5

(751

.0–1

421.

4)

0.00

5 DO

PAC:

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.

008

HVA:

DA

2,10

,10,

10

12.9

(9.0

–)

18.4

(14.

5–31

.9)

10.1

(7.3

–17.

1)

16.3

(12.

0–29

.4)

n.s.

Hip

poca

mpu

s

DA

3,5,

11,1

0 12

.0 (1

0.3–

) 22

.1 (1

6.5–

37.3

) 5.

8 (4

.8–1

8.7)

20

.3 (7

.2–2

9.2)

n.

s.

DOPA

C 3,

5,11

,10

10.5

(4.5

–)

12.8

(5.7

–17.

7)

9.2

(6.3

–15.

8)

8.0

(5.6

–13.

8)

n.s.

HV

A 3,

5,11

,10

180.

2 (1

38.3

–)

149.

5 (1

20.5

–270

.6)

206.

5 (1

41.9

–315

.0)

268.

3 (1

64.7

–482

.1)

n.s.

DO

PAC:

DA

3,5,

11,1

0 0.

9 (0

.4–)

0.

5 (0

.3–1

.1)

1.2

(0.4

–2.3

) 0.

6 (0

.3–1

.1)

n.s.

HV

A:DA

3,

5,11

,10

13.5

(11.

6–)

10.6

(5.3

–14.

8)

33.0

(15.

8–36

.8)

18.5

(15.

3–27

.0)

(0.0

19)

BA11

/12

orbi

tofr

onta

l co

rtex

DA

4,5,

11,1

0 6.

7 (5

.1–1

1.4)

11

.7 (6

.4–4

6.8)

5.

0 (3

.2–8

.3)

5.8

(3.8

–9.5

) n.

s.

DOPA

C 4,

6,11

,10

7.6

(2.1

–23.

5)

7.0

(3.8

–23.

8)

7.6

(6.1

–9.9

) 8.

7 (5

.0–1

2.2)

n.

s.

HVA

4,6,

11,1

0 17

1.8

(89.

2–20

8.8)

11

3.3

(95.

3–15

3.0)

17

1.3

(121

.7–2

11.7

) 17

0.1

(106

.4–2

32.3

) n.

s.

DOPA

C:DA

4,

5,11

,10

1.3

(0.2

–3.3

) 0.

5 (0

.3–1

.5)

1.9

(1.3

–2.2

) 2.

2 (0

.9–3

.7)

n.s.

HV

A:DA

4,

5,11

,10

26.4

(8.9

–41.

5)

11.1

(4.2

–14.

8) *

* 38

.2 (2

5.8–

53.7

) **

33.4

(26.

7-47

.0)

0.01

3

Cing

ulat

e gy

rus

DA

3,8,

11,1

0 22

4.2

(7.9

–)

55.5

(40.

6–21

1.2)

**

10.5

(3.8

–13.

0) *

* 9.

6 (3

.0–1

7.4)

<0

.001

DO

PAC

3,8,

11,1

0 21

.9 (5

.3–)

18

.4 (5

.7–2

5.3)

12

.9 (6

.8–1

7.2)

7.

6 (4

.9–1

4.0)

n.

s.

HVA

3,8,

11,1

0 12

8.5

(107

.4–)

18

1.6

(84.

5–26

6.0)

21

6.2

(178

.4–2

61.5

) 23

0.4

(174

.9–3

22.5

) n.

s.

DOPA

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

.001

HV

A:DA

3,

8,11

,10

0.7

(0.6

–)

1.6

(1.1

–5.1

) **

23.4

(15.

7–44

.5) *

* 24

.7 (1

7.3–

98.1

) <0

.001

197

Tabl

e S2

(con

tinue

d)

Br

ain

regi

on

Com

poun

d/ra

tio

N

DS

(n=4

) D

S+AD

(n=1

7)

EOAD

(n=1

1)

Cont

rols

(n=1

0)

P-va

lue

Thal

amus

DA

3,11

,11,

10

95.9

(6.2

–)

41.3

(25.

1–74

.7)

14.7

(7.8

–19.

9)

16.0

(8.8

–29.

6)

n.s.

DOPA

C 3,

11,1

1,10

23

.9 (2

.0–)

17

.7 (1

1.1–

33.7

) 15

.0 (9

.3–1

7.8)

14

.1 (8

.9–1

9.3)

n.

s.

HV

A 3,

11,1

1,10

32

4.4

(188

.5–)

33

3.5

(213

.7–4

47.2

) 38

8.1

(336

.4–6

67.1

) 47

8.3

(433

.7–6

43.5

) n.

s.

DO

PAC:

DA

3,11

,11,

10

0.2

(0.2

–)

0.5

(0.1

–1.5

) 1.

1. (0

.7–2

.1)

1.0

(0.5

–1.3

) n.

s.

HV

A:DA

3,

11,1

1,10

17

.4 (1

.9–)

8.

8 (3

.4–1

7.8)

*

39.7

(25.

9–56

.9) *

30

.2 (2

4.7–

50.5

) 0.

010

Basal ganglia

Caud

ate

nucl

eus

DA

3,16

,11,

10

4297

.2 (1

845.

8–)

2395

.2 (1

178.

5–27

84.8

) **

4721

.2 (3

403.

8–69

05.9

) **

3965

.1 (2

987.

2–44

20.5

) 0.

003

DOPA

C 3,

16,1

1,10

22

9.6

(139

.4–)

28

5.6

(245

.1–5

60.3

) 47

4.1

(250

.1–7

47.1

) 28

1.8

(153

.3–8

66.9

) n.

s.

HVA

3,16

,11,

10

2732

.0 (2

231.

3–)

3145

.6 (1

522.

7–37

56.0

) *

4681

.2 (3

714.

5–56

40.3

) *

4372

.3 (3

564.

4–68

18.8

) 0.

014

DOPA

C:DA

3,

16,1

1,10

0.

1 (0

.0–)

0.

2 (0

.1–0

.3)

0.1

(0.1

–0.2

) 0.

1 (0

.1–0

.2)

n.s.

HV

A:DA

3,

16,1

1,10

0.

6 (0

.4–)

1.

3 (1

.0–1

.5)

1.0

(0.6

–1.5

) 1.

5 (1

.0–2

.0)

n.s.

Glo

bus p

allid

us

DA

2,8,

11,1

0 25

98.7

(206

.6–)

45

1.5

(211

.7–1

263.

4)

343.

8 (1

34.4

–137

2.9)

48

3.5

(185

.4–6

65.0

) n.

s.

DOPA

C 2,

8,11

,10

208.

3 (2

8.0–

) 13

1.7

(61.

8–14

1.5)

*

39.5

(19.

5–85

.8) *

20

.1 (1

0.5–

34.7

) 0.

004

HVA

2,8,

11,1

0 59

96.8

(526

1.9–

) 34

72.1

(230

9.5–

5576

.1)

3964

.2 (3

679.

9–43

37.5

) 38

23.6

(347

0.1–

4657

.9)

n.s.

DO

PAC:

DA

2,8,

11,1

0 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 HV

A:DA

2,

8,11

,10

13.4

(1.3

–)

6.7

(3.8

–21.

5)

10.9

(3.3

–21.

0)

8.1

(5.9

–29.

5)

n.s.

Puta

men

DA

3,15

,11,

10

4988

.6 (2

454.

6–)

4077

.4 (2

458.

4–56

73.9

) 60

20.7

(445

6.2–

7864

.4)

5016

.3 (3

818.

4–62

36.3

) n.

s.

DOPA

C 3,

15,1

1,10

20

7.5

(165

.1–)

61

1.6

(274

.7–8

51.6

) 42

1.9

(235

.7–6

25.7

) 20

2.2

(119

.0–2

99.7

) 0.

008

HVA

3,15

,11,

10

6280

.9 (4

930.

2–)

5438

.0 (4

273.

9–83

62.6

) 85

14.1

(736

2.8–

9140

.4)

8194

.4 (5

785.

5–95

92.9

) n.

s.

DOPA

C:DA

3,

15,1

1,10

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 HV

A:DA

3,

15,1

1,10

1.

3 (0

.8–)

1.

6 (1

.0–2

.4)

1.3

(1.1

–2.0

) 1.

6 (1

.2–2

.3)

n.s.

Subs

tant

ia

nigr

a

DA

2,14

,11,

10

164

(126

.8–)

15

7.2

(89.

0–29

2.4)

**

572.

7 (2

84.9

–623

.7) *

* 62

4.0

(295

.7–9

36.8

) <0

.001

DO

PAC

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.

2–10

1.0)

0.

005

HVA

2,14

,11,

10

3061

.7 (2

010.

5–)

2421

.8 (1

995.

7–30

07.8

) **

3799

.4 (3

461.

0–43

85.5

) **

4331

.0 (3

241.

6–53

49.9

) <0

.001

DO

PAC:

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.

001

HVA:

DA

2,14

,11,

10

18.1

(15.

9–)

17.0

(9.6

–23.

4) *

8.

3 (5

.3–1

2.9)

*

8.2

(4.8

–9.7

) 0.

008

Metencephalon

Locu

s co

erul

eus

DA

0,10

,10,

10

– 22

.2 (1

6.9–

38.4

) 28

.6 (2

1.1–

46.7

) 34

.9 (3

0.7–

56.6

) n.

s.

DOPA

C 0,

10,1

0,10

–

12.3

(8.1

–19.

2) *

* 39

.0 (2

3.6–

71.3

) **

55.5

(33.

1–98

.6)

<0.0

01

HVA

0,10

,10,

10

– 73

0.2

(482

.0–1

003.

5)

1009

.7 (9

05.7

–144

3.4)

13

51.0

(119

5.0–

1482

.2)

<0.0

01

DOPA

C:DA

0,

10,1

0,10

–

0.5

(0.3

–0.6

) *

1.1

(0.9

–1.6

) *

1.4

(0.7

–2.0

) 0.

002

HVA:

DA

0,10

,10,

10

– 27

.8 (2

2.3–

32.0

) 34

.3 (2

4.7–

48.8

) 35

.2 (2

4.5–

48.7

) n.

s.

Cere

bellu

m

DA

2,9,

11,1

0 32

.1 (8

.4–)

15

.5 (1

2.2–

22.1

) **

3.6

(1.5

–8.6

) **

3.6

(3.2

–5.4

) <0

.001

DO

PAC

2,9,

11,1

0 35

.1 (6

.4–)

16

.4 (1

1.3–

26.8

) *

8.2

(5.8

–11.

1) *

8.

0 (6

.0–8

.3)

0.00

5 HV

A 2,

9,11

,10

112.

3 (8

2.8–

) 10

6.9

(77.

3–12

2.4)

10

5.6

(62.

5–13

2.7)

80

.4 (6

4.4–

142.

3)

n.s.

DO

PAC:

DA

2,9,

11,1

0 1.

0 (0

.8–)

1.

4 (0

.7–1

.5)

2.3

(1.0

–4.6

) 2.

3 (0

.9–2

.6)

n.s.

HV

A:DA

2,

9,11

,10

6.2

(2.5

–)

6.0

(5.2

–6.9

) **

28.7

(12.

1–44

.9) *

* 28

.5 (1

8.5–

32.8

) <0

.001

M

onoa

min

es a

nd m

etab

olite

s (n

g/g

tissu

e) a

nd t

he c

orre

spon

ding

rat

ios

are

roun

ded

off t

o on

e de

cim

al a

nd e

xpre

ssed

as

med

ian

(50%

) with

the

inte

rqua

rtile

ran

ge (2

5%-7

5%) b

etw

een

brac

kets

. A

Krus

kall-

Wal

lis te

st w

as u

sed

to c

ompa

re th

e fo

ur g

roup

s. S

igni

fican

t P-v

alue

s (<

0.01

5) a

re p

rovi

ded.

Tho

se in

ital

ics

betw

een

brac

kets

are

no

long

er re

gard

ed s

igni

fican

t aft

er c

orre

ctio

n (P

<0.0

15).

Post

-hoc

Man

n-W

hitn

ey U

tes

ts: D

S vs

DS+

AD, D

S vs

con

trol

s (#

<0.0

15) a

nd D

S+AD

vs

EOAD

(*<0

.015

; **<

0.00

1). A

bbre

viat

ions

: BA,

Bro

dman

n ar

ea; D

A, d

opam

ine;

DS,

Dow

n sy

ndro

me

with

out

neur

opat

holo

gic

AD d

iagn

osis;

DS+

AD, D

own

synd

rom

e w

ith n

euro

path

olog

ic A

D di

agno

sis; D

OPA

C, 3

-4-d

ihyd

roxy

phen

ylac

etic

aci

d; E

OAD

, ear

ly-o

nset

Alz

heim

er’s

dise

ase;

HVA

, hom

ovan

illic

aci

d;

n.s.

, not

sign

ifica

nt.

198

Supp

lem

enta

ry m

ater

ial,

Tabl

e S3

: Ser

oton

ergi

c sy

stem

- co

mpa

rison

of p

ost-

mor

tem

con

cent

ratio

ns a

nd ra

tios b

etw

een

the

grou

ps

Brai

n re

gion

Co

mpo

und/

ratio

N

D

S (n

=4)

DS+

AD (n

=17)

EO

AD (n

=11)

Co

ntro

ls (n

=10)

P-

valu

e Neocortex

BA7

su

perio

r pa

rieta

l lob

ule

5-HT

2,

13,1

1,10

16

.7 (1

4.5–

) 10

.1 (6

.6–1

4.5)

5.

2 (3

.9–1

1.0)

10

.5 (4

.9–1

6.5)

n.

s.

5-HI

AA

2,13

,11,

10

92.8

(70.

4–)

40.2

(30.

4–71

.1)

55.3

(40.

5–93

.7)

106.

4 (7

5.3–

158.

9)

0.00

3 5-

HIAA

:5-H

T 2,

13,1

1,10

5.

5 (4

.9–)

5.

5 (2

.6–8

.2)

9.0

(5.1

–27.

5)

16.7

(8.2

–18.

6)

(0.0

17)

HVA:

5-HI

AA

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.

001

BA9/

10/4

6

pref

ront

al

cort

ex

5-HT

4,

14,1

1,10

17

.8 (1

3.2–

30.6

) 28

.7 (1

5.3–

49.8

) *

11.1

(6.1

–15.

0) *

11

.7 (7

.4–1

6.7)

0.

009

5-HI

AA

4,14

,11,

10

81.1

(64.

6–12

2.2)

62

.2 (3

3.9–

87.4

) *

144.

7 (1

10.5

–186

.6) *

16

4.1

(128

.2–2

15.6

) 0.

001

5-HI

AA:5

-HT

4,14

,11,

10

5.9

(3.2

–8.3

) # 4.

2 (2

.3–9

.2) *

* 15

.0 (1

3.1–

32.6

) **

16.3

(10.

9–19

.8) #

<0.0

01

HVA:

5-HI

AA

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

BA17

oc

cipi

tal p

ole

(V1)

5-HT

3,

7,11

,10

16.4

(3.0

–)

8.3

(3.4

–11.

3)

7.0

(4.1

–21.

5)

18.6

(6.6

–24.

2)

n.s.

5-

HIAA

3,

7,11

,10

126.

0 (9

3.6–

) 92

.1 (4

6.9–

144.

1)

95.6

(57.

5–14

3.6)

20

0.8

(154

.3–2

63.9

) 0.

008

5-HI

AA:5

-HT

3,7,

11,1

0 8.

9 (7

.7–)

13

.7 (1

1.0–

14.6

) 12

.7 (6

.7–2

0.4)

13

.9 (8

.9–2

4.5)

n.

s.

HVA:

5-HI

AA

3,7,

11,1

0 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

BA22

su

perio

r te

mpo

ral g

yrus

5-HT

3,

10,1

1,10

11

.7 (8

.9–)

5.

6 (3

.2–1

0.4)

4.

2 (1

.7–7

.7)

7.4

(3.8

–11.

9)

n.s.

5-

HIAA

3,

10,1

1,10

11

9.1

(98.

7–)

84.0

(60.

4–13

1.7)

*

303.

2 (1

19.6

–450

.2) *

17

1.5

(121

.7–3

20.6

) 0.

004

5-HI

AA:5

-HT

3,10

,11,

10

10.2

(8.3

–)

16.9

(12.

5–20

.5) *

67

.8 (2

8.0–

147.

7) *

23

.5 (1

8.0–

36.7

) 0.

002

HVA:

5-HI

AA

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.

005

Limbic system

Amyg

dala

5-HT

2,

10,1

0,10

59

.3 (1

1.7–

) 33

.0 (1

8.6–

49.6

) *

121.

1 (5

5.1–

148.

6) *

24

4.9

(221

.7–2

97.2

) <0

.001

5-

HIAA

2,

10,1

0,10

19

7.9

(162

.9–)

14

1.3

(102

.8–2

27.1

) **

522.

4 (3

34.9

–795

.2) *

* 99

9.8

(754

.5–1

270.

4)

<0.0

01

5-HI

AA:5

-HT

2,10

,10,

10

8.0

(2.2

–)

5.2

(3.5

–6.5

) 4.

7 (3

.8–7

.9)

4.2

(2.3

–4.9

) n.

s.

HVA:

5-HI

AA

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.

014

Hip

poca

mpu

s

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.

3–11

1.2)

0.

003

5-HI

AA

3,5,

11,1

0 14

1.3

(110

.6–)

47

.7 (4

3.1–

213.

4) *

33

6.1

(257

.8–4

76.2

) *

383.

9 (2

79.5

–717

.0)

0.00

4 5-

HIAA

:5-H

T 3,

5,11

,10

4.2

(2.4

–)

5.0

(4.1

–6.0

) 9.

2 (5

.0–1

1.0)

5.

3 (3

.4–7

.3)

n.s.

HV

A:5-

HIAA

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

.019

)

BA11

/12

orbi

tofr

onta

l co

rtex

5-HT

4,

6,11

,10

16.5

(13.

6–25

.7)

8.3

(3.9

–10.

9)

13.6

(8.7

–20.

3)

21.5

(13.

4–26

.0)

(0.0

18)

5-HI

AA

4,6,

11,1

0 98

.2 (8

5.4–

189.

7)

56.7

(35.

3–78

.5) *

* 23

8.5

(183

.5–3

44.3

) **

229.

9 (1

68.9

–328

.3)

<0.0

01

5-HI

AA:5

-HT

4,6,

11,1

0 6.

5 (5

.5–7

.5)

7.4

(6.4

–8.9

) *

21.0

(14.

4–28

.1) *

13

.3 (8

.5–2

2.1)

0.

003

HVA:

5-HI

AA

4,6,

11,1

0 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.

001

Cing

ulat

e gy

rus

5-HT

3,

8,11

,10

21.7

(3.7

–)

14.8

(7.6

–28.

7)

32.2

(25.

3–41

.6)

44.7

(29.

5–52

.4)

(0.0

48)

5-HI

AA

3,8,

11,1

0 66

.0 (3

4.5–

) # 10

2.6

(58.

5–19

1.8)

**

357.

3 (2

81.2

–379

.3) *

* 38

7.2

(313

.7–4

75.7

) # <0

.001

5-

HIAA

:5-H

T 3,

8,11

,10

3.0

(2.1

–)

6.5

(4.7

–9.1

) 11

.6 (8

.7–1

3.1)

9.

1 (6

.5–1

3.3)

(0

.044

) HV

A:5-

HIAA

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

.001

Thal

amus

5-HT

3,

11,1

1,10

18

4.9

(158

.1–)

88

.4 (7

0.9–

177.

9)

150.

3 (1

06.7

–188

.9)

204.

5 (1

69.0

–226

.6)

(0.0

26)

5-HI

AA

3,11

,11,

10

673.

7 (5

08.8

–)

689.

3 (4

12.4

–895

.8) *

* 15

84.5

(123

7.1–

1952

.8) *

* 15

25.8

(116

8.1–

1946

.8)

<0.0

01

5-HI

AA:5

-HT

3,11

,11,

10

3.6

(3.2

–)

5.3

(4.2

–8.7

) 9.

7 (7

.3–1

3.1)

8.

9 (5

.7–9

.4)

0.00

9 HV

A:5-

HIAA

3,

11,1

1,10

0.

4 (0

.3–)

0.

5 (0

.4–0

.7)

0.3

(0.2

–0.5

) 0.

3 (0

.2–0

.4)

(0.0

46)

199

Tabl

e S3

(con

tinue

d)

Br

ain

regi

on

Com

poun

d/ra

tio

N

DS

(n=4

) D

S+AD

(n=1

7)

EOAD

(n=1

1)

Cont

rols

(n=1

0)

P-va

lue

Basal ganglia

Caud

ate

nucl

eus

5-HT

3,

16,1

1,10

12

1.5

(33.

1–)

55.8

(35.

6–97

.0) *

* 16

8.3

(139

.5–2

30.2

) **

240.

0 (1

88.0

–285

.2)

<0.0

01

5-HI

AA

3,16

,11,

10

170.

9 (7

6.6–

) 21

7.6

(106

.3–2

84.2

) **

537.

2 (3

62.3

–727

.8) *

* 57

9.6

(441

.0–7

84.9

) <0

.001

5-

HIAA

:5-H

T 3,

16,1

1,10

2.

3 (1

.4–)

2.

8 (2

.4–4

.6)

2.7

(2.4

–3.7

) 2.

7 (1

.6–4

.4)

n.s.

HV

A:5-

HIAA

3,

16,1

1,10

16

.0 (5

.4–)

11

.5 (9

.3–1

4.3)

8.

4 (6

.8–1

2.2)

7.

5 (5

.2–9

.6)

(0.0

20)

Glo

bus p

allid

us

5-HT

2,

8,11

,10

149.

7 (8

9.7–

) 97

.4 (7

0.5–

120.

4) *

17

1.9

(117

.6–2

07.7

) *

161.

8 (1

40.3

–215

.5)

(0.0

22)

5-HI

AA

2,8,

11,1

0 10

09.2

(384

.6–)

43

9.5

(329

.9–9

78.8

) *

984.

5 (8

64.7

–140

7.3)

*

1320

.2 (1

019.

2–16

14.4

) (0

.015

) 5-

HIAA

:5-H

T 2,

8,11

,10

6.0

(4.3

–)

5.0

(3.0

–8.9

) 7.

1 (4

.4–8

.0)

8.3

(5.3

–9.6

) n.

s.

HVA:

5-HI

AA

2,8,

11,1

0 10

.4 (3

.2–)

6.

2 (4

.6–1

2.9)

4.

0 (2

.7–4

.9)

3.1

(2.3

–3.6

) 0.

012

Puta

men

5-HT

3,

15,1

1,10

18

9.5

(43.

9–)

77.8

(35.

2–15

9.6)

*

189.

2 (1

53.9

–219

.8) *

21

9.7

(200

.5–3

26.3

) <0

.001

5-

HIAA

3,

15,1

1,10

26

0.0

(231

.7–)

37

2.7

(203

.7–6

69.5

) **

790.

9 (6

38.7

–102

6.1)

**

998.

4 (7

81.9

–140

0.3)

<0

.001

5-

HIAA

:5-H

T 3,

15,1

1,10

5.

3 (0

.9–)

4.

3 (3

.5–8

.0)

4.2

(3.8

–5.3

) 4.

6 (2

.7–6

.1)

n.s.

HV

A:5-

HIAA

3,

15,1

1,10

21

.3 (6

.8–)

15

.4 (1

0.7–

19.2

) 9.

4 (7

.8–1

4.1)

6.

9 (5

.0–1

0.0)

0.

006

Subs

tant

ia

nigr

a

5-HT

2,

14,1

1,10

20

8.3

(167

.6–)

36

4.1

(243

.1–4

20.4

) 48

7.2

(389

.2–5

31.2

) 46

1.0

(404

.1–5

88.8

) 0.

009

5-HI

AA

2,14

,11,

10

1270

.8 (1

158.

6–)

1838

.2 (1

135.

2–23

72.0

) 23

16.6

(186

4.9–

3398

.5)

2749

.0 (2

085.

0–33

63.6

) (0

.024

) 5-

HIAA

:5-H

T 2,

14,1

1,10

6.

5 (4

.7–)

5.

0 (3

.7–7

.3)

4.9

(3.7

–5.6

) 4.

9 (4

.5–7

.3)

n.s.

HV

A:5-

HIAA

2,

14,1

1,10

2.

5 (1

.5–)

1.

5 (1

.3–1

.7)

1.6

(1.2

–1.8

) 1.

5 (1

.2–2

.1)

n.s.

Metencephalon

Locu

s co

erul

eus

5-HT

0,

10,1

0,10

–

379.

1 (2

53.6

–606

.9)

420.

1 (2

33.1

–579

.0)

490.

9 (3

15.6

–658

.4)

n.s.

5-

HIAA

0,

10,1

0,10

–

3676

.6 (2

045.

7–45

79.4

) 37

27.1

(297

8.6–

4976

.3)

4959

.5 (3

690.

3–69

69.7

) n.

s.

5-HI

AA:5

-HT

0,10

,10,

10

– 7.

1 (6

.5–8

.1)

9.2

(8.5

–14.

0)

10.7

(8.2

–13.

2)

(0.0

24)

HVA:

5-HI

AA

0,10

,10,

10

– 0.

2 (0

.2–0

.4)

0.3

(0.2

–0.3

) 0.

3 (0

.2–0

.4)

n.s.

Cere

bellu

m

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)

<0.0

01

5-HI

AA

2,9,

11,1

0 30

.8 (2

1.9–

) 41

.7 (3

3.8–

56.5

) **

206.

0 (9

2.8–

301.

4) *

* 98

.5 (7

3.7–

207.

2)

<0.0

01

5-HI

AA:5

-HT

2,9,

11,1

0 0.

7 (0

.4–)

1.

5 (1

.1–3

.3) *

* 41

.8 (1

5.7–

97.1

) **

32.9

(22.

0–44

.0)

<0.0

01

HVA:

5-HI

AA

2,9,

11,1

0 3.

7 (3

.6–)

2.

1 (1

.6–2

.9) *

* 0.

6 (0

.4–1

.2) *

* 0.

8 (0

.4–1

.1)

<0.0

01

Mon

oam

ines

and

met

abol

ites

(ng/

g tis

sue)

and

the

cor

resp

ondi

ng r

atio

s ar

e ro

unde

d of

f to

one

deci

mal

and

exp

ress

ed a

s m

edia

n (5

0%) w

ith t

he in

terq

uart

ile r

ange

(25%

-75%

) bet

wee

n br

acke

ts.

A Kr

uska

ll-W

allis

test

was

use

d to

com

pare

the

four

gro

ups.

Sig

nific

ant P

-val

ues

(<0.

015)

are

pro

vide

d. T

hose

in it

alic

s be

twee

n br

acke

ts a

re n

o lo

nger

rega

rded

sig

nific

ant a

fter

cor

rect

ion

(P<0

.015

). Po

st-h

oc M

ann-

Whi

tney

U t

ests

: DS

vs

DS+A

D, D

S vs

con

trol

s (#

<0.0

15)

and

DS+

AD v

s EO

AD (

*<0.

015;

**<

0.00

1).

Abbr

evia

tions

: BA

, Br

odm

ann

area

; 5-

HIAA

, 5-

hydr

oxyi

ndol

eace

tic a

cid;

5-

HT, s

erot

onin

; DS,

Dow

n sy

ndro

me

with

out

neur

opat

holo

gic

AD d

iagn

osis;

DS+

AD, D

own

synd

rom

e w

ith n

euro

path

olog

ic A

D di

agno

sis; E

OAD

, ear

ly-o

nset

Alz

heim

er’s

dise

ase;

HVA

, hom

ovan

illic

ac

id; n

.s.,

not s

igni

fican

t.

200

university of groningen down & alzheimer dekker, alain daniel

Documents