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The Neurobiology of Aging and Alzheimer Disease in Down Syndrome. https://doi.org/10.1016/B978-0-12-818845-3.00011-6
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CHAPTER 2
Alzheimer’s neuropathology in Down
syndrome: From gestation to old age
Elliott J. Mufsona,b,∗, Jennifer C. Miguela, and Sylvia E. Pereza
aDepartment of Neurobiology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center,
Phoenix, AZ, United States, bDepartment of Neurology, Barrow Neurological Institute, St. Joseph’s
Hospital and Medical Center, Phoenix, AZ, United States
∗Corresponding author: elliott.mufson@barrowneuro.org
Introduction
At the turn of the twentieth century, Dr. Alois Alzheimer described a form of progressive
presenile dementia in a female patient named Auguste Deter, who developed memory loss
and died at the age of 56 [1]. Postmortem neuropathology revealed extensive brain shrinkage
and accumulation of senile plaques (SPs) and neurofibrillary tangles (NFTs) [2], now
considered the defining lesions of the disease bearing his name. SPs accumulate in the
extracellular matrix and contain insoluble fibrils of amyloid beta (Aβ) protein fragments, which
are derived from the larger transmembrane amyloid precursor protein (APP) through serial
cleavage by beta-site APP cleaving enzyme 1 (BACE1) and the γ-secretase complex [3–5].
NFTs are composed of argyrophilic aggregates of several hyperphosphorylated epitopes of the
protein tau [6, 7]. These pathological protein aggregates display a β-pleated sheet conformation
and are thought to interfere with cytoskeletal integrity, which disrupts synaptic and neuronal
function. In over 99% of individuals, the onset of Alzheimer’s disease (AD) occurs in late
adulthood, usually after the age of 65 [8]. In a small portion of people (<1%), the disease
displays an autosomal dominant pattern of inheritance (“familial AD,” FAD), resulting from
one of three different gene mutations, APP, presenilin 1 (PS1), or presenilin 2 (PS2) and
manifests much earlier [8]. Interestingly, genetic analysis of tissue from the brain of Auguste
Deter revealed a PSEN1 mutation [1, 9], which was consistent with her early age of disease
onset. Genotyping for APOE ε4, a major risk factor for AD, revealed that Augusta Deter was an
ε3/ε3 carrier [1].
Although late onset sporadic AD is the leading cause of dementia in the United States, affecting
an estimated 5.4 million people and is predicted to afflict 13 million people in the USA by 2050
https://doi.org/10.1016/B978-0-12-818845-3.00011-6
12 Chapter 2
[8], the field lacks a true animal model that fully recapitulates the pathobiology underlying the
disease. Currently, premortem clinically and postmortem neuropathologically
well-characterized human brain tissue provides the gold standard for defining the
pathophysiology of AD. Although similar to AD, individuals with Down syndrome (DS)
develop SP and NFT pathology and, dementia as they age, there is a discrepancy between the
prevalence of AD pathology (about 100% by age 40) and the prevalence of dementia (about
2%–5% by age 40 and 70% by age 70) in adults with DS [10–12] (see Chapters 1, 13, and 15).
This population provides a unique resource for the investigation of the location, temporal
course, and clinical association of the cellular and molecular neuropathology related to the
development of AD.
As described in Chapter 1, in 1866, the Cornish physician John L. Down published an article
entitled “Mental affections of childhood and youth,” which described the external and innate
characteristics of people referred to as a “mongoloid idiot.” Down’s grandson was born with
this condition and displayed a phenotype (described by Dr. Down) that included a round face,
oblique eyes, flat nape, thin eyebrows, a small pug nose, and thick cleaved tongue [13]. In 1965
the World Health Organization officially confirmed the eponym for this disorder as Down
syndrome (DS). Although Down assumed that parental tuberculosis was the cause of this
disorder [14], genetic analysis revealed that DS was due to an extra copy of chromosome
21 (Ch21) [15]. This chromosome harbors the gene encoding APP, that results in an
overproduction of the Aβ peptide and is associated with the onset of FAD (<50 years of age)
[16–18]. Several groups linked the gene for the APP to a locus on the proximal portion of the
long arm of Ch21 [19–21], which then narrowed the location of APP to Ch21 [19, 20].
Behaviorally, DS is characterized by intellectual disability attributed to a full or partial extra
copy of human Ch21 (HSA21) [15] that accounts for 95% of the chromosomal anomalies in this
disorder, or lesser frequent genotypes associated with a translocation of Ch21 onto another
chromosome (4%) and mosaicism where some cells exhibit an extra copy of Ch21 (1%). DS
occurs in 1 in 800 births in the United States and affects approximately 6 million people
worldwide. The extra copy of Ch21 results in developmental alterations that distinguishes DS
from the neurotypical brain.
Prenatal brain pathology in DS
During gestation, the DS brain appears smaller than its neurotypical counterpart (Fig. 1). In the
neurotypical cortex the lateral sulcus (Sylvian fissure), which first appears on the superolateral
surface as a minor indentation, is observed as early as gestational weeks 13.5 in DS (Fig. 1E). At
these early developmental ages, the corpus callosum and callosal sulcus appear on the medial
surface of the hemisphere, although these structures are more evident in the neurotypical brain
(Fig. 1A0 and E0). During normal brain embryology, the lateral sulcus gradually becomes
deeper, at the same time as the frontal, parietal, and temporal opercula are rapidly expanding
Fig. 1
(A–G) Photographs of the lateral and medial cerebral surface showing development of the Sylvian/
lateral fissure (A, C, D, E, F, G), central (C, D, G), callosal (A0, E), cingulate (B, G0), and superior
temporal (D, G) sulci in the neurotypical fetal brain at 17 (A, A0), 18 (B), 21 (C), and 24 (D) weeks
compared to Down syndrome (DS) 13.5 (E, E0), 20 (F), and 26 (G, G0) gestational weeks. Note the
delayed and less visible central sulcus (CS) (B, G) and Sylvian fissure (SyF) (A, C, D, F, G) between the
neurotypical and DS fetus. Panel G0 shows the calcarine fissure (CaF), posterior occipital sulci (POS),
the corpus callosum (CC), fornix (fx), diencephalon (Di), and cerebellum (Cb). (H–K) Images
showing diffuse APP/Aβ immunoreactivity (H), absence of Aβ40 (I), Aβ42 (J), and AT8 tau
(K) reactivity in the frontal cortex of a 1-year-old female DS case. Abbreviations: CaS, callosal sulcus;
CiS, cingulate sulcus; IHF, interhemispheric fissure (longitudinal cerebral fissure); Ins, insula; POCS,
postcentral sulcus; PreCS, precentral sulcus. Sections in panels I and J were counterstained with cresyl
violet. Scale bar in G0 equals to 5mm in E, 7mm in E0 and G, 8mm in F and, the scale bar in K¼25μm,
15μm in I and 100μm in H.
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 13
14 Chapter 2
(Fig. 1C and D) compared to a less well-developed narrow lateral fissure seen in DS gestational
weeks 20 and 26 (Fig. 1F and G). At about week 21 of gestation, the central sulcus appears as a
small groove in the dorsal region of the normal cortex (Fig. 1C), which was not evident in DS
(Fig. 1F). However, the central sulcus was clearly visible in the dorsolateral cortex at
gestational week 26 in DS (Fig. 1G). At 24weeks’ gestation, the neurotypical cortex displays a
pre- and postcentral sulci as well as a developing superior temporal sulcus (Fig. 1D) that are not
clearly visible at embryonic week 26 in DS (Fig. 1G). At fetal week 17 the medial surface of the
neurotypical brain displayed cortical invagination indicative of the callosal fissure and the
developing corpus callosum (Fig. 1A0), which was less well defined at gestational week 13.5 in
DS (Fig. 1F0). The medial surface of the neurotypical cortex alsorevealed indentations
indicative of a developing calcarine and parietooccipital sulcus (Fig. 1B) similar to that seen at
26 weeks in the DS brain (Fig. 1G0) [22]. We were able to identify the cerebellum at DS fetal
week 13.5 (Fig. 1E, E’) and 26 (Fig. 1G, G’). The developmentally altered trisomy brain
indicated by delayed central nervous systemmaturation associated with prenatal arrest of neuro
and synaptogenesis [23, 24] may be partially a consequence of delayed cortical development. It
is important to note these developmental differences in the brains of people with DS, since these
regions are similar to those later affected by aging and AD.
Adult brain pathology in DS
Gross evaluation of the adult DS brain revealed abnormalities that include a reduction in brain
weight, altered configuration, less numbers of gyri and depth of cerebral sulci, stumpy/
shortened appearance of the frontal and temporal lobes, hypoplasia of the brainstem and
cerebellum (Fig. 2). Coronal sections of the DS brain revealed ventricular enlargement (Fig. 2),
cortical and hippocampal shrinkage (Fig. 2) [25–28], as well as alterations of cortical
lamination. Increased cortical thickness has been reported in sensory (Brodmann areas in 1, 3b),
middle frontal, and orbital cortex [29] in DS. Although these changes may play a role in defects
in somatic, memory, and olfactory responses in people with DS, there is variability in these
alterations across cortical regions. In 1948 George Jervis reported that individuals with DS
displayed dementia premortem and a postmortem neuropathological phenotype that included
SP and NFT similar to that described 60 years earlier by Dr. Alzheimer [2, 30]. Other studies
confirmed these pathological findings and showed a similar topography of these lesions
between DS and euploid AD cases [31]. Individuals with DS also exhibit characteristics of
premature aging and are at a higher risk for developing dementia of the AD type several decades
earlier than patients with sporadic AD [10, 32–34] (see Chapters 1, 13, and 15). Despite
parallels between AD and DS, current transgenic mouse models do not truly replicate these
disorders and the consequence of trisomy in nonhuman species is not consistent with the
genotype or phenotype of DS [35]. Therefore this chapter will provide an overview of the
similarities and differences in the cellular and molecular pathobiology of the human DS brain
from gestation to older ages in contrast to AD.
Fig. 2
Lateral view of the cerebral hemisphere from an aged healthy control (HC) subject (A), Alzheimer’s
disease (AD) (B) and Down syndrome (DS) (C) brain. Note the widening of sulci and the narrowing of
gyri in AD compared to the aged healthy control and DS case and the shrinkage of the DS cerebellum
(C) compared to the control (A) and AD (B) brains. (D–J) Rostral to caudal coronal hemibrain slabs
from a 59-year-old female DS case with dementia showing ventricular hypertrophy and gray matter
reduction. (K) Coronal slab showing a reduction in the size of the hippocampal formation, entorhinal
cortex, and an enlarged lateral ventricle in this 59-year-old female with DS and dementia.
Abbreviations: Cd, caudate; Ent, entorhinal cortex; fi, fimbria; fx, fornix; HP, hippocampus; LV, lateral
ventricle; Pt, putamen; SN, substantia nigra; Th, thalamus.
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 15
Cortical amyloid in AD and DS
Although all cases of DS display AD pathology, only about two-thirds develop dementia [10]
(see Chapters 1, 13, and 15). The biology underlying this disconnect between the presence of
AD neuropathology and presence of dementia is an underinvestigated area, primarily due to the
lack of autopsy cases that are clinically well characterized. In AD andDS, an increase in soluble
Aβ species precedes plaque deposition [34, 36, 37], and occurs as early as 21 gestational weeks
in DS [36, 37]. In a recent study, amyloid plaques were not seen at 0.01, 1.6, and 3months of age
in the frontal and temporal cortex or brainstem in DS [38]. In our ongoing developmental
investigations, we were not able to immunohistochemically visualize Aβ40, Aβ42, or Aβ
plaques-like structures in the frontal cortex at 6months, 1 year (Fig. 1H–K), and in a 3-year-old
DS case. However, an occasional diffuse starburst deposit displaying immunoreactivity against
16 Chapter 2
the 6E10 antibody, which recognizes amyloid precursor protein (APP) and Aβ was found in
these same cases (Fig. 1H). Interestingly, there is one other case report showing a similar
“starburst” pattern [39]. By contrast, no amyloid-like staining was seen in a 5-year-old
neurotypical case. These observations suggest that APP and not Aβ is an early event in the
developing DS cortex, which may be related to altered axonal pruning and neuronal
culling [40]. Although diffuse deposits of Aβ42 have been reported in people with DS between 8
and 27 years of age [38, 41, 42], an earlier time point than seen in AD [41–43], they appear to
have negligible effects upon neurons and their deposition is not associated with clinical
symptoms [44]. In DS cases aged 40–50 years, levels of cortical Aβ deposition are similar to
those observed in sporadic, late onset AD [11, 42, 45–49]. In addition to diffuse plaques, a few
cored plaques associated with dystrophic neurites (neuritic plaques) have been reported at age
19 and in a 30-year-old DS case [38], which are of neuropathological diagnostic significance in
AD. By age 40, people with DS exhibit Aβ plaque density and morphology similar to that seen
in AD [38].
Cerebellar amyloid in AD and DS
Although neuropathological reports suggest that the reduced size of the DS cerebellum leads to
a delay in fine motor development [25, 50], studies of AD neuropathology in this structure
during gestation are minimal. We found scattered diffuse amyloid-like plaques, some
containing a dense central core immunoreactive for APP/Aβ1–16 (6E10) in the white matter and
granular cell layer of the cerebellum at postnatal day 10 and between 4 and 20 weeks of age in
DS. Another study reported virtually no amyloid plaque pathology between 0 and 53 years of
age using the 4G8 antibody (APP/Aβ17–24), but beyond this age all cases displayed various
levels of Aβ pathology in DS [38]. The difference between earlier and a more current study [38]
may be related to the sequence specificity of the 4G8 antibody [51]. However, in both the adult
AD andDS cerebellum, amyloid deposition visualized using an antibody against the Aβ peptide
(Aβ4) appears as amorphous patches, which often occur perpendicular or parallel to the pial
surface within the molecular layer (ML) [52]. We recently investigated the deposition of
amyloid using antibodies that detect different epitopes of the Aβ sequence in cerebellar cortex
obtained from demented (average age 51.2; range 45–59) and nondemented (average age 50.3;
range 44–60) individuals with DS (average age 81.7; range 71–98) and healthy aged controls
(average age 70.9; 51–85). Cerebellar tissue reacted with an antibody that recognizes both APP
and Aβ (6E10), revealed patches of APP/Aβ in the ML that was greater in both DS groups
compared to AD and nondemented healthy subjects (Fig. 3A–D). Since the elevation of levels
of the long form of Aβ, Aβ42, compared to the short form, Aβ40, plays a role in the early events
underlying the pathogenesis of AD, we evaluated Aβ42 and Aβ40 immunoreactivity within
cerebellar tissue from these same adult cases. Aβ42, but not Aβ40 immunoreactivity, was found
in the cerebellum of both DS groups but to a lesser extent in AD (Fig. 3E–H). In DS, Aβ42
appeared as bands perpendicular to the pial surface within the ML (Fig. 3F and G).
AP
P/
Aβ
GL
ML
Aβ
42
HC DSD-
DSD+ AD
ML
GL
ML
GL
GL
GL
ML
ML
HC DSD-
DSD+ AD
A B
C D
E F
G H
Fig. 3
Photomicrographs showing APP/Aβ (A–D) and Aβ42 (E–H) immunolabeling in molecular layer (ML)
of the cerebellar cortex in aged healthy control (HC)(A, E), nondemented Down syndrome (DS)
(B, F), demented DS (G, C), and Alzheimer’s disease (AD) (D, H) subjects. Note the deposits
of APP/Aβ in DS without (DSD�, B), with dementia (DSD+, C), and AD (D), while Aβ42
immunoreactivity appeared as parallel bands reaching the pial surface within the ML in
DSD� compared to wider patches of immunoreactivity in DSD+ and the limited patches in AD.
APP/Aβ and Aβ42 immunostaining was not detected in HC subjects. Abbreviations: GL, granular
cell layer; ML, molecular layer. Scale bar¼100μm.
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 17
Interestingly, only scattered patches of APP/Aβ and Aβ42 were seen in the cerebellar ML in AD
(Fig. 3D and H). Both demented and nondemented individuals with DS had significantly higher
Aβ42 plaque loads in the ML compared to nondemented controls. Cerebellar Aβ42 loads in
demented individuals with DS were significantly increased compared to AD [53].
Striatal amyloid pathology in AD and DS
Although striatal plaques mainly exhibit a diffuse morphology, there are reports of cored/
neuritic plaques in this region in AD [54, 55]. Neuropathological studies revealed that Aβ42
positive plaques first develop in the striatum in DS and in select FAD types of PS1 mutation
carriers, as early as the second decade of life [42, 56]. Since APP cleavage products appear
more resistant to degradation, it is possible that they act as early seeds for the eventual
18 Chapter 2
formation of amyloid plaques in the adult DS brain. Postmortem reports indicate that amyloid in
the striatum appears 20–30 years later in life compared to the neocortex in DS [45, 46]. Data
derived from various studies of individuals with DS ranging in age from newborn to 76 years
[45, 46] revealed an absence of striatal amyloid in all cases aged 50 years or less. Studies
suggest that only 7% of cases over 55 years had a greater striatal amyloid load than found in
the temporal cortex, while 36% had a higher amyloid load in temporal cortex than the striatum
and 29% had similar amyloid loads in both regions [38]. These finding suggest that amyloid
pathology displays a regional development in people with DS similar to the Thal and Braak
phases seen in AD [57, 58], with neocortical regions including temporal neocortex affected
earlier than the striatum and other subcortical regions. By contrast, amyloid positron emission
tomography (PET) Pittsburgh Compound B (PiB) imaging studies reported that striatal amyloid
deposition precedes neocortical amyloid [59–62], which was related to cognitive decline in DS
[59] (see Chapter 9). When prevalence of amyloid positivity was assessed using PiB, both the
striatum and precuneus cortex showed the highest values in the youngest participants (age range
36–40years), suggesting that these brain regions are affected early by amyloid in DS [63]. It has
been suggested that striatal amyloid labeling may be a measure for pathology-based clinical
staging of AD, as increased sensitivity of [F-18]Flutemetamol PET was associated with higher
histological density thresholds of striatal amyloid deposits [64]. It remains to be determined
whether PET ligand imaging would reveal differences in brain amyloid load between
individuals with and without DS [59–61, 65]. Amyloid immunocytochemistry performed by
our group failed to reveal any significant differences in caudate or putamen amyloid plaque
load and number between demented and nondemented adults (40–60 year old) with DS [34].
Striatal plaque loads determined using either the 6E10 (APP/Aβ) antibody or X-34
histochemistry, which reveals β-pleated sheet structures, displayed higher mean values than Aβ
and 6-CN-PiB (tissue stain for the PiB compound) in nondemented DS cases [34]. Recently,
elevated striatal [C-11]PiB binding was found in 13 of 16 people with DS with cognitive
impairment or dementia, whereas similar labeling was observed in only 7 of 33 cognitively
stable individuals [59]. Further clinical pathological studies are needed to develop a striatal
amyloid signature that distinguishes demented from nondemented people with DS [55].
It is widely accepted that amyloid-related peptides are toxic resulting in the formation of
neuropil threads, dystrophic dendrites, and axons in the AD brain [66, 67]. Interestingly, we did
not observe tau, choline acetyltransferase (ChAT), and tyrosine hydroxylase dystrophic
neurites and neuropil threads (NTs) associated with striatal amyloid plaques in demented or
nondemented individuals with DS compared to frontal cortex (FC) plaques [34]. Not all striatal
ChAT positive interneurons located adjacent to Aβ42 plaques or containing NFTs displayed an
abnormal morphology in either DS phenotype (see Fig. 5E and F) compared to the shrunken
appearance of striatal cholinergic neurons in AD (see Fig. 5G). Striatal amyloid is an area that
requires further investigation using both imaging and histopathological methods to determine
its role in the pathobiology and onset of dementia in DS and AD [55].
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 19
Tau deposition in AD and DS
In addition to amyloid pathology, both the AD and DS brain develop age-related tau-containing
NFT and neuritic thread (NT) pathology (see Fig. 5) [12]. Tau, a microtubule protein associated
with normal cytoskeletal function, transitions from a relatively soluble state into filamentous
aggregates in AD and DS [68–71]. NFT pathology occurs at a later age than Aβ in DS [11,
72–74], exhibits an age-related pattern similar to AD [75], and NFT burden is tightly correlated
with cognitive decline and dementia in both AD and DS [34, 76–79].
Fetal tau in DS
Despite the age-associated onset of tau pathology in the adult AD and DS brain, tau deposition
in the fetal DS brain remains underinvestigated. However, different abnormal tau
phosphorylation events have been demonstrated during fetal development (14–28 gestation
weeks) using tau epitope-specific antibodies that revealed tau within white matter tracts (e.g.,
cerebellar peduncles and internal capsule) [80, 81] suggesting early axonal transport defects, a
common theme in tauopathies [82]. Interestingly, we observed a band of phosphorylated CP13
(Ser202) and PHF-1 (Ser396) tau immunoreactivity situated between the external granular (or
germinal) and molecular layers of the cerebellum during early postnatal development (Fig. 4).
Although this tau band is similar to that reported in the outer two-thirds of the molecular layer of
the hippocampus in individuals with DS over the age of 31 years, these profiles were not
positive for PHF-1 [48] as seen in the fetal cerebellum. Although the functional significance of
these bands of tau remains unclear, this fine dust-like immunoreactivity may represent synaptic
terminals related to neuronal sprouting or pruning during both development and aging [83].
Recently, using neuron-derived extracellular vesicles (NDEs) isolated from blood [84, 85],
increased levels of p-Tau and Aβ were found in NDE cargo obtained from children with DS as
early as 8 years of age, lending support to the suggestion that putative pathologic tau is initiated
in brain decades prior to symptom [86] and lesion onset.
Tau pathology in the adult AD and DS brain
Tangle bearing neurons appear within neocortical layer III, V, and VI pyramidal, entorhinal
cortex layers II and IV neurons. Cortical NFTs display a flame-like shape compared to a more
globose morphology associated with subcortical neurons such as those seen in the cholinergic
neurons of the nucleus basalis in both the adult DS and AD brain [87–89] (Fig. 6M, N, Q).
Recently, we reported that neuritic pathology, found in these regions, is an excellent correlate of
cognitive decline during the progression of AD suggesting that axonal and dendritic pathology
precedes neuron dysfunction [90]. Furthermore, we reported greater neuritic pathology in the
frontal cortex of demented compared to nondementedDS cases suggesting that axonal integrity
also plays a key role in dementia onset in DS [34]. These observations suggest that despite the
10d 4wk 9wk
ML
CP
13 PL
A B C
1d
PH
F-
1
IGL
ML
PL
IGL
ML
ML
PL
IGL
IGL
PL
IGL
EGL
IGL
EGL
* * *
D E F
G H I
J K L
Trisomy 21
Neurotypical
CP
13
PH
F-
1
4wk 9wk
Fig. 4
Photomicrographs of the cerebellar cortex showing a band of phosphorylated CP13 (A–C and G–I)
and PHF-1(D–F and J–L) tau immunostaining (black arrows) located between the external granular (or
germinal) cell layer andmolecular layer at 10days, 4 and 9weeks in DS, and at 1-day, 4 and 9weeks in
the neurotypical cerebellum. Note that cerebellar CP13 immunostaining is seen at early postnatal
stages in DS (A–C) but not in the neurotypical cerebellum (G–I). PHF-1 immunoreactivity is seen at
postnatal day 10, but not at weeks 4 or 9 (D–F) in the DS cerebellum. PHF-1 is observed at weeks 4
and 9 in the neurotypical cerebellum (J–L). All tissues in the figure were counterstained with Gill’s
hematoxylin. J–L *indicates external granule layer. Abbreviations: EGL, external granular (germinal)
layer; IGL, internal granular layer; ML, molecular layer; PL, Purkinje cell layer. The 25μm scale bar in
K applies to A–F, G, H and J panels and the 25μm scale bar in L applies to panel I.
20 Chapter 2
appearance of Aβ at an early age, NFT pathology is more closely linked to cognitive decline and
dementia in DS similar to AD [76–78]. Braak and colleagues have staged the progression of
NFT pathology in AD using the tau AT8 antibody. Braak staging revealed a progression of NFT
pathology that begins within the entorhinal and transentorhinal cortex (stage I) spreading to the
hippocampus (stage II), temporal cortex (stage III), then to other neocortical regions (stage IV),
and finally reaching visual association cortex (stage V–VI). Recently, this schema was
modified and stages prior to entorhinal cortex involving subcortical neurons containing tau
located within noradrenergic locus coeruleus (LC), serotonergic dorsal raphe (DRN), and
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 21
cholinergic nucleus basalis neurons [91, 92] were described as pretangle or prodromal stages
“a” and “b” [93, 94], independent of cortical tau pathology and these areas are implicated in
cognitive decline in AD [87, 95]. Recently, tau (AT8) positive NFTs or NTs were not found
within the entorhinal cortex, hippocampus, or neocortex in a subset of DS cases 35 years and
under plus a single 39, 50, and 60-year-old subject, which were considered Braak stage II–IV
[38] indicative of mild to moderate tangle pathology within the medial temporal lobe (MTL)
[96, 97]. Remaining cases over the age of 50 showed moderate to numerous NFTs in all brain
regions examined similar in appearance, distribution, and degree to AD, which were scored as
Braak stage V or VI [45] indicative of advanced MTL and neocortical NFT pathology [96] that
is associated with dementia in AD. Davidson and colleagues [38] reported tau pathology in LC
and DNR nuclei only after 35 years of age in DS. These investigators also evaluated the
substantia nigra, which contains dopaminergic neurons, and found no tau pathology in any case
less than 50 years of age. After this age NFTs and neuropil threads increased similar to that
described in older people with AD [98]. A detail application of Braak staging needs to be
applied to demented and nondemented individuals with DS.
Evolution of tau pathology in AD and DS
To better understand the development of NFTs in adults with AD, a linear model has been
proposed that can be tracked by antibodies that mark tau epitopes during the early, intermediate,
and late stages of NFT formation [71, 90, 99, 100]. This model has been applied to the evolution
of NFTs in the cortex, hippocampal complex, and cholinergic basal forebrain (CBF) in AD [71,
90, 100, 101]. Recently, this paradigm was used to determine whether changes in
posttranslational tau epitopes in NFTs differ in the frontal cortex (FC) and striatum between DS
without (DSD�) and with (DSD+) dementia. This study used antibodies against early tau
phosphorylation epitopes (pS422 and AT8), structural/conformational tau changes (Alz50 and
MC1), and tau truncation (Tau C3 andMN423) applied to FC layer V and VI pyramidal neurons
in DSD� and DSD+ cases (Fig. 5A–D). NFT and NT profiles positive for phosphorylation
(pS422) and truncation (TauC3) were more abundant compared to conformational (Alz50 or
MC1) NFTs in layer V and VI neurons in both DS groups. However, significant differences in
pS422 and TauC3 compared to Alz50 positive NFT densities were found in the DSD+ group. In
addition, pS422 positive NT numbers were significantly higher than MC1 and Alz50 positive
NTs and the number of AT8 reactive NTs was greater than those containingMC1 only in DSD+
cases. Furthermore, NFT counts revealed that only the number of NFTs and NTs containing
AT8was significantly higher in DSD+ compared to DSD�. By contrast, MN423-positive NFTs
were seen in 2/6 DSD� compared to 6/8 DSD+ cases mainly in layer II and III, with the
exception of two female DSD+ cases (59 and 45 years old) where NFTs were found in layer
V and VI. Triple-labeled NFTs (AT8+pS422+Alz50, TauC3+pS422+Alz50) and dual reacted
(TauC3+pS422) were more frequent in DSD+ compared to DSD� cases (Fig. 5A–D). The
number of dual-labeled FCNFTs (Alz50+pS422 and Alz50+TauC3) compared to triple-labeled
DSD+ ADDSD+
DSD+DSD+DSD- DSD-
DS DSD+ AD1yr 46yr 83yr
DSD+ DSD+ DSD+
DSD- DSD- DSD- DSD+
A B C D
E F G
H I J
K L M
N O P Q
DG
CA1
CA3
CA2
GCL
Fig. 5
(A–D) Confocal immunofluorescence merged images showing single pS422 (green), Alz50 (blue), AT8
(red), or TauC3 (red) and triple-labeled profiles (pink) frontal cortex layers V–VI profiles from a
44-year-old female nondemented subject with DS (A, C) and a 46-year-old demented male with DS
(B, D). Note manymore cortical pS422+Alz50+AT8 and pS422+Alz50+TauC3 positive neurofibrillary
tangles (NFTs) and neuropil threads (NTs) (pink) in the demented DS compared to the nondemented
DS case. The presence of single TauC3 positive NFTs (white arrow) was observed in both cases (C, D).
(E) Image showing Aβ42 immunoreactive (-ir) plaques (dark blue) and ChAT-ir neuron (brown) in the
putamen of a 46-year-old male demented subject with DS. Note the lack of dystrophy in ChAT-ir
neuron. (F) Immunofluorescence images showing striatal neurons double labeled (yellow) for ChAT
(red) and AT8 (green) in a 46-year-old male demented DS and a 76-year-old female AD case (G). Of
particular interest is the globose and shrunken appearance of the cholinergic tangle-bearing neuron
(white arrow) in AD compared to the relative normal morphology of a cholinergic perikarya displaying
NFT pathology in a demented subject with DS (F). Insets in F and G show a single AT8
immunofluorescence (green) striatal cholinergic neurons. (H–M)Nuclear nonphosphorylated TDP-43
(Continued)
22 Chapter 2
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 23
(AT8+pS422+Alz50 and TauC3+pS422+Alz50) positive neurons was lower in DSD� and
DSD+, respectively, suggesting that tau phosphorylation and truncation events precede
conformational events in FC neurons in DS.
Striatal NFTs containing tau phosphorylation, conformational and truncated (pS422, AT8,
Alz50, MC1, and TauC3) epitopes were detected in all DSD+ cases, whereas conformational
(Alz50 and MC1) NFTs were found in a subset of DSD� cases. MN423-positive profiles were
not detected in the striatum of either DS group. Since there were no significant differences in
pS422, AT8, TauC3, MC1, and Alz50 positive NFTs in the striatum between DS groups,
conformational alterations may be an early event in DS that is discontinuous as the amino
terminus is eliminated during the evolution of tau pathology [34]. In contrast, TauC3 NFT
density was significantly reduced compared to pS422, but comparableto Alz50 in the putamen
in both DS groups, revealing a less advanced stage of NFT pathology. In this study, several
striatal cholinergic interneurons displayed intracellular aggregates of tau (AT8) resembling
skeins of yarn (Fig. 5F) in both DS groups but the cellular morphology remained unchanged
compared to shrunken NFTs in DS and AD (Fig. 5G). These observations suggest that
cholinergic NFT containing interneurons were at an early pathological stage or were resistant to
tau toxicity.
Recent evidence suggests that prefibrillar amyloid oligomeric forms are a more likely
neurotoxic species of tau [101, 102]. In this regard, we examined the accumulation of tau
oligomeric species in CBF neurons during the progression of AD and found that tau oligomer
formation is an early event in tangle evolution [101, 102] suggesting that aberrant
phosphorylation primes the tau protein for additional phosphorylation and conformational
changes [90, 95] that facilitate oligomerization [96]. Whether similar events occur in striatal
neurons in individuals with DS requires investigation. In the future, the accumulation of tau
burden using PET imaging will assist in defining the role that tau plays in the onset of dementia
in both AD and DS. Rafii and colleagues [79] have shown a similar tau binding pattern and
progression in non-DS AD cases, which were related to cognitive decline (see also Chapter 9).
Fig. 5, cont’d
immunostaining in orbital cortex layers V and VI in a 1-year-old female with DS (H), 46-year-old male
DS with dementia (I), and an 83-year-old AD (J) case. TDP-43 immunostaining in hippocampal
subfields in a 46-year-old male DS case with dementia (K). High-power images of TDP43-ir
hippocampal granule (L) and CA1 pyramidal neurons (M) from panel K. (N–Q) Images showing Lewy
bodies (LB) and Lewy neurites (LN) in the hypothalamus (N), basomedial amygdala (O), and
entorhinal cortex (P) in a 46-year-old male DS without dementia and cortical amygdala (Q) in a
46-year-old male DS with dementia. Abbreviations: DG, dentate gyrus; DSD+, demented DS; DSD�,
nondemented DS; GCL, granule cell layer; CA, Cornus Ammonis. The 50μm scale bar in D and
F applies to A–C and G and inset panels, respectively; 25μm scale bar in J applies to H and I panels;
scale bars: E¼50μm, K¼500μm, N–Q¼25μm, and 25μm scale bar in M applies to panel L.
24 Chapter 2
Further longitudinal studies are needed to assess the interaction of tau with cognitive
impairment in DS.
Other pathologies in AD and DS
Transactive response DNA-binding protein 43 (TDP-43) inclusions in DS
TDP-43 was identified as a 414 amino acid (molecular mass 43kDa) binding partner to the TAR
regulatory element in HIV virus type 1. TDP-43 is a nuclear versatile RNA/DNA binding
protein involved in RNA-related metabolism, particularly in RNA splicing. Phosphorylated
TDP-43 proteins, in the form of neuronal cytoplasmic inclusions, a pathological hallmark of
amyotrophic lateral sclerosis (ALS) are also found in other neurodegenerative diseases
including in some, but not all AD (30%–40%) and FAD [103–105] cases. In both AD
phenotypes, phosphorylated TDP-43 inclusions appear mainly in temporal cortex,
hippocampus (e.g., dentate granule cells and CA pyramidal fields), subiculum, entorhinal
cortex, amygdala, and frontal cortex in more advanced AD [45, 104–107]. By contrast, a few
reports have described a small number of older DS cases with AD pathology (age range 54–62)
that contained TDP-43 inclusions in the hippocampus, amygdala, and temporal cortex similar
to AD [45, 105]. We observed nuclear nonphosphorylated TDP-43 immunostaining in layers
V and VI of the orbital cortex in a 1-year-old female with DS (Fig. 5H), a 46-year-old male DS
with dementia (Fig. 5I), and an 83-year-old AD (Fig. 5J) case. TDP-43 reactivity was also seen
in hippocampal subfields in a 46-year-old male DS case with dementia (Fig. 5K) including
granule (Fig. 5L) and CA1 pyramidal (Fig. 5M) neurons, while cytoplasmic nonphosphorylated
TDP-43 positive inclusions were not observed in these DS cases. Recently, a staging schema
was applied to TDP-43 distribution in AD [107] but has not yet been undertaken in DS. The
functional significance of cytoplasmic TDP-43 inclusions remains a highly debated question.
Some have argued that TDP-43 intracellular deposits in AD andDS are more related to an aging
process than to AD alone [105]. Although there is a strong relationship between these inclusions
and cognition in AD, only 30%–40% of AD and 7% of DS cases examined display TDP-43
immunoreactivity [105], arguing against a primary role in the onset of dementia in most people
with these disorders.
α-Synuclein inclusions in DS
Lewy bodies (LB), which contain ubiquitin and a-synuclein, are classic lesions of Parkinson’s
disease but are also found in AD and dementia with LBs (DLB) [108]. Tissue from DS
immunostained for α-synuclein revealed the greatest number of LBs and Lewy neurites (LN) in
the amygdala [108]. We found LBs to be concentrated in the basal medial, lateral, and
accessory basal amygdaloid nuclei as well as scattered Lewy Neurites (LNs) with a heavy
concentration in white matter tracts (Fig. 5O and Q). LBs have been reported, to a lesser degree,
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 25
in orbital frontal, entorhinal, and temporal cortex as well as in the substantia nigra, nucleus
basalis ofMeynert and hypothalamus (Fig. 5N and P), and an occasional LB inDS cases 50years
or older but not in very young cases [45, 108–110]. Although LBs occur mainly in neurons
lacking NFTs, an occasional dual-labeled neuron was reported in the amygdala and adjacent
periamygdaloid cortex in DS [108]. LBs were found in 50% of DS cases with AD pathology but
not in DSwithout AD and to a lesser degree in FAD suggesting a role for the APP gene as well as
trisomy in the biological processes driving the development of this pathology [108]. We found
LBpathology in both demented and nondemented adultswithDS aged 40–60 years (Fig. 5N–Q).
However, the factors that determine the formation of synuclein containing LB pathology and
their role in the behavioral/functional decline seen in DS remain unknown.
Cholinergic basal forebrain dysfunction in DS and AD
Of the many subcortical brain regions implicated in cognitive impairment the neurons located
within the nucleus basalis of Meynert (nbM), which innervate the entire cortical mantle [111,
112], display NFT pathology even prior to that seen in the transentorhinal and entorhinal cortex
in AD [94] have received extensive investigation in AD but to a lesser degree in DS [31,
113–121]. Outcomes from brain imaging studies have strengthened the importance of this
region in AD including altered basal forebrain signaling in relation to cognitive decline [122],
propagation of cortical atrophy early in the evolution of AD [123], as a presymptomatic
biomarker for AD [124, 125] and a predictor of memory impairment in AD [123]. The increase
in [F-18]FDG PET signal within the nucleus basalis in MCI compared to control and AD [126]
suggests a transient cholinergic upregulation, similar to the neuroplastic increase in ChAT
activity found in the frontal cortex [127] and hippocampus [128] in MCI. This renewed interest
in the CBF projection system demonstrates a critical need to better understand the mechanistic
factors driving the selective vulnerability of the CBF connectome and its association to
dementia in DS. Since there are numerous reviews that discuss the structural and functional
correlates of CBF degeneration in AD [87, 120, 129–133], here we will concentrate on the
pathobiology of this system in DS.
Prenatal cholinergic basal forebrain connectome in DS
The neurotypical human newborn brain displays an intact CBF system [134, 135]. However,
whether individuals with DS are also born with an intact CBF system remains an intriguing area
of research. To investigate the CBF system during gestation we examinedseveral neurotypical
and DS embryonic brains [134, 136]. Due to methodological caveats related to the use of ChAT
antibodies to visualize the CBF system in paraformaldehyde fixed fetal tissue, we employed
antibodies that recognize the low affinity nerve growth factor receptor (p75NTR) and the high
affinity (TrkA) cognate receptor for the trophic substance nerve growth factor (NGF), both are
excellent markers for the CBF connectome in fetal [134–136], young, aged, and diseased
26 Chapter 2
human brain [114, 137]. The neurotypical fetal human brain displayed extensive p75NTR
immunostaining at least at embryonic weeks 21 and 27 (Fig. 6A and B) [134, 136] and TrkA
reactivity at least to embryonic week 34 [134]. At these fetal ages, CBF p75NTR
immunoreactive neurons appear multipolar and display varicose beaded axons, dendrites
(Fig. 6D), as well as straight fibers (Fig. 6E). However, trisomy results in an altered cholinergic/
p75NTR phenotype during gestation including reduced reactivity within subregions of the CBF
at DS embryonic week 18 (Fig. 6C) suggesting a defect in the cellular machinery producing
p75NTR. Although many CBF neurons exhibit a similar morphology in DS to the neurotypical
brain at fetal week 18 (Fig. 6F), others appear dystrophic with blunted dendrites (Fig. 6G)
and fibers appearbrokenwith swollenprocesses (Fig. 6I).Atbothprenatal neurotypical (20weeks)
andDS (18weeks), p75NTR immunoreactive fibers coursedwithin the external capsule to reach the
subplate zone of the developing cortex (Fig. 6J and K). Although a similar distribution of p75NTR
immunoreactivity was seen in the subplate zone in the neurotypical and DS cortex (Fig. 6J and K)
the later displayed less neurons and swollen and blunted neurites (Fig. 6K). Recently, we
immunostained tissue containing the nbM from a 6-month-old female individual with DSwith
an antibody against p75NTR and observed large multipolar and elongated bipolar cholinergic
neurons (Fig. 6L) similar to that seen in normal (Fig. 6O) and aged (Fig. 6P) adults as well as
children with DS [26]. Although it remains to be determined whether numerical differences
exist in CBF neuron number and cellular phenotype between prenatal, newborn, young, and
aged neurotypical and DS cases, the current findings suggest that the embryonic DS CBF
projection system already displays structural alterations similar to the degenerative phenotype
seen in AD and older individuals with DS [115, 138].
Adult cholinergic basal forebrain connectome in AD and DS
Since the classic reports of CBF neuron and cholinergic cortical dysregulation and the
involvement of NGF and its cognate receptors in this process in AD, there have been numerous
reviews published on this topic [120, 129–133, 139–141]. By contrast, there are only a few
investigations of cholinotrophic basal forebrain dysfunction in adults with DS [11, 142]
compared to healthy aged controls using modern cytochemical and quantitative counting
methods. Our group performed qualitative and quantitative investigations of cholinotrophic
degeneration in DS (mean age 52.2 years) compared to aged healthy controls (mean age 66.4
years) [115, 138] (Fig. 6R–W). Unbiased counting estimates revealed an average of 245,282
TrkA positive neurons in aged controls compared to 130,140 in DS, indicating 47% fewer CBF
neurons in DS compared to aged controls (Fig. 6Z). The loss of CBF neurons within the nbM
was confirmed by Nissl staining [115] (Fig. 6X and Y), suggesting a frank loss of cells in DS
[119, 143]. This contrasts to observations showing a phenotypic downregulation of both
p75NTR and TrkA but not ChAT containing CBF neurons during the progression of AD [116,
144] suggesting that different mechanisms underlie CBF neuron degeneration between these
disorders. We also found reduced frontal cortex TrkA protein levels in DS compared to aged
Fig. 6
(A–C) Low-power images showing p75NTR immunoreactivity (-ir) within the basal forebrain at
gestationweeks21 (A)and27(B) in theneurotypical andDSweek18 (C) fetus. (D,E)p75NTR-ir profiles
within the basal forebrain at neurotypical embryonic week 20 (D), 27 (E), and DS week 18 (F, G).
Note the healthy appearing p75NTR-ir neurons (embryonicweek 20 and 27) (D, E) and fibers (week 20)
(H) in neurotypical compared to DS p75NTR-ir dystrophic neurons at embryonic week 18 (G) and
fibers (I). Also note the healthy appearing neuron in the DS case (F). (J, K) p75NTR-ir neurons
within the subplate zone of the neurotypical embryonic week 20 (J) and DS 18-week neocortex (K).
(Continued)
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 27
28 Chapter 2
controls similar to AD, which positively correlates with lower cognitive test scores [145].
Interestingly, we reported a de novo expression of multipolar cortical p75NTR immunoreactive
neurons in AD and DS [135, 136], suggesting cellular plasticity in the face of pathologic
degeneration in both disorders.
More recently, we observed an increase in cortical levels of the precursor molecule for NGF,
proNGF [146] but not the mature NGF protein in the human AD cortex [130, 147], which
preferentially binds to p75NTR and with lesser affinity to the TrkA receptor [147]. Coinciding
with the increase in proNGF there is a reduction in TrkA but not p75NTR cortical and
hippocampal protein levels during the onset of AD [148–150]. Together, these findings suggest
that the accumulation of proNGF combined with a reduction in TrkA indicates a shift in
survival to cell death signaling via retrograde transport resulting in CBF neuron dysfunction
possibly through apoptosis [130, 131, 147]. In this regard, proNGF isolated from AD cortex
induces apoptosis in neuronal cell cultures via an interaction with the p75NTR receptor by a
mechanism dependent upon γ-secretase shedding of the receptor [151]. Whether these
mechanisms occur in DS requires investigation.
Recently, the McGill group reported increased proNGF cortical levels in DS [152], suggesting
that similar mechanisms underlie NGF neurotrophic dysfunction in both AD and DS. However,
a different mechanism of action has been proposed that is related to defective metabolic
processing of proNGF into mature NGF [153, 154] that underlies the accumulation of proNGF
as opposed to a transport defect in DS and AD [130, 131, 147, 155]. This hypothesis is based
upon data showing a decrease in the plasminogen-tPA-neuroserpin metabolic loop in brain
tissue, which processes proNGF combined with enhanced matrix metallopeptidase 9 (MMP9)
Fig. 6, cont’d
Note the reduced neuronal number and shorten processes in the neocortical subplate zone in DS
compared to the neurotypical fetus. (L, M) Images of the double immunostained p75NTR/ChAT
(brown) and AT8 (black) nucleus basalis profiles in a 6-month-old female DS (L), 46-year-old male DS
without (M) and with dementia (N), 56- (O) and 77-year (P) old female control and a 79-year-old
female AD (Q) case. Note the lack of AT8 tau immunostaining in the nucleus basalis of a 6-month-old
DS (L) and both healthy controls (O, P) compared to the globose appearing p75NTR/AT8 positive
NFTs (black arrows) as well AT8-ir NTs in the nucleus basalis of DS subjects without (M) and with
dementia (N) and AD (Q). (R–W) TrkA-ir neurons within the anterior medial division of the nucleus
basalis in a control subject (R and S), Down’s cases with mild (T and U), and a severe reduction in
TrkA-positive neurons (V andW). X and Y, Images of anterior nucleus basalis neurons stained for Nissl
substance showing a greater number of Nissl-stained magnocellular neurons in the aged control
(X) relative to DS (Y). (Z) Histogram showing a significant reduction in the total number of TrkA-
immunopositive neurons in the nucleus basalis of individuals with DS, AD compared to controls.
Abbreviations: DSD+, demented DS; DSD�, nondemented DS; HC, healthy control. The 50μm scale
bar in C applies to A and B panels; scale bars: D, E, G, H¼50μm and C¼20μm; The 100μmscale bar
in K and W applies to J and S, U panels respectively; The 200μm scale bar in V applies to R and
T panels; scale bars: L¼100μm, O¼60μm, M¼40μm, N, X and Y¼50μm, P¼60μm, Q¼80μm.
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 29
activity, which underlies mature NGF degradation, resulting in reduced mNGF availability,
thereby increasing the imbalance between proNGF and mNGF via a different mechanism but
that again leads to loss of function in the CBF projection neurons in both DS and AD [133, 152,
156, 157]. Since proNGF is the major form of NGF found in the normal human brain, more
work is needed to determine the role that transport or metabolic loop defects or both play in
cholinotrophic dysfunction in both disorders.
Endosomal dysfunction in AD and AD
Another factor that may play a role in cholinotrophic neuron dysregulation is early endosomal
enlargement, which occurs even prior to amyloid and tau pathology in both AD and DS
with AD [158]. Since survival of CBF neurons is dependent upon retrograde transport of
proNGF/mNGF bound to its p75NTR and TrkA receptors, understanding deficits in signaling
endosomes that are required to support normal neuronal structure and function in AD and DS is
required.
Synaptic integrity in AD and DS
Since there are several excellent reviews of synaptic integrity during the progression of AD
[159, 160], we will briefly provide an overview of this topic in AD. Scheff and Price (2006)
investigated synaptic change in the hippocampal outer molecular layer in individuals with
amnestic MCI (aMCI) and early AD (eAD), which failed to show a significant difference
between the cognitive intact and aMCI cases, but eAD showed a significant loss in dentate
gyrus molecular layer synapses compared to control and aMCI [160]. Hippocampal stratum
radiatum revealed a significant 48% decline in CA1 pyramidal neurons in early and late stage
AD [161–164]. Others report a significant decline in synaptotagmin and synaptopodin in frontal
cortex compared to control and for synaptophysin and synaptopodin in parietal cortex [165].
Protein levels for the presynaptic vesicle marker, synaptophysin, were selectively decreased in
the superior temporal and inferior parietal cortex in severe AD compared to controls [166].
Cortical synaptotagmin levels remained stable from control to MCI to AD. Drebrin was
significantly reduced in several cortical areas in AD compared to controls but was
downregulated in MCI superior temporal cortex [166]. However, drebrin levels were
upregulated in the superior frontal cortex in MCI. Since drebrin is localized in postsynaptic
dendritic spines at excitatory synapses [167–169], it may play a role in synaptic plasticity and a
shift in drebrin levels may represent changes in dendritic spine density in AD.
Information about altered synaptic integrity in the fetal and adult DS brain is limited. A study of
fetal cortex revealed that synaptosomal nerve-associated protein 25 (SNAP 25) and αSNAP
were significantly reduced despite no neuronal loss at about gestation age 19weeks [170]. The
finding that drebrin, a marker for dendritic spines and the synaptosomal associated proteins
30 Chapter 2
alpha SNAP and SNAP 25 are significantly reduced indicates impaired synaptogenesis, which
may underlie degeneration of the dendritic tree and its arborization, which occurs from infancy
onward in the DS brain. Only in a few studies have synaptic proteins been examined in adult DS
brain. A loss of synapses, including a reduction of synaptophysin in adults with DS, indicates a
relationship between trisomy of APP and other genes near the APP locus and synaptic
dysfunction [171]. Recently, Rho GTPase effector p21-activated kinase 3 (PAK3) and Arp2,
proteins that promote filamentous actin stabilization, were examined using fluorescent
deconvolution tomography to determine postsynaptic PAK3 and Arp2 levels for parietal cortex
excitatory synapses in individuals with DS/AD and AD. Synaptic PAK3 was greatly reduced in
DS/AD and AD than controls, whereas Arp2 was also reduced in both disorders, but to a greater
extent in AD [172]. Since these proteins play a role in actin stabilization, which supports
synaptic plasticity, lessening of these proteins at the synapsemay be an early event in synaptic
spine dysfunction that effects cognitive decline in these disorders. FC synaptophysin protein
levels were significantly lower in DS/AD compared to DS alone but similar to AD, which was
related to various Aβmeasures [172]. This study also revealed that the gene for synaptojanin-1
(SYNJ1) located on chromosome 21, which is involved in synaptic vesicle recycling, was
overexpressed in DS/AD compared to DS alone and to an even greater degree than in sporadic
AD, suggesting that a synapse-related protein is differentially expressed in trisomy 21 [172].
Alterations in white matter (WM) tracts likely also play a role in defects in synaptic integrity.
DS cases as young as 35 years of age show loses in frontal cortex WM integrity that correlate
with cognitive function by neuroimaging [173], which is reminiscence of changes in WM
integrity seen during the progression of AD [174] (see Chapter 8).
Gene expression profiling in DS
During the last several years, gene expression profiling has been used to examine classes of
transcripts underlying the molecular pathogenesis that occurs during the progression of AD
within subcortical [102, 175–177] and limbic cortical structures [178–182]. By contrast, only a
few studies have investigated transcriptional alterations in the fetal and adult trisomy brain
using RNA sequencing (RNA-seq) and/or qPCR approaches [183, 184]. For example, the
transcriptome of the dorsolateral prefrontal cortex (DLPFC) from seven adults with DS (mean
age 58.6 years) and eight controls (mean age 47.9 years) was examined using gene profiling
[183]. Intriguingly, APP was not found to be significantly overexpressed in adult or fetal
DLPFC tissue but genes functionally linked to APP were upregulated in adult DS compared to
control brain, including chromosome 21 genes BACE2, S100B, and other genes not related to
chromosome 21 but associated with AD [e.g., apolipoprotein (ApoE), clusterin (CLU),
presenilin 1 (PSEN1), presenilin 2 (PSEN2), and microtubule-associated protein tau (MAPT)]
[185, 186]. These transcript changes suggest disturbances in the normal processing and function
of the amyloid precursor protein, which may predispose the DS brain to an increase in Aβ
production. In addition, overrepresented and dysregulated genes associated with developmental
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 31
pathways included lipid transport, cellular proliferation, and transcriptional regulation [185,
186]. Downregulated developmental genes were members of the distal-less family of genes
(DLX2) in both fetal and adult trisomy and DLX6 only in the adult DS brain. The DLX family
of genes play a key role in the development of forebrain GABAergic neurons [187], which
express deficits in DS cortical layers [188]. Other developmental genes upregulated were
related to Notch signaling (e.g., NOTCH2), which plays a role in early embryogenesis.
Cytoskeleton and vesicle function/trafficking transcripts were overrepresented similar to a
proteomic study of AD and DS brain tissue [183]. Lockstone et al. reported that cytoskeleton
organization and synaptic transmission genes were downregulated, suggesting that despite
enlarged vesicles and/or activated vesicle recycling processes, cells were unable to compensate
fully for synaptic dysregulation in DS [183]. Immune-related classes of genes were also
upregulated in the adult and fetal DS brain, which also may be linked to AD pathology.
Single cell expression profiling in DS
As indicated previously, cell expression data exist from only a few population-based profiling
studies using whole brain tissue homogenates from DS cases [183]. Toour knowledge, the
publication by our group was the first to employ single cell gene profiling of FC layer V and VI
neurons containing the pretangle marker pS422 obtained postmortem from adults with DS with
(DSD+, n¼12) and without (DSD�, n¼6) dementia ranging from 42 to 60 years of age [34].
Quantitative analysis revealed 64 transcripts associated with amyloid and cytoskeleton/tau,
glutamatergic, cholinergic, and monoaminergic metabolism, endocytosis, and intracellular
signaling that were significantly dysregulated between DS groups. Transcript levels related to
APP/Aβmetabolism including β-secretase (Bace1), γ-secretase components Nicastrin (Ncstn),
presenilin enhancer 2 (Psenen), anterior pharynx defective 1A subunit L(Aph1a), α-secretase
ADAM10 component (Adam10), and the calcium binding protein calsenilin (Kcnip3)-,
lipoprotein metabolism: very low density lipoprotein receptor (Vldlr), the apolipoprotein serum
amyloid A4 (Saa4) and the high density lipoprotein (Hdlbp), tau (Mapt5), prion (Prnp), and
parkin7 (Park7/Dj1) were significantly downregulated only in DSD+ (Fig. 7A). A lack of
differential regulation of APP expression in single FC neurons in DS is consistent with previous
findings seen in individual CBF [175, 179, 181, 189, 190] and hippocampal CA1 pyramidal
neurons in AD and with whole tissue homogenate profiling in DS [183]. Although expression
levels of the cholesterol carrier APOE, a major risk factor for AD, which is involved in Aβ
production via α-secretase regulation were comparable between DS groups [191], APOE
upregulation was reported in homogenates of prefrontal cortex between DS and controls [183].
Since APOE ε4 is associated with greater deposition of Aβ protein in adults with and without
DS [158, 192], this may explain the similarities in Aβ load reported in demented and
nondemented DS [34]. Cholesterol related genes Vldlr, Hdlbp, and Saa4 were only
downregulated in DSD+, suggesting that low cholesterol neuronal uptake might favor an APP
nonamyloidogenic pathway in DSD+ (Fig. 7A). By contrast, only a single tau isoform,Mapt5,
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0.0
0.2
0.4
0.6
0.8
1.0 DSD- 
DSD+ 
A B
C D
Fig. 7
(A–D) Histograms showing downregulation in mean value of relative density of mRNA transcripts related to (A) APP/Aβmetabolism [β-
secretase (Bace), γ-secretase (Ncstn, Psenen, Aph1a), α-secretase (Adam10), and calsenilin (Kcnip3)], lipoprotein metabolism (Vldlr, Saa,
Hdlbp), tau (Mapt5), prion (Prnp), parkin7 (Park7/Dj1), (B) glutamatergic neurotransmission [mGluR1 (Grm1) and 5 (mGluR5) (Grm5)
receptors, NMDA receptor subunits 2B (Grin2b) and 2D (Grin2d), Glutamate receptor ionotropic, kainate 2 (Grik2), AMPA ionotropic
glutamate receptor 3 (Gria3), transporters 1 (Slc1a3) and 3 (Slc1a1), glutamate receptor interacting protein 1 (Grip1) and 2 (Grip2) and
transglutaminase 1 (Tgm1)], (C) cholinergic [acetylcholinesterase (Ache), butyrylcholinesterase (Bche), muscarinic receptor M2 (Chrm2),
M4 (Chmr4), cholinergic receptor nicotinic alpha 2 (Chrna2), 3 (Chrna3), 4(Chrna4), and 7 (Chrna7) subunits] and monoaminergic [α1B
adrenergic receptor (Adra1b), serotonin receptor 1B (Htr1b), 2C (Htr2c), 3A (Htr3a), 7 (Htr7), monoamine oxidase A (Maoa) and
B (Maob), noradrenaline transporter (Slc6a2), serotonin transporter (Slc6a4), and tyrosine hydroxylase (Th)], neurotransmission and
(D) autoimmune regulator (Aire), FAM3B protein (Fam3b), homeobox 2 (Emx2) transcript factor, the autophagy protein 5 (Atg5), TNF
type-1 (Tradd), protein p53 (Tp53), caspase 6 (Casp6), BCL2-associated X (Bax), several cyclin proteins [cyclin B1 (Ccnb1), cyclin D1
(Ccnd1), and cyclin D2 (Ccnd2)], G-proteins [(Rgs2), (Rgs3), (Rgs4), (Rgs9), (Rgs10)], adenylate cyclases [(Adcy1), (Adcy6)], proto-
oncogene c-fos (Fos), Jun (Jun), protein fosB (Fosb), and transcript factor EB (Tfeb) mRNAs in frontal cortex pS422 positive NFTs in
demented compared to nondemented subjects with DS, while transcript expression levels for the Sirt6 gene were upregulated in frontal
cortex pS422 positive NFTs in demented compared to nondemented subjects with DS (Mann-Whitney rank sum test, P < .05).
34 Chapter 2
was differentially expressed betweenDSD� andDSD+ subjects. Taken together, these findings
suggest that APP amyloidogenic and nonamyloidogenic proteolytic pathways, but not the
expression of APP, are downregulated in the FC in DSD+ compared to DSD�. This may affect
APP protein augmentation, which is supported by our findings showing an increase in FC APP
plaque burden in DSD+ [34].
Glutamatergic genes including glutamate receptor 1 (mGluR) 1 (Grm1) and 5 (Grm5), NMDA
receptor subunits 2B (Grin2b) and 2D (Grin2d), kainate receptor 2 (Grik2), AMPA receptor 3
(Gria3), excitatory amino acid transporter 1 (Slc1a3) and 3 (Slc1a1), glutamate receptor
interacting protein 1 (Grip1) and 2 (Grip2), and transglutaminase 1 (Tgm1) transcript levels
were downregulated only in DSD+ (Fig. 7B). Similarly, the cholinergic genes
acetylcholinesterase (Ache), butyrylcholinesterase (Bche), muscarinic acetylcholine receptor
M2 (Chrm2) and M4 (Chmr4), cholinergic receptor nicotinic alpha 2 (Chrna2), 3 (Chrna3), 4
(Chrna4), and 7 (Chrna7) subunits as well as the monoaminergic-related neurotransmission
α1B adrenergic receptor (Adra1b), serotonin receptor 1B (Htr1b), 2C (Htr2c), 3A (Htr3a), 7
(Htr7), monoamine oxidase A (Maoa) and B (Maob), noradrenaline transporter (Slc6a2),
serotonin transporter (Slc6a4), and tyrosine hydroxylase (Th) were downregulated in DSD+
compared to DSD� (Fig. 7C). These findings suggest that dysregulation of ascending cortical
neurotransmitter systems plays a role in the molecular pathogenesis of dementia in DS, which is
translationally relevant, as these systems are likely targets for therapeutic intervention in DS as
well as AD.
Transcript levels of the autoimmune regulator (Aire) and FAM3B protein (Fam3b), both
located on chromosome 21, the development neocortical-related empty spiracles homeobox 2
(Emx2) transcript factor, the autophagy protein 5 (Atg5), were downregulated in DSD+
compared to DSD�. Cell survival/death transcripts tumor necrosis factor receptor (TNF) type-1
(Tradd), p53 (Tp53), caspase 6 (Casp6), BCL2-associated X (Bax), cell cycle cyclin proteins:
cyclin B1 (Ccnb1), cyclin D1 (Ccnd1), and cyclin D2 (Ccnd2), G-proteins: (Rgs2), (Rgs3),
(Rgs4), (Rgs9), (Rgs10)- and adenylate cyclase 1 (Adcy1) and 6 (Adcy6) transcripts were
downregulated in DSD+ vs DSD�. Proto-oncogenes and the transcript factor EB (Tfeb) were
significantly downregulated in DSD�. Expression levels of the epigenetic gene Sirt6 were
significantly upregulated in DSD+ compared to DSD (Fig. 7D). Together, these findings
indicate that genes related to the immune system and cellular homeostasis display a greater
decline in NFT-bearing FC neurons in DSD+ compared to DSD�.
Interestingly, no significant differences in transcript expression were seen for genes located on
chromosome 21 [e.g., amyloid precursor protein (App), Down syndrome critical region 1
(Dscr1)], as well as genes not-locatedon HSA21 [e.g., caveolin 2 (Cav2), drebrin (Dbn1),
doublecortin (Dcx), apolipoprotein E (Apoe), sirtuin 3 (Sirt3), and alpha synuclein (Snca)].
Interestingly, the dual specificity and tyrosine phosphorylation-regulated kinase1A (Dyrk1A)
gene was not changed between DS groups but was significantly upregulated in DS compared to
Alzheimer’s neuropathology in Down syndrome: From gestation to old age 35
controls [183], suggesting a one hit upregulation of Dyrk1A in DS compared to controls, which
remain unchanged between DSD� and DSD+. These observations suggest that another trisomy
21 gene or one found on another chromosome likely underlie the increase in tau pathology seen
in DSD+ [34]. Moreover, the differential genetic signature of FC pS422 bearing neurons
between DSD� and DSD+ suggests a possible genetic signature for dementia in DS that may
stimulate therapeutic interventions that arrest cognitive decline in this special population that
represents a unique genetic model of AD.
Summary
This chapter describes features of the DS brain suggesting that structurally and biochemically
there are important differences pre- and postnatally relative to neurotypically developing
brains. Compared to the neurotypical fetal brain, cortical sulcal development is delayed, the
CBF connectome displays degenerative events reminiscent of adults with AD, the cerebellar
cortex displays phosphorylated tau, and synaptic integrity is disrupted. Despite these
gestational abnormalities the fetal DS brain does not contain classic amyloid plaque or NFT
pathology. The adult temporal, frontal, and occipital poles appear blunted, cerebellum is
shrunken and displays patches of diffuse amyloid, ventricles are enlarged, neo and limbic
cortical regions are reduced in size (e.g., hippocampus), select striatal cholinergic interneurons
display a normal morphology despite containing NFT pathology, CBF neurons display globose
NFTs, and amyloid plaque deposition occurs prior to NFT pathology similar to AD. Cortical
amyloid plaque load is similar in both demented and nondemented DS but cortical NFT
pathology is greater and more advanced in DSD+. Cholinotrophic dysfunction is similar in both
AD and DS. Gene profiling of brain homogenates revealed numerous dysregulated transcripts
in DS vs control. Single cell gene array analysis of tau containing layer V and VI FC cortical
neurons revealed a downregulation of various transcripts in individuals with DSD+ compared
to DSD�. Other features of the aging DS brain are described in more depth in other chapters
and include neuroinflammation (Chapter 3), cerebrovascular pathology (Chapter 4), and
oxidative damage (Chapter 5). In combination, there is much work yet to be done to understand
the temporal events of AD pathogenesis in DS, in identifying key molecular pathways that in
turn, could serve as targets for future intervention studies to slow or prevent AD in DS.
Acknowledgments
Support by NIH P01AG14449, R01AG043375, R01AG061566, AZADC Consortium and the Bright Focus
Foundation CA2018010. We thank Laura Mahady for editing the manuscript.
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