PROJECT SUMMARY

We will test the hypothesis that choroid plexus epithelial cells (CPECs) are affected in late onset Alzheimer's
Disease (AD) associated with the type 4 isoform of apolipoprotein E (APOE4). At least one copy of APOE4 is
present in 56-65% of people with AD, and two copies are highly predictive of AD. APOE4 has been implicated
in faulty clearance of the toxic Aβ peptide associated with AD, as well as in numerous other AD-related
functions in various cell types. Rodent CPECs are known to remove toxins from the cerebrospinal fluid (CSF),
express high levels of APOE, and take up and transport Aβ peptides. However, human CPECs have not been
well studied, in part due to the difficulty in acquiring healthy human cells. Our laboratory has developed a
protocol to derive human CPECs from pluripotent stem cells, which provides the opportunity to study basic
human CPEC functions and their roles in AD and other diseases. We propose in Aim 1 to derive CPECs using
existing induced pluripotent stem cells (iPSCs) from APOE4/4 AD patients and from their isogenic APOE3/3
counterparts that have been edited using CRISP/Cas9. We will monitor the CPEC derivations for changes in
differentiation efficiency, then test the specific hypothesis that Aβ uptake is impaired in APOE4/4 CPECs. In
Aim 2, we will characterize the transcriptomes of individual human CPECs using single cell RNA sequencing
(scRNAseq) to look for gene expression changes in CPEC subpopulations that correspond to the Aβ uptake
findings from Aim 1. Based on a prior study that examined neurons, astrocytes, and microglia derived from
APOE isogenic iPSCs, we anticipate a broad spectrum of gene expression differences that are unique to
CPECs and predictive of altered functions. We will then validate scRNAseq findings by RT-qPCR and
immunostaining, then cross-validate in vivo by immunostaining postmortem choroid plexus specimens from
patients with known APOE genotypes. We envision this R21 proposal leading to subsequent R01 submissions
that extend this work to CPECs derived from additional isogenic pairs involving APOE and other AD risk
genes, to test additional emergent hypotheses regarding altered CPEC functions in AD, and to explore
potential therapeutic interventions to correct impaired CPEC functions.
PROJECT NARRATIVE

Late onset Alzheimer's Disease (AD) is a major health problem in the aging US population, and presence of
the type 4 isoform of apolipoprotein E (APOE4) is strongly associated with the disease. Choroid plexus
epithelial cells (CPECs), which express APOE at high levels, are likely to be involved in clearing the toxic beta-
amyloid peptides that cause AD, but human CPECs have not been available to test. We will use our unique
procedure to derive human CPECs from stem cells of AD patients carrying APOE4, and from patient cells that
have had their APOE gene "corrected" to the type 3 isoform. Using these cells, we will test the hypothesis that
APOE4 CPECs are deficient in beta-amyloid uptake and possess single cell transcriptomes that predict
deficient beta-amyloid clearance, thereby accelerating the onset and progression of AD.
SPECIFIC AIMS

As the most common cause of dementia affecting older adults, Alzheimer's disease (AD) has long been
an NIH priority. Despite years of research, a complete understanding of AD has been elusive, and treatment
options are limited. A small percentage of patients inherit an early-onset disease, having mutations in genes
related to amyloid precursor protein (APP) or enzymes that cleave APP to produce beta-amyloid peptides (Aβ),
which aggregate to form plaques in AD. A particular isoform of apolipoprotein E (APOE ε4 or APOE4) is found
in a large percentage of patients with the more prevalent late-onset AD, and APOE4 is implicated in altered
interactions with Aβ that are thought to reduce Aβ clearance.
Choroid plexus epithelial cells (CPECs) reside in all four brain ventricles, where they produce the
cerebrospinal fluid (CSF) and express high levels of APP, Aβ-processing enzymes, Aβ chaperones including
APOE, and Aβ transport proteins. Studies using rodent cells suggest that CPECs contribute significantly to Aβ
secretion into CSF, thus directly impacting the material (CSF) used for AD diagnostic workup. In addition, rat
and mouse CPECs take up and transport Aβ from the CSF to blood, suggesting an important role of CPECs
in removing Aβ from the CNS, possibly involving APOE. Rodent CPECs have also been identified as
targets of Aβ toxicity, consistent with the reduced CPEC thickness and dysfunction seen in AD patients.
Although rodent studies implicate CPECs in many important aspects of APOE and Aβ biology, the
ultimate goal of AD research is to develop drugs and therapies that will benefit humans. Because rodent
models only partially capture human biology and have not translated into effective therapies, there is great
need for research using human cells. So far, the extremely limited research on human CPECs in AD and other
diseases has been compromised by autolysis and small amounts of available material. Our lab has
developed efficient procedures to derive CPECs from human embryonic and induced pluripotent stem
cells (iPSCs), providing a unique opportunity to test hypotheses regarding CPEC roles in AD.
In preliminary studies, we readily observed fluorescent Aβ1-42 uptake by human embryonic stem cell-
derived CPECs, as well as CPEC gene expression signatures by single cell RNA sequencing (scRNAseq). In
this proposal, we utilize these approaches on isogenic iPSC lines derived by the UCI ADRC iPSC core and
colleagues. These iPSC lines were derived from two APOE4/4 patients with dementia (one male, one female),
which were converted to APOE3/3 lines using CRISPR/Cas9 technology. Using these isogenic lines, we will
measure Aβ uptake by CPECs to test the hypothesis that APOE4 CPECs are deficient in Aβ uptake. To
identify CPEC gene expression networks and pathways affected by APOE genotype, we will perform
scRNAseq with established UCI collaborators who have expertise in scRNAseq data acquisition and analysis.
Validation of APOE4-associated differences in gene expression will be sought using postmortem choroid
plexus from patients with known APOE genotypes. We envision that the results of these studies will form the
basis for subsequent R01 proposals testing AD-related functional changes and mechansims in CPECs, and
extending the isogenic iPSC approach to other AD risk genes highly expressed by CPECs.

Aim 1: Determine the impact of APOE isoform on Aβ uptake by human CPECs. We hypothesize that
APOE4/4 human CPECs will be deficient in Aβ uptake. For both patients, three independent 4/4 and 3/3 clonal
lines are available. We will perform CPEC derivations on these lines (12 derivations total) using a paired
experimental design that yokes the independent variable (APOE isoform). To assess the impact of APOE
genotype on CPEC differentiation, we will monitor CPEC derivations by morphology and immunostaining for
CPEC markers. After establishing an optimal combination of Aβ concentration and exposure time, we will
compare uptake of monomeric and fibrillated fluorescent Aβ peptides, including the toxic Aβ1-42, the more
benign Aβ1-40, and a scrambled peptide, at the single cell level using confocal microscopy and flow cytometry.

Aim 2: Determine the impact of APOE isoform on the transcriptomes of individual human CPECs. We
hypothesize that individual APOE4/4 human CPECs will have altered gene expression networks predictive of
deficient Aβ uptake. Cells from the paired derivations will be subjected to scRNAseq. Existing and novel
scRNAseq algorithms (e.g. SoptSC), as well as gene ontology and interaction databases, will be used to
identify CPEC networks and functions affected by APOE genotype, particularly those involved in Aβ clearance.
We will use immunostaining and Western blotting of postmortem choroid plexus with known APOE genotypes
from the UCI autopsy service and ADRC biorepository to cross-validate consistently large differences identified
by scRNAseq.
A. SIGNIFICANCE

Alzheimer's Disease (AD) and Apolipoprotein E isoform 4 (APOE4). In 2018, 5.7 million Americans are
estimated to be living with AD, the vast majority of whom develop a late-onset form of the disease that is
strongly associated with carrying the APOE4 allele [1]. Indeed, 56-65% of AD patients in the USA have at least
one copy of APOE4 [2, 3] and about 90% of homozygotes (APOE4/4) may develop AD [4]. Numerous
differences have been identified for the APOE4 protein compared to the neutral APOE3 and protective APOE2
forms, including differences in interactions with toxic beta-amyloid (Aβ) peptides, which aggregate to form the
plaques that define AD pathologically [5-10]. APOE4 is associated with reduced clearance of Aβ [8, 11], but
APOE4 also has been associated with increased production and secretion of Aβ [5, 12-14], suggesting
interactions with multiple Aβ-related pathways.
Choroid plexus epithelial cell (CPEC) involvement in
AD and Aβ biology. CPECs, which reside in all four brain
ventricles, are an understudied cell type that secretes
the cerebrospinal fluid (CSF) and many CSF proteins,
forms the blood-CSF barrier, transports nutrients and
toxins between the blood and CSF, and comprises a
gateway for immune cells [15] (Fig. 1). There is also
growing evidence, mostly from rodent studies, that
CPECs are involved in Aβ metabolism and AD.
FACS-sorted embryonic mouse CPECs show high
levels of expression of AD-related genes including
ApoE, the 96th most abundant transcript by bulk
RNAseq, and ApoE also is detected in the secretome of Figure 1: Human brain and choroid plexus anatomy.
(A) Modified Frank Netter illustration. Choroid plexus (red)
cultured mouse CPECs [16]. Based on public GEO resides in the brain ventricles. Other arrows indicate CSF
Profile data, ApoE expression by adult mouse choroid flow. (B) Microscopic anatomy. CPECs form a highly-
plexus (ChP) is the highest among brain regions (Fig. 2). polarized simple epithelium coupled by tight junctions.
The amyloid precursor protein (APP) and secretases
involved in Aβ cleavage are also abundant in CPECs
[16-31], and cells of the human ChP secrete Aβ [29, 32].
Aβ secretion by CPECs into the CSF would be clinically
relevant, since CSF Aβ composition and ratios have
emerged as early biomarkers for AD [1]. Rodent CPECs
also take up and transport Aβ peptides [33-46],
consistent with their high-level production of Aβ
chaperones such as Clusterin and Transthyretin (TTR) in
addition to ApoE [16, 27, 28, 47-52]. CPECs express
multiple receptors for Aβ, ApoE and Clusterin that likely
mediate their uptake (e.g. LRP1, LRP2, VLDLR,
ApoER2) [16, 28, 49, 53-58]. In addition to their
involvement in Aβ metabolism, CPECs and the ChP
exhibit pathological changes in both AD [30, 59-65] and Figure 2: ApoE expression in two strains of adult mice
(public GEO microarray data). Among brain regions, ApoE
mouse models of AD [66], and acute exposure of rodent expression is highest in the choroid plexus (arrows).
CPECs to Aβ peptides causes inflammatory responses https://www.ncbi.nlm.nih.gov/geoprofiles/14944887
[67-69], barrier disruption [68, 70], and oxidative damage
[70, 71].
Use of stem cell-derived neural cells to study AD. Most data on CPEC function in Aβ metabolism have
come from rodent studies, but AD is a uniquely human disease. Rodent models of AD involving overproduction
of Aβ only partially capture the clinically relevant biology of Aβ and AD, particularly its late onset form, which
limits their utility in developing therapies [72-74]. We identified 26 publications with insights relevant to AD,
which emerged by studying neurons, astrocytes, and microglia derived from human induced pluripotent stem
cells (iPSCs) [75-100]. These iPSCs were derived from AD patients and controls, or from control iPSCs that
were gene-edited to contain APP or presenilin mutations associated with familial AD. The majority of AD iPSC
studies have involved the rare, early onset, familial forms of AD involving mutations in APP or presenilins,
which cause increased Aβ production. Recently, however, isogenic iPSC lines were applied to study the more
prevalent APOE4-associated late onset AD [100]. This study utilized iPSCs from a control APOE3/3 subject,
which were edited to APOE4/4 using CRISPR/Cas9 technology while leaving the rest of the genome
unmodified [100]. After the isogenic iPSCs were differentiated into neurons, astrocytes, and microglia, RNAseq
revealed profound changes to the transcriptome associated with APOE genotype, and the changes were
specific to each cell type [100]. Cell type-specific functional differences between the APOE genotypes were
also evident, and these were largely validated using another isogenic pair of iPSCs from an AD patient, in
which the APOE4/4 genotype was edited to APOE3/3 [100]. Despite their likely importance in AD, CPECs were
not included in this or any other study using AD iPSCs. Having developed and patented a method for deriving
CPECs from pluripotent stem cells [101], we can address this omission using isogenic iPSC lines developed by
the UCI Alzheimer Disease Research Center (ADRC) iPSC core and collaborator Poon (see letter).

B. INNOVATION

Human CPEC biology. CPECs are remarkably understudied, in part due to past perceptions about their
"uninteresting" functions (e.g. buoyancy) and a lack of availability of living human cells. Despite the pivotal
roles that CPECs are now known to play in maintaining brain health, only 15 of the >15,000 abstracts at the
2017 Society for Neuroscience annual meeting contained "choroid plexus" as a keyword. The work proposed
here will not only represent the first human stem cell-based approach into how these cells contribute to AD, but
it also will vastly increase our basic knowledge of human CPEC biology. Our unique CPEC derivation
procedure [101], recently made more efficient, rapid, and simple, makes this new line of research possible.
CPEC heterogeneity. We propose to use scRNAseq to characterize differences between isogenic pairs of
derived CPECs. Microarray analyses have typified studies of differential gene expression by other cell types
derived from AD iPSCs [78, 85, 88, 100]. These "averaged" population analyses lose valuable information
regarding cell subtypes that invariably reside within tissues. Our use of scRNAseq should allow us to identify
CPEC subtypes, to discriminate them from other cell types in the iPSC cultures, and to identify the CPEC
subclasses that are most relevant to APOE, Aβ, and AD biology. CPEC heterogeneity is relatively unexplored
in other species as well. These findings should represent significant conceptual innovations for the field.

C. APPROACH

Preliminary Data
Improved CPEC derivation from human pluripotent
stem cells. Our original protocol involved BMP4
treatment of ES cell aggregates and resulted in
efficiencies of only 5-10% [101]. We have since
simplified and greatly improved the efficiency of these
derivations using monolayer cultures grown on feeder-
free (Matrigel) substrates followed by rapid neural
induction (Gibco) and BMP4 treatment. We optimized
culture conditions (clump sizes, densities, and ROCK
inhibitor duration),
neural induction (timing and
duration), and BMP4 exposure (concentration, timing,
and duration) (Fig. 3A). With the new protocol, CPECs
differentiated in relatively pure sheets and islands,
routinely comprising 30-60% of cells as judged visually
by immunostaining for mature CPEC markers including
TTR (cytoplasmic), AQP1 (apical microvilli), AE2
(basolateral membrane), CLDN1 (tight junctions),
ARL13B (multiple primary cilia, a distinctive feature of
CPECs [102]), OTX2 and LMX1A (nuclear). With
increasing time in culture (up to 60 days without
requiring replating), CPECs expressed progressively
higher levels of mature CPEC markers in more complex Figure 3: Improved CPEC protocol. (A) Schematic. (B-D)
3-D folds (Fig. 3B-D) reminiscent of the endogenous Edge of CPEC island with strong TTR and OTX2
ChP (Fig. 1B). CPECs in these folds exhibited expression (50-day derivation). (E) Confocal view of apical
appropriate apicobasal polarity based on staining for AQP1, basolateral AE2, and non-CPEC core (arrows; 20-
day). (F) Flow cytometry showing >90% of dissociated
AQP1, AE2 (Fig. 3E) and ARL13B (data not shown). In
viable single cells are TTR-positive CPECs (35-day).
the relatively minimal media we use, CPECs remain
healthy while many non-CPECs perish or fail to survive dissociation, such that CPECs often represent the vast
majority of viable dissociated cells by flow cytometry (Fig. 3F). The improved derivation protocol has now been
used by two collaborating labs to successfully derive CPECs from human iPSCs or ESCs (data not shown).
Further optimizations and enrichment strategies are being developed, but are not necessary for this proposal,
which focuses on assays with single cell resolution.
Robust Aβ1-42 uptake by derived human CPECs. CPECs
derived from H1 ESCs (APOE3/3, male) expressed APOE protein
(Fig. 4A) and took up monomeric fluorescently-tagged Aβ1-42 (10
μg/ml) after 24 hour incubation. Aβ uptake resulted in bright
intracellular fluorescence that was punctate (potentially endosomal)
in TTR-positive CPECs (Fig. 4B) in widespread and robust fashion.
Single cell RNA-seq of derived human CPECs. In collaboration
with Drs. Kessenbrock and Nie at UCI, we have begun to use
Figure 4: APOE expression and Aβ1-42
scRNAseq to study individual human CPECs and CPEC subtypes. uptake by derived human CPECs. (A) APOE
Figure 5 shows a 2-D projection of scRNAseq data after 35-day expression (red) in TTR-positive CPECs
derivation from H1 hESCs, dissociation using TrypLE Express, and (green). (B) Confocal orthogonal views of 5-
FACS sorting for viable single cells. TTR immunostains of residual FAM Aβ1-42 (green) uptake into the TTR-
cytospun cells after loading onto the 10X Genomics Chromium positive cytoplasm (red) of a human CPEC
(33-day derivation).
platform (UCI Genomics High Throughput Facility) confirmed that
the majority of loaded cells were CPECs. Shallow scRNAseq data
(8,668 cells, 40,118 mean reads/cell, 2,556 median genes/cell)
were analyzed using a "similarity matrix optimization for clustering,
lineage, and signaling inference" algorithm (SoptSC) developed by
the Nie lab, which, unlike other algorithms, provides a single
mathematical framework for identifying cell subpopulations, marker
genes, pseudotemporal order, and lineage relationships without
supervision [103]. Eigenvalue-based thresholding clearly defined
eight subpopulations: clusters 1-4 are presumptive CPECs,
whereas cluster 5 and clusters 6-8 represent neurons and cycling
neural progenitors, respectively (Fig. 5A). Pseudotemporal
ordering and lineage mapping suggest two CPEC lineages that
differ by age/birth order, but arise from a common progenitor
(cluster 2) (Fig. 5B,C), presumably an early pre-neurogenic
neuroepithelial cell [101]. While speculative, the two human CPEC
lineages may reflect the known lineage differences between
forebrain (lateral ventricle) and hindbrain (fourth ventricle) CPECs
in mice [16]. Not surprisingly, based on the pseudotemporal
ordering (Fig. 5B), the four CPEC clusters varied in their degrees
of expression of functional genes involved in secretion, transport,
and barrier formation. Importantly, the CPEC clusters also
expressed APOE and other Aβ-related genes (e.g. TTR, APP,
CLU) at variable levels and were particularly high in the oldest
CPECs (cluster 1).

Aim 1: Determine the impact of APOE isoform on Aβ uptake by

human CPECs
Rationale. APOE is directly implicated in Aβ uptake by other
cells [5], and H1-derived human CPECs (APOE3/3, male) express
APOE and readily take up Aβ1-42 (Fig. 4). Rodent CPECs also take
up Aβ1-40 [33-46], and secreted ApoE plays a likely role in this
uptake. ApoE is produced and secreted by rodent ChP [50, 52], Figure 5: SpotSC analysis of scRNAseq
APOE binds Aβ peptides [5, 6, 8-10, 104], and rodent CPECs data (35-day derivation). (A) Cluster analysis.
express high levels of ApoE receptors that mediate Aβ uptake [16, (B) Pseudotemporal ordering. (C) Lineage
28, 49, 53-58]. Both binding and the consequence to Aβ depend on analysis. Clusters 1-4 are the putative CPECs.
Cluster 5 is neuronal, while clusters 6-8 are
APOE isoform and its lipidation [5, 104, 105]. In turn, lipoprotein cycling neural progenitors.
composition differs by cell of origin, and CSF lipoproteins differ
from those released by astrocytes [106], raising the possibility of a CPEC origin for APOE in the CSF.
Astrocytes derived from APOE4/4 iPSCs are deficient in Aβ1-42 uptake compared to isogenic APOE3/3
astrocytes [100]. We therefore hypothesize that APOE4/4 human CPECs will be deficient in Aβ uptake.
Design and Methods. The isogenic iPSCs proposed for this study were derived from two living members of
the UCI ADRC longitudinal cohort (one male, one female), both of whom have dementia and APOE4/4
genotypes. Fibroblasts grown from their skin biopsies were reprogrammed to iPSCs using the 4-Yamanaka
factor CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher) by the ADRC iPSC core directed by
consultant Blurton-Jones (see letter). The two patient iPSC lines were then gene-edited to APOE3/3 genotype
using CRISP/Cas9 technology by collaborator Poon (see letter). From each original iPSC line, three separate
edited (APOE3/3) and sham-reprogrammed unedited clones (APOE4/4) were propagated and validated by
sequencing, karyotype, and pluripotency testing.
To maximize rigor, reproducibility, and analytical power for the APOE-based hypotheses, human CPEC
derivations will be performed as yoked pairs for the independent variable (APOE isoform). That is, one
APOE3/3 and one APOE4/4 clonal iPSC line from one patient will be cultured and processed in parallel. Using
this paired design, we will conduct 6 paired derivations, or 12 human CPEC derivations in total (2 patients, 2
genotypes, 3 clones each), for Aβ uptake (Aim 1) and scRNAseq studies (Aim 2).
The iPSCs will be differentiated into CPECs using our now-established protocol (Fig. 3A), described here:
iPSCs are grown in feeder-free conditions (Matrigel-coated 4-well chamber slides) and seeded at an optimal
density (2-4 clumps/mm2) of optimally-sized clusters (100-200 cells/clump) in TeSR E8 medium containing 10
μM ROCK inhibitor until clusters reach an average diameter of 150-200 μm, after which they are switched to
Gibco neural induction medium (NIM); this represents "day 0". After 1 day in NIM, when the earliest
neuroepithelial progenitors have begun to emerge, we add recombinant human BMP4 (R&D Systems) at 10
ng/ml in NIM. On day 5 (4 days in BMP4), NIM is changed to CPEC medium (DMEM/F12, heparin, non-
essential amino acids, N2), also containing 10 ng/ml BMP4. BMP4 is present for a total of 15 days, at which
point it is omitted. Cells are fed daily using half-volume media changes.
CPEC differentiation analysis: At days 20, 35, and 50 (Fig. 3), cells grown in parallel chamber slides will be
fixed and immunostained to monitor the progress of CPEC differentiation. Double labelling will be performed
for each reaction and detected using fluorescently-labeled secondary antibodies (Alexa 488 and 555).
Antibodies compatible for double labelling include TTR-CLDN1 (mature CPEC tight junctions), TTR-CK18
(early CPEC intermediate filaments), TTR-SOX2 (early CPEC/progenitor nuclei), TTR-NESTIN (early
CPEC/progenitor intermediate filaments), TTR-OTX2 (mature CPEC nuclei), AQP1-AE2 (mature CPEC apical
and basolateral membranes), and TTR-APOE (Figs. 3,4). Staining of isogenic pairs will be evaluated for
possible differences in maturation (disappearance of early markers, appearance of mature markers, degree of
polarity, 3-D complexity) and derivation efficiency (TTR-positive cell numbers or area).
Aβ uptake studies: Our first task will be to establish an optimal combination of Aβ concentration and
exposure time that yields high signal:noise at a steady state. H1-derived CPECs at day 50 (Fig. 3B-D) will be
exposed to varying concentrations (0.5, 1, 2, 4, 8 μM) of 5-FAM-labelled human Aβ1-42 (AnaSpec) for varying
amounts of time (6, 12, 24, 48, 72 hours), which span the successful 2 uM and 24 hour time point tested
initially (Fig. 4). Aβ1-42 uptake into TTR co-labelled CPECs will be examined by epifluorescent and confocal
microscopy, and suitable concentration-duration combinations will be determined qualitatively. If necessary,
fluorescence intensities will be quantitatively measured using ImageJ or Imaris software on microscopic
images, or by flow cytometry of dissociated cells (Fig. 3F). Additional durations will be tested, as needed, if a
steady state is not observed.
Using the selected concentration-duration combination, we will add FAM-labeled Aβ peptides (Fig. 4) to the
paired isogenic cultures at day 50. We will use the toxic human Aβ1-42 peptide, the more benign Aβ1-40 used in
many rodent CPEC studies, and a scrambled version of Aβ1-42 as a control for non-specific uptake. Peptides
will be prepared as monomers [46] or fibrillated oligomers with consultant Blurton-Jones, who generates
fibrillated Aβ oligomers routinely (see letter). CPEC uptake will be evaluated by epifluorescent and confocal
microscopy after 4% paraformaldehyde fixation, TTR immunostaining, and DAPI counterstaining (Fig. 4B). We
will first assess qualitatively whether Aβ intensities and intracellular patterns differ by APOE isoform. For
quantification, flow cytometry will be used primarily, with microscopic image analysis (as described earlier)
used for secondary confirmation. For flow cytometry, cells will be dissociated with TrypLE Express, incubated
with LIVE/DEAD fixable far red dead cell stain (ThermoFisher), fixed, then TTR immunostained (Fig. 3F). We
will characterize and compare statistically (2-factor ANOVA: isoform x peptide) fluorescence intensities of TTR-
positive CPECs for 3 derivations per line. We will also collect culture media to measure residual fluorescence
[100] and to potentially assess for cleavage via electrophoresis.
 Potential pitfalls and alternatives. 1) Differences in CPEC differentiation due to APOE isoform are not
anticipated, since ChP and CPECs do not grossly differ among AD patients, and such differences are not
generally seen with AD iPSCs, although there are exceptions [81, 88]. If differences are observed, CPECs of
similar maturity based on immunocytochemical profile can be compared. 2) False negative or noisy results
may arise due to CPEC immaturity, a very common feature of stem cell-derived neurons [74] and other cells.
This would likely be resolved by obtaining kinetic uptake data with much higher temporal resolution. To this
end, we would measure fluorescent Aβ uptake using an IncuCyte S3 Live Cell Analysis system in collaboration
with consultant Blurton-Jones (see letter) once adequate human CPEC enrichment (>90% purity) is achieved;
FACS enrichment for live, TTR+ cells may suffice, at least for initial IncuCyte studies (Fig. 3F). Further CPEC
maturation could also be explored using longer culture times and easily-manipulated maturation factors such
as oxygen and glucose levels.

Aim 2: Determine the impact of APOE isoform on the transcriptomes of individual human CPECs
Rationale. Prior work on neurons, astrocytes, and microglia derived from APOE isogenic iPSCs showed
profound and widespread transcriptome differences based on APOE isoform, and APOE4 has been associated
with differing Aβ production and aggregation, suggesting gene expression changes that broadly affect Aβ
metabolism. Transcriptome differences in APOE isogenic CPECs are therefore highly likely, including those
that are predictive of deficient Aβ uptake by APOE4/4 CPECs, the hypothesis associated with Aim 1.
Design and Methods. We will use the same rigorous yoked pair design for deriving CPECs from APOE
isogenic iPSCs and for their analysis, as done in Aim 1. Derived CPECs will be dissociated using TrypLE
Express, washed, and FACS sorted for live single cells prior to scRNAseq. Replicate samples will be
performed (3 derivations from 3 clones of each genotype); such replicates have been shown to have a greater
impact on identifying differential gene expression than depth of sequencing of individual samples [107].
scRNAseq: As done previously with co-investigator Kessenbrock (Fig. 5), ~10,000 single cells will be
captured using a 10X Genomics Chromium platform, and next-generation sequencing on an Illumina 4000
system will be performed to a target depth of 30-50K reads/cell. Sequence alignment will be performed using
10X Genomics Cell Ranger software. If the smallest CPEC cluster (cluster 1 in Fig. 5A) continues to be
adequately represented (>100 cells), we will sequence together the barcoded libraries from each yoked pair
(~5000 cells/pool at 30-50K reads/cell), which would be preferable experimentally and economically.
Data will be analyzed with co-investigators Kessenbrock and Nie. Cluster, pseudotime, lineage (Fig. 5),
and gene expression (violin plots, heat maps, and other representations of CPEC identity, functional, and Aβ-
related genes) will be analyzed using his SoptSC approach [103] and other software and statistical packages
available to the Nie lab, such as DAVID [107] to identify networks and pathways. Differential gene expression
of individual CPEC clusters will also be specifically analyzed. For each subpopulation from each of the 6
derivations for a given patient, we will use the DEseq2 [108] statistical component bundled with Seurat [109]
from the Satija lab (https://satijalab.org/seurat/) to identify genes differentially expressed with respect to the
APOE genome. These data will then be assessed across the three yoked pairs for each patient, then across
patients to determine whether isogenic pairs reveal consistent differences. As a positive control, we will
investigate the neurons in our derivations (cluster 5 in Fig. 5), since APOE genotype was previously found to
alter neuronal transcriptomes based on bulk RNAseq data [100]. Changes will be compared to reported bulk
transcriptome data for AD patient and control ChP, mouse AD models, and cell types derived from AD and
APOE isogenic iPSCs.
Validation of scRNAseq data: A similar yoked approach for the independent variable (APOE isoform) will
be applied for these studies. Among the CPEC functional and Aβ-related genes showing the largest
differences by scRNAseq, 5-10 will be selected for validation by RT-qPCR and immunocytochemistry of
separate paired CPEC derivations. Like immunocytochemistry, semi-quantitative (relative) RT-qPCR is
performed routinely in the PI's lab [101, 113].
To cross-validate for relevance in vivo, up to 5 immunocytochemical differences validated in the human
CPEC derivations will be examined using postmortem ChP (fixed and frozen) from AD and control patients of
known APOE genotype. Both immunostaining (CPEC specificity and semi-quantitative single cell analysis) and
Western blotting (quantitative population analysis) will be performed. Postmortem ChP is collected by staff of
the UCI ADRC neuropathology core, which the PI co-directs. The ADRC neuropathology core maintains an
extensive tissue repository, including frozen and fixed ChP, from patients with relatively short postmortem
intervals (generally 2-6 hours) along with known APOE genotypes, sexes, ages, and clinical data; for deceased
patients, neuropathology reports and scoring of AD pathologies (Aβ plaques and others) are also available.
Additional postmortem ChP samples from younger, including perinatal, control patients are also routinely
collected by the autopsy service of the Department of Pathology & Laboratory Medicine, which the PI chairs.
Formalin-fixed paraffin embedded (FFPE) autopsy ChP or other tissues will be used for APOE genotyping
[108]. We will compare ChP from AD patients with APOE4/4, 3/3, and 3/4 genotypes, as well as from age-
matched controls with APOE3/3 and 3/4 genotypes (control APOE4/4 genotypes are rare and are not currently
represented in the UCI ADRC repository). We also will stain perinatal ChP samples of known APOE genotype,
which may better reflect the young CPECs derived in culture. We will select cases from roughly equal numbers
of males and females for which race and other metadata are known.
Frozen tissue will be sectioned (20 μm thickness) in a cryostat and post-fixed in 4% paraformaldehyde in
the PI's lab. FFPE tissue will be sectioned using a microtome (4-5 μm thickness) and stained with
commercially-available antibodies to the candidate proteins. Fluorescent (frozen sections) or HRP-conjugated
secondary antibodies (FFPE sections) will then be applied and visualized, as done routinely in the PI's lab or
by the pathology services core. CPEC staining intensity will be scored (0-3+ scale) by multiple observers
blinded to disease status or APOE genotype. Western blotting will involve homogenizing frozen ChP,
determining protein concentrations, adding Laemmli sample buffer (Bio-Rad) and electrophoresing on Mini-
Protean TGX gels (Bio-Rad) in Tris Glycine SDS buffer (Bio-Rad). Samples will be transferred onto 0.45 μm
nitrocellulose membranes (Bio-Rad) in Tris Glycine transfer buffer. Candidate proteins will be detected using
appropriate antibodies and Westernsure reagents, followed by scanning and quantification using an Odyssey
IR scanner (LI-COR Biosciences), as done previously in our lab [109, 110].
Anticipated results, pitfalls, and alternatives. Given the breadth of transcriptome differences in APOE
isogenic neurons, astrocytes, and microglia [100], we anticipate many differences in the isogenic CPECs.
Likely candidates for altered pathways include lipid metabolism and tissue development, as found in the other
isogenic cell types [100]. Based on ChP autopsy and mouse findings, additional differences may involve
proteasomes, mitochondria, oxidation-reduction, and barrier proteins [30, 64-66, 68-71, 111, 112]. The
scRNAseq differences that validate at the protein level in derived human CPECs are likely to cross-validate in
APOE4/4 and 3/3 ChP in vivo. In those cases, it will be interesting to determine examine APOE3/4 ChP for an
intermediate phenotype, which would suggest a gene dosage effect, and control APOE3/3 and 3/4 cases to
determine whether the presence of AD interacts with APOE genotype to determine CPEC phenotypes.
1) Aβ-relevant changes in rarer transcripts will be missed using the more common shallow scRNAseq
approach we propose. If important rarer transcripts are strongly suspected, deeper sequencing on fewer cells
can be easily performed. 2) While the possibility to two CPEC lineages (Fig. 5) is quite interesting, this could
mask Aβ uptake findings in Aim 1, where we treat CPECs as a single cell class. These can be resolved once
antibody markers for different CPEC subtypes are defined in Aim 2. Even without such markers, we could take
advantage of the regional CPEC differences in culture (e.g. enhanced TTR and APOE expression at the
periphery of CPEC islands) (Figs. 3B,4A) and look for regional differences in Aβ uptake. Another alternative
would be to dissociate and pool human CPECs to homogenize subtype representation prior to applying Aβ. 3)
Isogenic pairs from the two patients may show different rather than similar changes, which could be due to sex
or epistasis involving other AD risk factor genes [118]. Our long-term goals (future R01 proposals) would
include characterizing such differences by extending this work to additional isogenic APOE pairs available
through a new CRISPR/Cas9 iPSC core at UCI, through other investigators, or available commercially. 4)
While proposed studies do not require CPEC enrichment, future bulk assays will. In addition to other candidate
enrichment approaches (Fig. 3F), the scRNAseq data can be mined for cell surface antigens that would enable
positive and negative CPEC enrichment by FACS or MACS. 5) A general pitfall for the AD field is that the
major risk factor for AD is aging, while derivatives of pluripotent stem cells are inherently young. Despite these
concerns, many AD-related phenotypes have emerged from studying neural cells derived from AD patient and
control pluripotent stem cells [75-100], which may relate to APOE and Aβ metabolism being lifelong processes
that lead to preclinical differences in APOE4 subject [114-117]. Nonetheless, accelerated aging in vitro is
possible using, for example, using reactive oxygen species or forced expression of progerin [73, 74, 113].

Timeline
We would begin to derive CPECs at the beginning of
Year 1 and assess their progress on an ongoing basis as
they provide material for Aβ uptake (Aim 1) and scRNAseq
studies (Aim 2), which would begin as soon as paired CPEC
derivations are completed. Characterization of postmortem
tissues and other validations would follow the completion of
the first analyses of scRNAseq data.
BIBLIOGRAPHY

1. Alzheimer's Association, Alzheimer's Disease facts and figures. Alzheimer Dement, 2018. 14: p. 367-429.
2. Mayeux, R., A.M. Saunders, S. Shea, S. Mirra, D. Evans, A.D. Roses, B.T. Hyman, B. Crain, M.X. Tang, and C.H.
Phelps, Utility of the apolipoprotein E genotype in the diagnosis of Alzheimer's disease. Alzheimer's Disease
Centers Consortium on Apolipoprotein E and Alzheimer's Disease. N Engl J Med, 1998. 338(8): p. 506-11.
3. Ward, A., S. Crean, C.J. Mercaldi, J.M. Collins, D. Boyd, M.N. Cook, and H.M. Arrighi, Prevalence of apolipoprotein
E4 genotype and homozygotes (APOE e4/4) among patients diagnosed with Alzheimer's disease: a systematic
review and meta-analysis. Neuroepidemiology, 2012. 38(1): p. 1-17.
4. Corder, E.H., A.M. Saunders, W.J. Strittmatter, D.E. Schmechel, P.C. Gaskell, G.W. Small, A.D. Roses, J.L. Haines,
and M.A. Pericak-Vance, Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late
onset families. Science, 1993. 261(5123): p. 921-3.
5. Kanekiyo, T., H. Xu, and G. Bu, ApoE and Abeta in Alzheimer's disease: accidental encounters or partners?
Neuron, 2014. 81(4): p. 740-54.
6. LaDu, M.J., M.T. Falduto, A.M. Manelli, C.A. Reardon, G.S. Getz, and D.E. Frail, Isoform-specific binding of
apolipoprotein E to beta-amyloid. J Biol Chem, 1994. 269(38): p. 23403-6.
7. Petrlova, J., H.S. Hong, D.A. Bricarello, G. Harishchandra, G.A. Lorigan, L.W. Jin, and J.C. Voss, A differential
association of Apolipoprotein E isoforms with the amyloid-beta oligomer in solution. Proteins, 2011. 79(2): p.
402-16.
8. Tai, L.M., T. Bilousova, L. Jungbauer, S.K. Roeske, K.L. Youmans, C. Yu, W.W. Poon, L.B. Cornwell, C.A. Miller, H.V.
Vinters, L.J. Van Eldik, D.W. Fardo, S. Estus, G. Bu, K.H. Gylys, and M.J. Ladu, Levels of soluble apolipoprotein
E/amyloid-beta (Abeta) complex are reduced and oligomeric Abeta increased with APOE4 and Alzheimer disease
in a transgenic mouse model and human samples. J Biol Chem, 2013. 288(8): p. 5914-26.
9. Sanan, D.A., K.H. Weisgraber, S.J. Russell, R.W. Mahley, D. Huang, A. Saunders, D. Schmechel, T. Wisniewski, B.
Frangione, A.D. Roses, and W.J. Strittmatter, Apolipoprotein E associates with beta amyloid peptide of
Alzheimer's disease to form novel monofibrils. Isoform apoE4 associates more efficiently than apoE3. J Clin
Invest, 1994. 94(2): p. 860-9.
10. Hashimoto, T., A. Serrano-Pozo, Y. Hori, K.W. Adams, S. Takeda, A.O. Banerji, A. Mitani, D. Joyner, D.H. Thyssen,
B.J. Bacskai, M.P. Frosch, T.L. Spires-Jones, M.B. Finn, D.M. Holtzman, and B.T. Hyman, Apolipoprotein E,
especially apolipoprotein E4, increases the oligomerization of amyloid beta peptide. J Neurosci, 2012. 32(43): p.
15181-92.
11. Castellano, J.M., J. Kim, F.R. Stewart, H. Jiang, R.B. DeMattos, B.W. Patterson, A.M. Fagan, J.C. Morris, K.G.
Mawuenyega, C. Cruchaga, A.M. Goate, K.R. Bales, S.M. Paul, R.J. Bateman, and D.M. Holtzman, Human apoE
isoforms differentially regulate brain amyloid-beta peptide clearance. Sci Transl Med, 2011. 3(89): p. 89ra57.
12. Dafnis, I., C. Raftopoulou, C. Mountaki, E. Megalou, V.I. Zannis, and A. Chroni, ApoE isoforms and carboxyl-
terminal-truncated apoE4 forms affect neuronal BACE1 levels and Abeta production independently of their
cholesterol efflux capacity. Biochem J, 2018. 475(10): p. 1839-1859.
13. Huang, Y.A., B. Zhou, M. Wernig, and T.C. Sudhof, ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP
Transcription and Abeta Secretion. Cell, 2017. 168(3): p. 427-441 e21.
14. Vincent, B. and J.D. Smith, Astrocytes down-regulate neuronal beta-amyloid precursor protein expression and
modify its processing in an apolipoprotein E isoform-specific manner. Eur J Neurosci, 2001. 14(2): p. 256-66.
15. Lun, M.P., E.S. Monuki, and M.K. Lehtinen, Development and functions of the choroid plexus-cerebrospinal fluid
system. Nat Rev Neurosci, 2015. 16(8): p. 445-57.
16. Lun, M.P., M.B. Johnson, K.G. Broadbelt, M. Watanabe, Y.J. Kang, K.F. Chau, M.W. Springel, A. Malesz, A.M.
Sousa, M. Pletikos, T. Adelita, M.L. Calicchio, Y. Zhang, M.J. Holtzman, H.G. Lidov, N. Sestan, H. Steen, E.S.
Monuki, and M.K. Lehtinen, Spatially heterogeneous choroid plexus transcriptomes encode positional identity
and contribute to regional CSF production. J Neurosci, 2015. 35(12): p. 4903-16.
17. Mita, S., E.A. Schon, and J. Herbert, Widespread expression of amyloid beta-protein precursor gene in rat brain.
Am J Pathol, 1989. 134(6): p. 1253-61.
18. Tu, G.F., T. Cole, B.R. Southwell, and G. Schreiber, Expression of the genes for transthyretin, cystatin C and beta
A4 amyloid precursor protein in sheep choroid plexus during development. Brain Res Dev Brain Res, 1990. 55(2):
p. 203-8.
19. Coria, F., A. Moreno, A. Torres, I. Ahmad, and J. Ghiso, Distribution of Alzheimer's disease amyloid protein
precursor in normal human and rat nervous system. Neuropathol Appl Neurobiol, 1992. 18(1): p. 27-35.
20. Ohta, M., T. Kitamoto, T. Iwaki, T. Ohgami, M. Fukui, and J. Tateishi, Immunohistochemical distribution of
amyloid precursor protein during normal rat development. Brain Res Dev Brain Res, 1993. 75(2): p. 151-61.
21. Cribbs, D.H., L.S. Chen, S.M. Bende, and F.M. LaFerla, Widespread neuronal expression of the presenilin-1 early-
onset Alzheimer's disease gene in the murine brain. Am J Pathol, 1996. 148(6): p. 1797-806.
22. Kim, K.S., J. Wegiel, V. Sapienza, J. Chen, H. Hong, and H.M. Wisniewski, Immunoreactivity of presenilin-1 in
human, rat and mouse brain. Brain Res, 1997. 757(1): p. 159-63.
23. Sasaki, A., M. Iijima, H. Yokoo, M. Shoji, and Y. Nakazato, Human choroid plexus is an uniquely involved area of
the brain in amyloidosis: a histochemical, immunohistochemical and ultrastructural study. Brain Res, 1997.
755(2): p. 193-201.
24. Yan, X.X., T. Li, C.M. Rominger, S.R. Prakash, P.C. Wong, R.E. Olson, R. Zaczek, and Y.W. Li, Binding sites of
gamma-secretase inhibitors in rodent brain: distribution, postnatal development, and effect of deafferentation. J
Neurosci, 2004. 24(12): p. 2942-52.
25. Patel, S., S. O'Malley, B. Connolly, W. Liu, R. Hargreaves, C. Sur, and R.E. Gibson, In vitro characterization of a
gamma-secretase radiotracer in mammalian brain. J Neurochem, 2006. 96(1): p. 171-8.
26. Crossgrove, J.S., E.L. Smith, and W. Zheng, Macromolecules involved in production and metabolism of beta-
amyloid at the brain barriers. Brain Res, 2007. 1138: p. 187-95.
27. Marques, F., J.C. Sousa, G. Coppola, F. Gao, R. Puga, H. Brentani, D.H. Geschwind, N. Sousa, M. Correia-Neves,
and J.A. Palha, Transcriptome signature of the adult mouse choroid plexus. Fluids Barriers CNS, 2011. 8(1): p. 10.
28. Janssen, S.F., S.J. van der Spek, J.B. Ten Brink, A.H. Essing, T.G. Gorgels, P.J. van der Spek, N.M. Jansonius, and
A.A. Bergen, Gene expression and functional annotation of the human and mouse choroid plexus epithelium.
PLoS One, 2013. 8(12): p. e83345.
29. Liu, F., Z.Q. Xue, S.H. Deng, X. Kun, X.G. Luo, P.R. Patrylo, G.M. Rose, H. Cai, R.G. Struble, Y. Cai, and X.X. Yan,
gamma-secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a
putative origin of CSF Abeta. Eur J Neurosci, 2013. 37(10): p. 1714-25.
30. Bergen, A.A., S. Kaing, J.B. ten Brink, B. Netherlands Brain, T.G. Gorgels, and S.F. Janssen, Gene expression and
functional annotation of human choroid plexus epithelium failure in Alzheimer's disease. BMC Genomics, 2015.
16: p. 956.
31. Herr, U.M., P. Strecker, S.E. Storck, C. Thomas, V. Rabiej, A. Junker, S. Schilling, N. Schmidt, C.M. Dowds, S.
Eggert, C.U. Pietrzik, and S. Kins, LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric
and Dimeric State. Front Mol Neurosci, 2017. 10: p. 118.
32. Kalaria, R.N., D.R. Premkumar, A.B. Pax, D.L. Cohen, and I. Lieberburg, Production and increased detection of
amyloid beta protein and amyloidogenic fragments in brain microvessels, meningeal vessels and choroid plexus
in Alzheimer's disease. Brain Res Mol Brain Res, 1996. 35(1-2): p. 58-68.
33. Zlokovic, B.V., C.L. Martel, E. Matsubara, J.G. McComb, G. Zheng, R.T. McCluskey, B. Frangione, and J. Ghiso,
Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a
complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers. Proc Natl
Acad Sci U S A, 1996. 93(9): p. 4229-34.
34. Martel, C.L., J.B. Mackic, E. Matsubara, S. Governale, C. Miguel, W. Miao, J.G. McComb, B. Frangione, J. Ghiso,
and B.V. Zlokovic, Isoform-specific effects of apolipoproteins E2, E3, and E4 on cerebral capillary sequestration
and blood-brain barrier transport of circulating Alzheimer's amyloid beta. J Neurochem, 1997. 69(5): p. 1995-
2004.
35. Carro, E., J.L. Trejo, T. Gomez-Isla, D. LeRoith, and I. Torres-Aleman, Serum insulin-like growth factor I regulates
brain amyloid-beta levels. Nat Med, 2002. 8(12): p. 1390-7.
36. Monro, O.R., J.B. Mackic, S. Yamada, M.B. Segal, J. Ghiso, C. Maurer, M. Calero, B. Frangione, and B.V. Zlokovic,
Substitution at codon 22 reduces clearance of Alzheimer's amyloid-beta peptide from the cerebrospinal fluid and
prevents its transport from the central nervous system into blood. Neurobiol Aging, 2002. 23(3): p. 405-12.
37. Carro, E., C. Spuch, J.L. Trejo, D. Antequera, and I. Torres-Aleman, Choroid plexus megalin is involved in
neuroprotection by serum insulin-like growth factor I. J Neurosci, 2005. 25(47): p. 10884-93.
38. Crossgrove, J.S., G.J. Li, and W. Zheng, The choroid plexus removes beta-amyloid from brain cerebrospinal fluid.
Exp Biol Med (Maywood), 2005. 230(10): p. 771-6.
39. Carro, E., J.L. Trejo, A. Gerber, H. Loetscher, J. Torrado, F. Metzger, and I. Torres-Aleman, Therapeutic actions of
insulin-like growth factor I on APP/PS2 mice with severe brain amyloidosis. Neurobiol Aging, 2006. 27(9): p.
1250-7.
40. Behl, M., Y. Zhang, and W. Zheng, Involvement of insulin-degrading enzyme in the clearance of beta-amyloid at
the blood-CSF barrier: Consequences of lead exposure. Cerebrospinal Fluid Res, 2009. 6: p. 11.
41. Behl, M., Y. Zhang, A.D. Monnot, W. Jiang, and W. Zheng, Increased beta-amyloid levels in the choroid plexus
following lead exposure and the involvement of low-density lipoprotein receptor protein-1. Toxicol Appl
Pharmacol, 2009. 240(2): p. 245-54.
42. Fujiyoshi, M., M. Tachikawa, S. Ohtsuki, S. Ito, Y. Uchida, S. Akanuma, J. Kamiie, T. Hashimoto, K. Hosoya, T.
Iwatsubo, and T. Terasaki, Amyloid-beta peptide(1-40) elimination from cerebrospinal fluid involves low-density
lipoprotein receptor-related protein 1 at the blood-cerebrospinal fluid barrier. J Neurochem, 2011. 118(3): p.
407-15.
43. Gu, H., Z. Zhong, W. Jiang, E. Du, R. Dodel, M.R. Farlow, W. Zheng, and Y. Du, The role of choroid plexus in IVIG-
induced beta-amyloid clearance. Neuroscience, 2014. 270: p. 168-176.
44. Chen, R., C.P. Chen, and J.E. Preston, Effects of transthyretin on thyroxine and beta-amyloid removal from
cerebrospinal fluid in mice. Clin Exp Pharmacol Physiol, 2016. 43(9): p. 844-50.
45. Costa, A.R., H. Marcelino, I. Goncalves, T. Quintela, J. Tomas, A.C. Duarte, A.M. Fonseca, and C.R. Santos, Sex
Hormones Protect Against Amyloid-beta Induced Oxidative Stress in the Choroid Plexus Cell Line Z310. J
Neuroendocrinol, 2016. 28(9).
46. McIntee, F.L., P. Giannoni, S. Blais, G. Sommer, T.A. Neubert, A. Rostagno, and J. Ghiso, In vivo Differential Brain
Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Abeta protein. Front Aging Neurosci, 2016.
8: p. 223.
47. Aronow, B.J., S.D. Lund, T.L. Brown, J.A. Harmony, and D.P. Witte, Apolipoprotein J expression at fluid-tissue
interfaces: potential role in barrier cytoprotection. Proc Natl Acad Sci U S A, 1993. 90(2): p. 725-9.
48. Page, K.J., R.D. Hollister, and B.T. Hyman, Dissociation of apolipoprotein and apolipoprotein receptor response to
lesion in the rat brain: an in situ hybridization study. Neuroscience, 1998. 85(4): p. 1161-71.
49. Xu, Q., Y. Li, C. Cyras, D.A. Sanan, and B. Cordell, Isolation and characterization of apolipoproteins from murine
microglia. Identification of a low density lipoprotein-like apolipoprotein J-rich but E-poor spherical particle. J Biol
Chem, 2000. 275(41): p. 31770-7.
50. Xu, Q., A. Bernardo, D. Walker, T. Kanegawa, R.W. Mahley, and Y. Huang, Profile and regulation of
apolipoprotein E (ApoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the
ApoE locus. J Neurosci, 2006. 26(19): p. 4985-94.
51. Fujiyoshi, M., S. Ohtsuki, S. Hori, M. Tachikawa, and T. Terasaki, 24S-hydroxycholesterol induces cholesterol
release from choroid plexus epithelial cells in an apical- and apoE isoform-dependent manner concomitantly with
the induction of ABCA1 and ABCG1 expression. J Neurochem, 2007. 100(4): p. 968-78.
52. Achariyar, T.M., B. Li, W. Peng, P.B. Verghese, Y. Shi, E. McConnell, A. Benraiss, T. Kasper, W. Song, T. Takano,
D.M. Holtzman, M. Nedergaard, and R. Deane, Glymphatic distribution of CSF-derived apoE into brain is isoform
specific and suppressed during sleep deprivation. Mol Neurodegener, 2016. 11(1): p. 74.
53. Kim, D.H., H. Iijima, K. Goto, J. Sakai, H. Ishii, H.J. Kim, H. Suzuki, H. Kondo, S. Saeki, and T. Yamamoto, Human
apolipoprotein E receptor 2. A novel lipoprotein receptor of the low density lipoprotein receptor family
predominantly expressed in brain. J Biol Chem, 1996. 271(14): p. 8373-80.
54. Dietrich, M.O., C. Spuch, D. Antequera, I. Rodal, J.G. de Yebenes, J.A. Molina, F. Bermejo, and E. Carro, Megalin
mediates the transport of leptin across the blood-CSF barrier. Neurobiol Aging, 2008. 29(6): p. 902-12.
55. Pascale, C.L., M.C. Miller, C. Chiu, M. Boylan, I.N. Caralopoulos, L. Gonzalez, C.E. Johanson, and G.D. Silverberg,
Amyloid-beta transporter expression at the blood-CSF barrier is age-dependent. Fluids Barriers CNS, 2011. 8: p.
21.
56. Matsumoto, K., Y. Chiba, R. Fujihara, H. Kubo, H. Sakamoto, and M. Ueno, Immunohistochemical analysis of
transporters related to clearance of amyloid-beta peptides through blood-cerebrospinal fluid barrier in human
brain. Histochem Cell Biol, 2015. 144(6): p. 597-611.
57. Spuch, C., D. Antequera, C. Pascual, S. Abilleira, M. Blanco, M.J. Moreno-Carretero, J. Romero-Lopez, T. Ishida,
J.A. Molina, A. Villarejo, F. Bermejo-Pareja, and E. Carro, Soluble Megalin is Reduced in Cerebrospinal Fluid
Samples of Alzheimer's Disease Patients. Front Cell Neurosci, 2015. 9: p. 134.
58. Storck, S.E., S. Meister, J. Nahrath, J.N. Meissner, N. Schubert, A. Di Spiezio, S. Baches, R.E. Vandenbroucke, Y.
Bouter, I. Prikulis, C. Korth, S. Weggen, A. Heimann, M. Schwaninger, T.A. Bayer, and C.U. Pietrzik, Endothelial
LRP1 transports amyloid-beta(1-42) across the blood-brain barrier. J Clin Invest, 2016. 126(1): p. 123-36.
59. Serot, J.M., M.C. Bene, B. Foliguet, and G.C. Faure, Morphological alterations of the choroid plexus in late-onset
Alzheimer's disease. Acta Neuropathol, 2000. 99(2): p. 105-8.
60. Serot, J.M., M.C. Bene, and G.C. Faure, Comparative immunohistochemical characteristics of human choroid
plexus in vascular and Alzheimer's dementia. Hum Pathol, 1994. 25(11): p. 1185-90.
61. Miklossy, J., R. Kraftsik, O. Pillevuit, D. Lepori, C. Genton, and F.T. Bosman, Curly fiber and tangle-like inclusions
in the ependyma and choroid plexus--a pathogenetic relationship with the cortical Alzheimer-type changes? J
Neuropathol Exp Neurol, 1998. 57(12): p. 1202-12.
62. Wen, G.Y., H.M. Wisniewski, and R.J. Kascsak, Biondi ring tangles in the choroid plexus of Alzheimer's disease and
normal aging brains: a quantitative study. Brain Res, 1999. 832(1-2): p. 40-6.
63. Serot, J.M., M.C. Bene, and G.C. Faure, Choroid plexus, aging of the brain, and Alzheimer's disease. Front Biosci,
2003. 8: p. s515-21.
64. Krzyzanowska, A., I. Garcia-Consuegra, C. Pascual, D. Antequera, I. Ferrer, and E. Carro, Expression of regulatory
proteins in choroid plexus changes in early stages of Alzheimer disease. J Neuropathol Exp Neurol, 2015. 74(4): p.
359-69.
65. Stopa, E.G., K.Q. Tanis, M.C. Miller, E.V. Nikonova, A.A. Podtelezhnikov, E.M. Finney, D.J. Stone, L.M. Camargo, L.
Parker, A. Verma, A. Baird, J.E. Donahue, T. Torabi, B.P. Eliceiri, G.D. Silverberg, and C.E. Johanson, Comparative
transcriptomics of choroid plexus in Alzheimer's disease, frontotemporal dementia and Huntington's disease:
implications for CSF homeostasis. Fluids Barriers CNS, 2018. 15(1): p. 18.
66. Mesquita, S.D., A.C. Ferreira, F. Gao, G. Coppola, D.H. Geschwind, J.C. Sousa, M. Correia-Neves, N. Sousa, J.A.
Palha, and F. Marques, The choroid plexus transcriptome reveals changes in type I and II interferon responses in a
mouse model of Alzheimer's disease. Brain Behav Immun, 2015. 49: p. 280-92.
67. Mesquita, S.D., A.C. Ferreira, A.M. Falcao, J.C. Sousa, T.G. Oliveira, M. Correia-Neves, N. Sousa, F. Marques, and
J.A. Palha, Lipocalin 2 modulates the cellular response to amyloid beta. Cell Death Differ, 2014. 21(10): p. 1588-
99.
68. Brkic, M., S. Balusu, E. Van Wonterghem, N. Gorle, I. Benilova, A. Kremer, I. Van Hove, L. Moons, B. De Strooper,
S. Kanazir, C. Libert, and R.E. Vandenbroucke, Amyloid beta Oligomers Disrupt Blood-CSF Barrier Integrity by
Activating Matrix Metalloproteinases. J Neurosci, 2015. 35(37): p. 12766-78.
69. Liu, C.B., R. Wang, Y.F. Yi, Z. Gao, and Y.Z. Chen, Lycopene mitigates beta-amyloid induced inflammatory
response and inhibits NF-kappaB signaling at the choroid plexus in early stages of Alzheimer's disease rats. J Nutr
Biochem, 2018. 53: p. 66-71.
70. Vargas, T., D. Antequera, C. Ugalde, C. Spuch, and E. Carro, Gelsolin restores A beta-induced alterations in
choroid plexus epithelium. J Biomed Biotechnol, 2010. 2010: p. 805405.
71. Vargas, T., C. Ugalde, C. Spuch, D. Antequera, M.J. Moran, M.A. Martin, I. Ferrer, F. Bermejo-Pareja, and E. Carro,
Abeta accumulation in choroid plexus is associated with mitochondrial-induced apoptosis. Neurobiol Aging,
2010. 31(9): p. 1569-81.
72. Ashe, K.H. and K.R. Zahs, Probing the biology of Alzheimer's disease in mice. Neuron, 2010. 66(5): p. 631-45.
73. Mungenast, A.E., S. Siegert, and L.H. Tsai, Modeling Alzheimer's disease with human induced pluripotent stem
(iPS) cells. Mol Cell Neurosci, 2016. 73: p. 13-31.
74. Sullivan, S.E. and T.L. Young-Pearse, Induced pluripotent stem cells as a discovery tool for Alzheimers disease.
Brain Res, 2017. 1656: p. 98-106.
75. Yagi, T., D. Ito, Y. Okada, W. Akamatsu, Y. Nihei, T. Yoshizaki, S. Yamanaka, H. Okano, and N. Suzuki, Modeling
familial Alzheimer's disease with induced pluripotent stem cells. Hum Mol Genet, 2011. 20(23): p. 4530-9.
76. Israel, M.A., S.H. Yuan, C. Bardy, S.M. Reyna, Y. Mu, C. Herrera, M.P. Hefferan, S. Van Gorp, K.L. Nazor, F.S.
Boscolo, C.T. Carson, L.C. Laurent, M. Marsala, F.H. Gage, A.M. Remes, E.H. Koo, and L.S. Goldstein, Probing
sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature, 2012. 482(7384): p. 216-
20.
77. Shi, Y., P. Kirwan, J. Smith, G. MacLean, S.H. Orkin, and F.J. Livesey, A human stem cell model of early Alzheimer's
disease pathology in Down syndrome. Sci Transl Med, 2012. 4(124): p. 124ra29.
78. Kondo, T., M. Asai, K. Tsukita, Y. Kutoku, Y. Ohsawa, Y. Sunada, K. Imamura, N. Egawa, N. Yahata, K. Okita, K.
Takahashi, I. Asaka, T. Aoi, A. Watanabe, K. Watanabe, C. Kadoya, R. Nakano, D. Watanabe, K. Maruyama, O.
 Hori, S. Hibino, T. Choshi, T. Nakahata, H. Hioki, T. Kaneko, M. Naitoh, K. Yoshikawa, S. Yamawaki, S. Suzuki, R.
Hata, S. Ueno, T. Seki, K. Kobayashi, T. Toda, K. Murakami, K. Irie, W.L. Klein, H. Mori, T. Asada, R. Takahashi, N.
Iwata, S. Yamanaka, and H. Inoue, Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated
with intracellular Abeta and differential drug responsiveness. Cell Stem Cell, 2013. 12(4): p. 487-96.
79. Woodruff, G., J.E. Young, F.J. Martinez, F. Buen, A. Gore, J. Kinaga, Z. Li, S.H. Yuan, K. Zhang, and L.S. Goldstein,
The presenilin-1 DeltaE9 mutation results in reduced gamma-secretase activity, but not total loss of PS1 function,
in isogenic human stem cells. Cell Rep, 2013. 5(4): p. 974-85.
80. Duan, L., B.J. Bhattacharyya, A. Belmadani, L. Pan, R.J. Miller, and J.A. Kessler, Stem cell derived basal forebrain
cholinergic neurons from Alzheimer's disease patients are more susceptible to cell death. Mol Neurodegener,
2014. 9: p. 3.
81. Lee, J.K., H.K. Jin, M.H. Park, B.R. Kim, P.H. Lee, H. Nakauchi, J.E. Carter, X. He, E.H. Schuchman, and J.S. Bae, Acid
sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease. J
Exp Med, 2014. 211(8): p. 1551-70.
82. Liu, Q., S. Waltz, G. Woodruff, J. Ouyang, M.A. Israel, C. Herrera, F. Sarsoza, R.E. Tanzi, E.H. Koo, J.M. Ringman,
L.S. Goldstein, S.L. Wagner, and S.H. Yuan, Effect of potent gamma-secretase modulator in human neurons
derived from multiple presenilin 1-induced pluripotent stem cell mutant carriers. JAMA Neurol, 2014. 71(12): p.
1481-9.
83. Mahairaki, V., J. Ryu, A. Peters, Q. Chang, T. Li, T.S. Park, P.W. Burridge, C.C. Talbot, Jr., L. Asnaghi, L.J. Martin,
E.T. Zambidis, and V.E. Koliatsos, Induced pluripotent stem cells from familial Alzheimer's disease patients
differentiate into mature neurons with amyloidogenic properties. Stem Cells Dev, 2014. 23(24): p. 2996-3010.
84. Muratore, C.R., H.C. Rice, P. Srikanth, D.G. Callahan, T. Shin, L.N. Benjamin, D.M. Walsh, D.J. Selkoe, and T.L.
Young-Pearse, The familial Alzheimer's disease APPV717I mutation alters APP processing and Tau expression in
iPSC-derived neurons. Hum Mol Genet, 2014. 23(13): p. 3523-36.
85. Sproul, A.A., S. Jacob, D. Pre, S.H. Kim, M.W. Nestor, M. Navarro-Sobrino, I. Santa-Maria, M. Zimmer, S. Aubry,
J.W. Steele, D.J. Kahler, A. Dranovsky, O. Arancio, J.F. Crary, S. Gandy, and S.A. Noggle, Characterization and
molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors. PLoS One, 2014. 9(1):
p. e84547.
86. Chang, C.Y., S.M. Chen, H.E. Lu, S.M. Lai, P.S. Lai, P.W. Shen, P.Y. Chen, C.I. Shen, H.J. Harn, S.Z. Lin, S.M. Hwang,
and H.L. Su, N-butylidenephthalide attenuates Alzheimer's disease-like cytopathy in Down syndrome induced
pluripotent stem cell-derived neurons. Sci Rep, 2015. 5: p. 8744.
87. Moore, S., L.D. Evans, T. Andersson, E. Portelius, J. Smith, T.B. Dias, N. Saurat, A. McGlade, P. Kirwan, K.
Blennow, J. Hardy, H. Zetterberg, and F.J. Livesey, APP metabolism regulates tau proteostasis in human cerebral
cortex neurons. Cell Rep, 2015. 11(5): p. 689-96.
88. Hossini, A.M., M. Megges, A. Prigione, B. Lichtner, M.R. Toliat, W. Wruck, F. Schroter, P. Nuernberg, H. Kroll, E.
Makrantonaki, C.C. Zouboulis, and J. Adjaye, Induced pluripotent stem cell-derived neuronal cells from a sporadic
Alzheimer's disease donor as a model for investigating AD-associated gene regulatory networks. BMC Genomics,
2015. 16: p. 84.
89. Young, J.E., J. Boulanger-Weill, D.A. Williams, G. Woodruff, F. Buen, A.C. Revilla, C. Herrera, M.A. Israel, S.H.
Yuan, S.D. Edland, and L.S. Goldstein, Elucidating molecular phenotypes caused by the SORL1 Alzheimer's disease
genetic risk factor using human induced pluripotent stem cells. Cell Stem Cell, 2015. 16(4): p. 373-85.
90. Balez, R., N. Steiner, M. Engel, S.S. Munoz, J.S. Lum, Y. Wu, D. Wang, P. Vallotton, P. Sachdev, M. O'Connor, K.
Sidhu, G. Munch, and L. Ooi, Neuroprotective effects of apigenin against inflammation, neuronal excitability and
apoptosis in an induced pluripotent stem cell model of Alzheimer's disease. Sci Rep, 2016. 6: p. 31450.
91. Reddy, K., C.L. Cusack, I.C. Nnah, K. Khayati, C. Saqcena, T.B. Huynh, S.A. Noggle, A. Ballabio, and R. Dobrowolski,
Dysregulation of Nutrient Sensing and CLEARance in Presenilin Deficiency. Cell Rep, 2016. 14(9): p. 2166-2179.
92. Woodruff, G., S.M. Reyna, M. Dunlap, R. Van Der Kant, J.A. Callender, J.E. Young, E.A. Roberts, and L.S.
Goldstein, Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial
Alzheimer's Disease Mutations. Cell Rep, 2016. 17(3): p. 759-773.
93. Brownjohn, P.W., J. Smith, E. Portelius, L. Serneels, H. Kvartsberg, B. De Strooper, K. Blennow, H. Zetterberg, and
F.J. Livesey, Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem
Cell Models of Alzheimer's Disease. Stem Cell Reports, 2017. 8(4): p. 870-882.
94. Ochalek, A., B. Mihalik, H.X. Avci, A. Chandrasekaran, A. Teglasi, I. Bock, M.L. Giudice, Z. Tancos, K. Molnar, L.
Laszlo, J.E. Nielsen, B. Holst, K. Freude, P. Hyttel, J. Kobolak, and A. Dinnyes, Neurons derived from sporadic
 Alzheimer's disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B
activation. Alzheimers Res Ther, 2017. 9(1): p. 90.
95. Ortiz-Virumbrales, M., C.L. Moreno, I. Kruglikov, P. Marazuela, A. Sproul, S. Jacob, M. Zimmer, D. Paull, B. Zhang,
E.E. Schadt, M.E. Ehrlich, R.E. Tanzi, O. Arancio, S. Noggle, and S. Gandy, CRISPR/Cas9-Correctable mutation-
related molecular and physiological phenotypes in iPSC-derived Alzheimer's PSEN2 (N141I) neurons. Acta
Neuropathol Commun, 2017. 5(1): p. 77.
96. Jones, V.C., R. Atkinson-Dell, A. Verkhratsky, and L. Mohamet, Aberrant iPSC-derived human astrocytes in
Alzheimer's disease. Cell Death Dis, 2017. 8(3): p. e2696.
97. Oksanen, M., A.J. Petersen, N. Naumenko, K. Puttonen, S. Lehtonen, M. Gubert Olive, A. Shakirzyanova, S.
Leskela, T. Sarajarvi, M. Viitanen, J.O. Rinne, M. Hiltunen, A. Haapasalo, R. Giniatullin, P. Tavi, S.C. Zhang, K.M.
Kanninen, R.H. Hamalainen, and J. Koistinaho, PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte
Pathology in Alzheimer's Disease. Stem Cell Reports, 2017. 9(6): p. 1885-1897.
98. Birnbaum, J.H., D. Wanner, A.F. Gietl, A. Saake, T.M. Kundig, C. Hock, R.M. Nitsch, and C. Tackenberg, Oxidative
stress and altered mitochondrial protein expression in the absence of amyloid-beta and tau pathology in iPSC-
derived neurons from sporadic Alzheimer's disease patients. Stem Cell Res, 2018. 27: p. 121-130.
99. Young, J.E., L.K. Fong, H. Frankowski, G.A. Petsko, S.A. Small, and L.S.B. Goldstein, Stabilizing the Retromer
Complex in a Human Stem Cell Model of Alzheimer's Disease Reduces TAU Phosphorylation Independently of
Amyloid Precursor Protein. Stem Cell Reports, 2018. 10(3): p. 1046-1058.
100. Lin, Y.T., J. Seo, F. Gao, H.M. Feldman, H.L. Wen, J. Penney, H.P. Cam, E. Gjoneska, W.K. Raja, J. Cheng, R. Rueda,
O. Kritskiy, F. Abdurrob, Z. Peng, B. Milo, C.J. Yu, S. Elmsaouri, D. Dey, T. Ko, B.A. Yankner, and L.H. Tsai, APOE4
Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in
Human iPSC-Derived Brain Cell Types. Neuron, 2018. 98(6): p. 1294.
101. Watanabe, M., Y.J. Kang, L.M. Davies, S. Meghpara, K. Lau, C.Y. Chung, J. Kathiriya, A.K. Hadjantonakis, and E.S.
Monuki, BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived
neuroepithelial progenitors. J Neurosci, 2012. 32(45): p. 15934-45.
102. Narita, K. and S. Takeda, Cilia in the choroid plexus: their roles in hydrocephalus and beyond. Front Cell Neurosci,
2015. 9: p. 39.
103. Wang, S.M., A.; Nie, Q., SoptSC: Similarity matrix optimization for clustering, lineage, and signaling inference.
bioRxiv, 2017. 168922.
104. Tokuda, T., M. Calero, E. Matsubara, R. Vidal, A. Kumar, B. Permanne, B. Zlokovic, J.D. Smith, M.J. Ladu, A.
Rostagno, B. Frangione, and J. Ghiso, Lipidation of apolipoprotein E influences its isoform-specific interaction
with Alzheimer's amyloid beta peptides. Biochem J, 2000. 348 Pt 2: p. 359-65.
105. LaDu, M.J., T.M. Pederson, D.E. Frail, C.A. Reardon, G.S. Getz, and M.T. Falduto, Purification of apolipoprotein E
attenuates isoform-specific binding to beta-amyloid. J Biol Chem, 1995. 270(16): p. 9039-42.
106. LaDu, M.J., S.M. Gilligan, J.R. Lukens, V.G. Cabana, C.A. Reardon, L.J. Van Eldik, and D.M. Holtzman, Nascent
astrocyte particles differ from lipoproteins in CSF. J Neurochem, 1998. 70(5): p. 2070-81.
107. Rapaport, F., R. Khanin, Y. Liang, M. Pirun, A. Krek, P. Zumbo, C.E. Mason, N.D. Socci, and D. Betel,
Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data. Genome Biol,
2013. 14(9): p. R95.
108. Love, M.I., W. Huber, and S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with
DESeq2. Genome Biol, 2014. 15(12): p. 550.
109. Butler, A., P. Hoffman, P. Smibert, E. Papalexi, and R. Satija, Integrating single-cell transcriptomic data across
different conditions, technologies, and species. Nat Biotechnol, 2018. 36(5): p. 411-420.
110. Huang, D.W., B.T. Sherman, Q. Tan, J.R. Collins, W.G. Alvord, J. Roayaei, R. Stephens, M.W. Baseler, H.C. Lane,
and R.A. Lempicki, The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to
functionally analyze large gene lists. Genome Biol, 2007. 8(9): p. R183.
111. Ghebremedhin, E., H. Braak, E. Braak, and J. Sahm, Improved method facilitates reliable APOE genotyping of
genomic DNA extracted from formaldehyde-fixed pathology specimens. J Neurosci Methods, 1998. 79(2): p. 229-
31.
112. Hong, T., E.S. Fung, L. Zhang, G. Huynh, E.S. Monuki, and Q. Nie, Semi-adaptive response and noise attenuation
in bone morphogenetic protein signalling. J R Soc Interface, 2015. 12(107).
113. Watanabe, M., E.S. Fung, F.B. Chan, J.S. Wong, M. Coutts, and E.S. Monuki, BMP4 acts as a dorsal telencephalic
morphogen in a mouse embryonic stem cell culture system. Biol Open, 2016. 5(12): p. 1834-1843.
114. Perez-Gracia, E., R. Blanco, M. Carmona, E. Carro, and I. Ferrer, Oxidative stress damage and oxidative stress
responses in the choroid plexus in Alzheimer's disease. Acta Neuropathol, 2009. 118(4): p. 497-504.
115. Rueli, R.H., A.C. Parubrub, A.S. Dewing, A.C. Hashimoto, M.T. Bellinger, E.J. Weeber, J.H. Uyehara-Lock, L.R.
White, M.J. Berry, and F.P. Bellinger, Increased selenoprotein P in choroid plexus and cerebrospinal fluid in
Alzheimer's disease brain. J Alzheimers Dis, 2015. 44(2): p. 379-83.
116. Campos, P.B., B.S. Paulsen, and S.K. Rehen, Accelerating neuronal aging in in vitro model brain disorders: a focus
on reactive oxygen species. Front Aging Neurosci, 2014. 6: p. 292.
117. Dean, D.C., 3rd, B.A. Jerskey, K. Chen, H. Protas, P. Thiyyagura, A. Roontiva, J. O'Muircheartaigh, H. Dirks, N.
Waskiewicz, K. Lehman, A.L. Siniard, M.N. Turk, X. Hua, S.K. Madsen, P.M. Thompson, A.S. Fleisher, M.J.
Huentelman, S.C. Deoni, and E.M. Reiman, Brain differences in infants at differential genetic risk for late-onset
Alzheimer disease: a cross-sectional imaging study. JAMA Neurol, 2014. 71(1): p. 11-22.
118. Lautner, R., P.S. Insel, T. Skillback, B. Olsson, M. Landen, G.B. Frisoni, S.K. Herukka, H. Hampel, A. Wallin, L.
Minthon, O. Hansson, K. Blennow, N. Mattsson, and H. Zetterberg, Preclinical effects of APOE epsilon4 on
cerebrospinal fluid Abeta42 concentrations. Alzheimers Res Ther, 2017. 9(1): p. 87.
119. Pletnikova, O., Y. Kageyama, G. Rudow, K.D. LaClair, M. Albert, B.J. Crain, J. Tian, D. Fowler, and J.C. Troncoso,
The spectrum of preclinical Alzheimer's disease pathology and its modulation by ApoE genotype. Neurobiol
Aging, 2018. 71: p. 72-80.
120. Knickmeyer, R.C., J. Wang, H. Zhu, X. Geng, S. Woolson, R.M. Hamer, T. Konneker, W. Lin, M. Styner, and J.H.
Gilmore, Common variants in psychiatric risk genes predict brain structure at birth. Cereb Cortex, 2014. 24(5): p.
1230-46.
121. Combarros, O., M. Cortina-Borja, A.D. Smith, and D.J. Lehmann, Epistasis in sporadic Alzheimer's disease.
Neurobiol Aging, 2009. 30(9): p. 1333-49.