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Understanding the role of APP and DYRK1A in human brain pericytes

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Understanding the role of APP and DYRK1A in human brain pericytes

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Content Understanding the role of APP and DYRK1A in
human brain pericytes

Varsha Neelakantan
1
Mentor: Ruchi Bajpai
1,2

1
Department of Biochemistry & Molecular Medicine, Keck School of Medicine, USC
2: Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, USC

In Partial Fulfilment of the Requirements for the Degree
Master Of Science
Biochemistry and Molecular Medicine

August 2019

2

TABLE OF CONTENTS
Abstract………………………….……………..…....……………………………………6
Introduction ………………………….……………..…....………………………………7
1. Alzheimer’s Disease: an incurable, progressive neurodegenerative disease………….7
2. Blood-Brain Barrier defects persist before onset of disease pathology………….…....8
3. Components of the Blood-Brain Barrier…………………………………………...….9
4. Importance of pericyte-endothelial crosstalk in BBB and disease…..………………..11
5. ADAD and sAD pericytes have structural and functional defects ………………..….13
6. Rationale for choosing candidate genes ………………………….……………..…....15
7. Hypothesis………………………….……………..…....………………………….….16
Materials and Methods………………………….……………..…....…………………..17
Results ……………..….……………..…....…………………..………………………….20
Section I: Knocking down APP and DYRK1A did not affect ANGPT1 expression
and NG2 localization
1.1 Treatment with DYRK1A inhibitors…………….……………………………......20
1.2 Strategy for knocking down candidate genes………………….………………….22
Confirmation of knockdown………………….…………………………………...22
1.3 Effect of PSEN1 knockdown on primary pericytes………..…………………..….23
1.4 Knocking down APP and DYRK1A does not affect PC structure and
migration…………………………………..…………………………………...….25
1.5 Knocking down APP, APOE and DYRK1A show no alterations in PC specific
protein expression/protein localization ………………………………………..….26
Section II: APP and DYRK1A affects PC-EC Crosstalk independent of ANGPT1
2.1 APP and DYRK1A knockdown affects PC mediated endothelial junction
plakoglobin expression …………...………………………………………..…...…27
2.2 APP and DYRK1A knockdown affects PC mediated endothelial tube
formation………………………………………………………………………..… 29
…20
…27
3
Section III: RNA seq analysis of primary human pericytes……………......………............32
Future Directions …………………………………………………………….…….……....36

Discussion…………………………………………………………….………….....….......37
Citations……………………………………………………………………………............39
4
LIST OF FIGURES

Fig: Schematic of the proposed hypothesis. DYRK1A and APP affect PC-EC crosstalk in an
unknown mechanism.

Fig1: BBB breakdown in the hippocampus during normal aging and aging associated with AD

Fig2: Vascular Two-Hit hypothesis in AD

Fig3: Pericyte deficiency accelerates Ab pathology in APP
sw/0
Pdgfrb
+/-
mice.

Fig4: Pericytes in the brain: Structure, localization, and function

Fig5: Denovo defects in PSEN1-ADAD pericytes

Fig6: Pericytes treated with DYRK1A inhibitors FINDY and HARMINE for three days.

Fig7: Representative confocal images shPSEN1 1°PC
Fig8: Representative confocal images shAPP, shAPOE and shDYRK1A pericytes stained with
ANGPT1 and NG2

Fig9: Violin Plots showing no variation in PC size.

Fig10: Scratch assay showing migration of pericytes into the scratch area at two time points

Fig11: Lentiviral-mediated transduction method used to knockdown candidate genes

Fig12: Pericytes cultured with endothelial cells and IF on ki67, γ catenin and VE cadherin

Fig13: Quantification of the total γ-catenin and VE cadherin fluorescence signal, and number
of ki67 +ve cells.

Fig14: Pericytes cultured with endothelial cells in matrigel showing tube-like structure
formation and branching points.

Fig15: RNA sequencing analysis for Sporadic AD pericytes.

Fig16: Volcano plots of the sAD pericytes in comparison with WT

5
LIST OF TABLES
Table1: Primary pericytes samples (Controls and sAD) used for RNA seq analysis
Table2: Gene ontology terms for the commonly downregulated genes

Table3: PANTHER pathways for the commonly downregulated genes

ABBREVIATIONS

AD - Alzheimer’s Disease
ADAD - Autosomal Dominant AD
sAD - sporadic AD
fAD - familial AD
CNS - Central Nervous System
NVU - Neurovascular Unit
BBB - Blood-Brain Barrier
PC – Pericytes
iPSC - induced Pluripotent Stem Cells
hESC - human Embryonic Stem Cells
NCC - Neural Crest Cells
EC - Endothelial Cells
cPC - cranial Pericytes derived from pluripotent stem cells in a dish
1
o
PC - primary Pericytes isolated from post-mortem brain tissue sample
HBMvEC - Human Brain Microvessel Endothelial Cells
PSEN1 - Presenilin1
PSEN2 - Presenilin2
APP - Amyloid Precursor Protein
DYRK1A - Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A
APOE - Apolipoprotein E
ANGPT1 - Angiopoeitin1
γ-cat - Gamma Catenin (also known as Plakoglobin)
Aβ- Amyloid Beta
shRNA – Short Hairpin RNA
6
ABSTRACT

Cerebrovascular dysfunction and leaky blood-brain barrier have emerged as early indicators of
Alzheimer's disease (AD). Animal studies have shown that defective pericytes (PC) accelerate
Alzheimer’s-like pathology in models of AD. Our lab has shown that Alzheimer’s disease
pericytes show structural and functional defects and impaired pericyte-endothelial cell
crosstalk. What is not known is, how are PC functions regulated and what are the gene
mutations that could cause pericyte specific defects. Various studies including sporadic AD
cases, analysis of patients with Down’s syndrome or GWAS, have revealed a cohort of gene
variants associated with AD. However, their role, if any in pericyte biology or vascular health
is not known. Here we test the function of two genes Amyloid Precursor Protein (APP) and
Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) in primary
human pericytes using shRNA mediated knockdown. While this did not affect pericyte
viability, proliferation, migration or expression of common PC specific proteins, they resulted
in a consistent and reproducible defect in PC-EC crosstalk. Understanding what loss of function
of these genes does to pericytes will give insights on PC specific defects as well as serve as
potential targets for treating AD.

Fig: Graphical Summary. DYRK1A and APP disrupt PC-EC crosstalk resulting in defective
EC functions via as yet undefined mechanism. (A) Normal pericyte interaction with endothelial
Cells (B) PC expressing shDYRK1A and shAPP interaction with endothelial cells.

7
INTRODUCTION

Alzheimer’s Disease: an incurable, progressive neurodegenerative disease:
Alzheimer’s Disease (AD) is an aging associated neurodegenerative disorder characterized by
neuronal loss and cognitive decline often leading to dementia and is currently incurable. AD is
the sixth leading cause of death in the United States and it is estimated that about 5.8 million
Americans of age 65 or older may have dementia caused by AD. Although age is considered
to be the primary risk factor for developing AD, it is known to be multifactorial with recent
studies pointing to environmental factors, lifestyle and diet also contributing to AD
progression. Genetically Alzheimer's disease can be categorized into two major forms: the first
is an Autosomal Dominant AD (ADAD), caused by the inheritance or occurrence of disease-
causing mutations. ADAD is often characterized as early onset AD (EOAD), where patients
are diagnosed with AD between 30-60 years of age. Predominant cause of ADAD are
alterations in Presenilin1/2 (PSEN1, PSEN2), and Amyloid precursor protein (APP) and
constitute a minority (<5% ) of all the Alzheimer’s disease cases (Statistics from NIH/NIA,
Alzheimer's Association and [3]). PSEN1/2 are part of the γ secretase complex that is involved
in cleaving APP to produce Aβ. Aggregates of Aβ result in the senile plaques, a hallmark
feature of AD. About 95% of all cases of AD, fall into the second category: sporadic AD or
LOAD (Late onset Alzheimer’s Disease) which could develop due to a combination of genetic,
environmental and lifestyle effects [4]. The genetic basis of LOAD is more complicated
because the susceptibility of developing the disease appears to be due to a combination of
common but less penetrant genetic factors. Formation of Neurofibrillary Tau Tangles (NFT)
and accumulation of amyloid plaques are hallmarks of AD and are considered to be a crucial
step in the disease cascade, that leads to cognitive decline and dementia. Senile plaques are
extracellular deposits composed primarily of 40-42 amino acid long Aβ, which is derived by
proteolytic cleavages of the Amyloid Precursor Protein (APP) [5]. For a long time research on
AD was based on the premise that the biochemical phase (which includes abnormal Aβ and tau
phosphorylation) occurred first which lead to the cellular phase which included neuronal death,
microglial dysfunction and BBB alterations, finally leading to the clinical phase of cognitive
dysfunction [6]. Inflammation is also considered to be one of the central mechanism of AD
progression. Acute neuroinflammation is the natural response of the brain to foreign toxic
elements but chronic inflammation appears to be a detrimental process, resulting in progressive
neurodegeneration [7]. An imbalance between anti- and pro-inflammatory responses causes
chronic neuroinflammation and this sustained inflammation in the brains of people with AD is
8
associated with neuronal loss and is also considered to exacerbate both Aβ and NFT
pathologies. The brain is one of the most vascularized regions in the body with capillaries
occupying approximately 85% of cerebral vessel length becoming an essential part of the BBB
[8, 9]. Neurons require blood vessels for oxygen and nutrient supply but also for the removal
of other toxic substances [10]. Over the last few years, an increasing amount of research points
towards the fact that BBB dysfunction and persists in AD.

Blood-Brain Barrier defects persist before onset of disease pathology:

A meta-analysis done on aging and microvascular diseases revealed that in over 31 BBB
permeability studies (approximately 1953 individuals) there was a significant increase in BBB
permeability with age [11]. Another recent study done with Dynamic Contrast-Enhanced
(DCE) MRI, used blood-to-brain transfer constant, Ktrans to quantify the subtle changes in the
BBB permeability during normal aging and preclinical AD stages and in people with MCI
(Mild Cognitive Impairment) in the hippocampal region [12]. Hippocampal atrophy is a
symptom observed in many diseases, namely AD, depression, schizophrenia, epilepsy,
hypertension, and MRI based measures of atrophy in the hippocampus is predictive of
progression of MCI to AD [13, 14]. The results from this study shows that BBB breakdown
occurs during normal aging but is much more severe in patients with MCI and AD (Fig1) [12].
Pathological breakdown of the BBB is characterized by structural and functional changes in
the blood vessels leading to pro-inflammatory responses, impaired endothelial contacts,
increased transcytosis of neurotoxins and perivascular accumulation of toxic products [8, 15,
16].

9
Fig1: BBB breakdown in the hippocampus during normal aging and aging associated with AD
using high-resolution DCE- MRI. BBB Ktrans maps within the left hippocampus in young (23–
47 years) and older (55–91 years) individuals with no cognitive impairment (NCI), as well as
in older MCI and AD patients (Modified from the original figure by Montagne A in [12, 17])
This dysfunction of the BBB often develops before neurodegeneration and persists as the
disease progresses. Many recent studies have further supported this theory, that defects in the
brain vasculature occur much before the disease progresses to the stage of neuronal death.
These results further supported the two-hit vascular hypothesis in AD proposed by Dr.Berislav
Zlokovic which states that cerebrovascular damage is the first hit, that initiates the disease
cascade promoting neuronal death, neurodegeneration and accumulation of A (Hit two) [10,
18]. Vascular dysfunction may influence amyloidogenic pathway leading to improper
clearance of A as well as increased production and accumulation of A.

Fig2: Vascular Two-Hit hypothesis in AD proposed by Dr.Berislav Zlokovic [18].
The restrictive architecture of the BBB tightly regulates the paracellular diffusion of small
molecules and neurotoxic materials molecules due to the presence of tight junctions and
adherens junctions between endothelial cells lining the blood vessels [10, 19]. Therefore
defects in any one of the components of the BBB could lead to a dysfunctional barrier.
Components of the Blood-Brain Barrier:
To have proper structural and functional brain connectivity, synaptic activity and information
processing, it is essential to have a highly coordinated activity of the multiple cell types that
constitute the NVU (endothelial cells, pericytes, smooth muscle cells, glial cells and neurons)
[20]. Endothelial cells of the brain are different from other vascular endothelial cells because
it can regulate the passage of molecules and cells to and from the neural parenchyma [21].
10
BBB leakiness or breakdown could be due to degradation of the basement membrane,
excessive transcytosis, as well as from environmental factors and cardiovascular risks.
Disruption of the barrier will allow the entry of blood derived neurotoxins like fibrinogen,
thrombin, and plasminogen as well as neurotoxic haemoglobin and iron (Fe
2+
), causing
production of reactive oxygen species eventually leading to further breakdown, eventually
leading to a compromised brain immunity and neurodegeneration [20]. Pericytes are mural
cells found as elongated contractile processes spanning 2-3 endothelial cells in blood micro
vessels. Pericytes were first described as a population of contractile cells surrounding
endothelial cells by French scientist Charles Rouget in 1873. Later, Zimmerman studied these
cells further and named them initially as “Rouget cells” and coined the term “pericytes”
because of their peri-endothelial location [22]. Brain pericytes originate from a
neuroectodermal progenitor called Neural crest cells (NCC) unlike pericytes found in almost
all other parts of the body that are derived from mesodermal precursors. While PC from
different tissues share common properties, depending on their localization, they widely differ
in surface markers, morphology and number [22-24]. Thus, it is essential to determine specific
features of neural crest derived brain pericytes that are critical for maintaining BBB integrity.

Pericytes surrounding the endothelial cells are involved in many functions critical for vascular
homeostasis. And loss of pericytes is shown in many diseases, including diabetic retinopathy,
tumour angiogenesis and AD [25]. Pericyte-deficient mice models (Pdgfrb
+/-
mice) show age
dependent pericyte loss and BBB disruption to plasma proteins as well as accelerated Aβ
Fig3: The neurovascular unit and the
structures that contribute to the BBB. The
primary structures associated with BBB
function illustrated include the endothelial
cells, tight junctions, pericytes, and
astrocytic foot processes. [2]

11
accumulation (Fig3) [26]. PDGFRβ (platelet-derived growth factor) is a receptor that is present
in pericytes and is essential for pericyte survival and proliferation, shown by PDGFR K/O
mouse models [27, 28]. PDGFB is secreted by endothelial cells and the ligand/receptor pair of
PDGF/PDGFRB is essential for angiogenesis and blood vessel maturation because lack of this
signaling mechanism not only leads to PC loss but also capillary dilation and rupture [28, 29].
Pericyte loss accelerates Aβ accumulation, leads to neuronal loss and triggers hyper-
phosphorylation of tau proteins and causes tau pathology in pericyte deficient APP
sw/0
mice
*

[30].

Fig4: Pericyte deficiency accelerates Ab pathology in APP
sw/0
Pdgfrb
+/-
mice. In vivo
multiphoton microscopy of cortical angiograms with Texas-Red-conjugated dextran (red) and
methoxy-X04-positive amyloid (green) [30]

Importance of Pericyte-Endothelial Crosstalk in BBB and disease:
Crosstalk between endothelial cells and pericytes is essential for maintaining the BBB. Ligands
secreted by pericytes such as Angiopoietin 1(ANGPT1) , CXCL12 and their interaction with
EC receptors like Tie2 as well as the presence of receptors such as PDGFRβ, TGFRβ in
pericytes facilitates the EC-PC crosstalk [22]. While this signaling pathway is endothelial to
mural cell, another important pathway necessary for PC-EC crosstalk is the Angiopoietin1/Tie2
(also known as TEK) receptor pathway, which is necessary for blood vessel formation and/or

*
APPsw mice express human APP with the Swedish double mutation under the control of the hamster prion
promoter. Pericyte-deficient mice were made by disrupting the PDGFRβ gene. APPsw/0; Pdgfrβ+/- the double
mutant develops elevated Aβ, amyloid plaques, and tau pathology including hyperphosphoylated tau and early
conformational changes in the protein that are thought to be precursors to mature tangles.
12
stability [22, 29]. Thus, angiogenic defects may arise as newly formed vessels may be leaky
due to widened inter-endothelial junctions, lack of junction proteins and recruitment of
pericytes [31, 32].

Fig5: Pericytes and EC interactions at the BBB [33, 34]
The PC-EC crosstalk determines properties of the BBB and signaling pathways that limit EC
proliferation, regulate transport across EC monolayer and enhance EC interactions.
Ultrastructural studies demonstrate that endothelial cells in the brain are different from those
in peripheral tissue, predominantly because of the tight junction proteins present between the
brain ECs [31]. The tight junctions (TJ), comprising of VE-Cadherin, N-Cadherin, adherens
junctions (AJ) comprising of β catenin, plakoglobin, occludin, ZO1 and gap junctions
(Connexin family), function together in maintaining the tight barrier. The tight junctions are
present in the apical positions and mainly function to restrict para cellular transport but the
adherens junctions are also involved in maintaining vessel morphogenesis and stability and a
dynamic interaction between these protein serves as the barrier [35]. Small molecules like ions
and glucose are transported to the brain by transporters/carriers present on the plasma
membrane of the endothelial cells and macromolecular transport takes place through para
cellular transport maintained by an equilibrium between the endothelial cell contacts and
adhesive forces between the junctions [36]. The adhesive properties of TJ and AJ proteins also
depends on the phosphorylation states on the Ser-, Thr-, and Tyr- residues, which affects their
interaction, distribution and transmembrane localization [35]. Therefore, it is clear that junction
proteins are essential for the tightly regulated endothelial barrier, and defects in the barrier
could arise due to the inability of pericytes to mediate EC contacts or mutations in the TJ/AJ
proteins itself. Defects in any of the cell type present in the NVU could lead to BBB
dysfunction. For example, although other CNS diseases like Huntington’s Disease, Parkinson’s
Disease and Amyotrophic lateral sclerosis have completely different clinical pathology, they
13
have defects/mutations in endothelial TJ proteins that are a significant determinant of
vasculature permeability, HD patients and mice models of HD have a reduction in Claudin5.
In addition, the increased capillary density (vessels 5–10 μm in diameter) and reduced numbers
of larger micro vessels (10–20 μm in diameter) implies that there could be angiogenic defects
too, showing that BBB is compromised and that the cerebral vasculature is considerably
disrupted in HD [8, 31, 32]. Studies in ALS patients have shown that there is reduced
endothelial cell integrity in spinal cord sections seen by a significant reduction in occluding
and ZO1. There was also decreased perivascular occludin and collagen IV, as well
as astrocyte end-feet dissociation from the endothelium, suggesting that blood-brain barrier/
blood-spinal cord Barrier is impaired in ALS patients [15, 37]. Thinning of the endothelial
layer, reduced expression of TJ markers including Claudin5, ZO1, occluding and VE Cadherin
and basement membrane changes have been reported in brain tissues from patients with
Parkinson’s disease [38]. These results suggests that vascular defects may precede neuronal
damage and that neurodegeneration may be a consequence or a secondary event to brain
vasculature dysfunction. Compromised BBB could be a critical event in CNS disorders. But it
is still unclear whether pericyte defects or dysfunctional PC-EC crosstalk are what caused the
barrier defect in these patients. To stop the BBB from becoming dysfunctional, it is first
important to understand how they become dysfunctional. Thus, studying genes associated with
disease pathology will go a long way towards understanding more about the BBB functioning
as well as the vascular two-hit hypothesis in AD.
ADAD and sAD Pericytes have structural and functional defects:
Griffin and Bajpai [39] have derived cranial pericytes in vitro from iPS/ES cells via an NCC
intermediate. Our lab has used iPSC derived pericytes from patients carrying the ‘Jalisco
mutation’ (who develop the AD usually in their 40s) to model Familial Alzheimer's Disease.
This is a PSEN1
A431E
mutation that leads to the formation of a defective γ secretase and shows
pericyte specific defects, including structural and functional defects [39, 40]. Griffin et al. have
shown that one of the key mediators in pericyte function ANGPT1, is downregulated in early
pericytes and is conserved in all ADAD cases analyzed in Rat model of AD. Early passage
cPCs are indicative of “early pericytes” or pericytes that exist at birth. This study points to the
fact that PC specific defects persists from birth in ADAD Patients, much before developing
AD Pathology. ADAD pericytes have morphological defects, contractility defects, and there is
dysfunctional PC-EC crosstalk. PSEN1 mutant pericytes when cultured with HBmVEC show
a defect in inhibiting EC proliferation and defect in establishing EC contacts due to lack of
14
junction protein expression (PECAM and γ-catenin). Due to this, there was increased
permeability of the ECs and increased transcytosis across the “barrier” modelled invitro by a
transwell assay. It was also seen that these pericytes were not able to facilitate tube formation
by endothelial cells (Fig5). Taken together this implies that PSEN1/ γ secretase activity and
ANGPT1 signaling is important for normal pericyte functioning [40].

Then the question arose as to whether only pericytes from familial AD patients exhibited such
defects. Primary pericytes from three unrelated, sporadic AD (LOAD) patients exhibited
similar NG2 mislocalization, reduced EC-junctions and defective tube formation. When cPCs
were generated invitro from fibroblasts from the same patients, they also showed PC specific
defects albeit to a slightly weaker extent compared to ADAD cPCs. Together these data suggest
that undetermined genetic factors can impact pericyte biology thus contributing to disease
Fig6: Representative images of
PC defects seen in ADAD
pericytes (a) Loss of ANGPT1
(b) NG2 mislocalization in
iPSEN* cPC but not in its
CRISPR corrected control (c)
Diminished EC-EC contact
showing γ-catenin staining when
cultured with iPSEN* cPC
compared to CRISPR corrected
control (d) Tube formation
defect.
(a) (b)
(c)
(d)
15
progression. Since APP is one of the targets of γ secretase and a causative gene for ADAD, it
remains to be seen whether PSEN1/ γ secretase mutation induced pericytes defects are
mediated via APP and Aβ or if there are any additional roles for APP in pericyte biology.

Rationale for choosing candidate genes:
DYRK1A is a protein kinase that belongs to the highly conserved family of kinases and is
known to play a major role in cell proliferation, differentiation and nervous system
development [42, 43]. Pathogenic variants in DYRK1A resulting in haploinsufficiency of the
gene have been reported to cause a range of developmental defects including intellectual
disability, microcephaly and distinct faces [44]. This was also recapitulated in animal models,
where homozygous DYRK1A
-/-
mice showed embryonic mortality, but heterozygous
DYRK1A
+/-
mice showed growth retardation, reduced brain and body size and behavioural
defects [43]. While it is clear that DYRK1A plays an important role in development, whether
it has a role in terminally differentiated cells of the brain such as pericytes still remains unclear.
Some of the mechanistic insights on AD has been through DS-AD patients who have a Chr21
trisomy. At least 75% of DS patients who live beyond 40 years of age develop AD and
dementia [45-47]. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1A) a
gene that maps to human chromosome 21 and also happens to fall within the Down Syndrome
(DS) critical region. DYRK1A is a kinase that is involved in the phosphorylation of two key
proteins involved in AD APP at Thr668 and Tau at Thr212 residue [48]. In these patients
hyperphosphorylation of Tau at Thr212 residue by DYRK1A is reported to lead to NFT
formation [48]. APP phosphorylation at Thr668 alters its turnover and processing, making it
more stable – enhancing cleavage of APP by BACE1 [48, 49]. Elevated levels of DYRK1A is
seen in brains of DS patients and higher levels of APP and phosphoAPP are seen in transgenic
mice overexpressing DYRK1A [50]. There is also evidence which suggests that increased
accumulation of Aβ is what leads to the overexpression of DYRK1A and hence tau
hyperphosphorlation. This results in a vicious cycle of whether Aβ accumulation is a cause or
consequence [48]. In this context, DYRK1A inhibition has been shown to rescue AD like
defects, particularly lowering the levels of phosphorylated tau, so DYRK1A inhibition has been
considered as potential therapy for AD and DS associated AD [48, 51].
One of the most important gene variant that has been confirmed as a risk factor for late onset
AD/ Sporadic AD is the 4 allele of ApolipoproteinE (APOE) [45]. ApoE has three alleles (2,
16
3, and 4), the most frequent of which is the 3 allele. The APOE4 is a significant genetic risk
factor for AD and this isoform differs from the other two by an arginine at residue 112 which
leads to relative instability compared to APOE2 and APOE3 [46, 47]. Studies have shown that
neurovascular dysfunction may be present in APOE4 carriers before cognitive decline [46, 48].
Post-mortem human brain tissue analysis using biomarkers for BBB reveals that APOE4
carriers had accelerated pericytes loss compared to APOE3 carriers. Moreover, in APOE4
transgenic mice, astrocyte-secreted apoE4 leads to MMP9 mediated degradation of the BBB
tight junction and basement membrane proteins. APOE2 and APOE3 maintained normal BBB
integrity in transgenic mice by suppressing the CypA–MMP-9 pathway [48-50]. When it
comes to the role of APOE in pericytes, LRP1/APOE pathway in pericytes have an important
function in clearing Aβ aggregates [52] and it has been reported that knocking down APOE in
pericytes affects its mobility through a RhoA Protein-mediated Pathway [53].
Hypothesis:
While AD transgenic mice models have shown that inhibition of DYRK1A reduces
tauopathies, Aβ pathology and improves cognitive abilities, their effect on cells of the BBB are
not known. Thus, we hypothesized that loss of function of DYRK1A will not affect pericyte
function or it may improve the barrier. APOE4 allele has been associated with pericyte loss in
AD patients. This may be due to loss of function or gain of function of APOE which results in
toxicity to PC. We wanted to determine if loss of function of APOE affected pericytes, and this
will also help elucidate what are the functions of APOE in pericytes since its role so far has
been identified predominantly in astrocytes. γ secretase or PSEN1 cleavage products of APP
includes Aβ and APP Intracellular Domain (AICD) which is involved in signaling pathways
[41]. Our lab has characteriszd many pericyte specific defects in PSEN1 mutant ADAD
pericytes. We wanted to know if these defects were mediated by APP processing and we
expected that knocking down APP in pericytes will give similar defects as PSEN1 mutations
did because knocking down will result in lesser Aβ and AICD. So, for this project we have
chosen one ADAD (APP) and two sAD (APOE and DYRK1A) associated risk genes for further
analysis in primary brain pericytes.
17
MATERIALS AND METHODS
Cell culture
All cells were grown at 37°C with 5% CO2. The 1°PC (from a healthy 22-year-old) was
obtained from ScienCell. Cells were passaged using Trypsin-EDTA (0.05%) and then spun
down (900 rpm for 3 minutes).
Transfection and Infection of Virus/shRNA
HEK 293T cells was grown in in DMEM with 10% FBS. The plasmid of interest (10ug) was
added along with the packaging vectors pCMV-VSVG (2ug), pPAX2(1ug),
CMVR8.74(1ug) to 500ul DMEM and mixed in a tube. In another tube 36 μl PEI was added
to 500 μl DMEM. The two tubes were incubated for 15 minutes at room temperature, then
mixed and incubated for 20 minutes at room temperature and added to the 293Ts. After
approximately 16 hours, the media was discarded and Ultraculture media (Lonza, #12-725F)
with 2X glutamine was added. Viral supernatant was collected the next day and concentrated
by spinning at 15,000 rpm at 4
o
C overnight in an ultracentrifuge. Virus was resuspended in
remaining media by shaking on ice for about 6 hours. To infect pericytes, the cells were lysed
using trypsin and the pellet was resuspended in minimal volume of media (300 μl). 50 μl of
Viral supernatant was added to 0.5 million cells along with polybrene to a final concentration
of 10 μg/mL. The cells were incubated at 37
o
C for 45 minutes and then transferred to a plate
with media. Media is changed the next day. Doxycycline is added at a final concentration of 2
μg/ml and checked for RFP expression.
EC-PC Co-Culture
Human Brain Microvascular Endothelial cells (HBMECs) obtained from Sciencell (cat#1000)
were grown on fibronectin-coated plates using endothelial cell medium(Sciencell Cat#1001).
ECs and PCs were grown on glass coverslips at a ratio of 3:1 (EC:PC) in endothelial cell
medium for 3 days and then fixed for Immunofluorescence assays.
Immuno-fluorescence
Cells were plated on glass coverslips coated with fibronectin and grown to 75% confluency.
Before fixing the cells, the media is aspirated and quickly washed once with PBS and then
fixed using 4% Paraformaldehyde for about 15 minutes. The cells can then be stored in PBS
18
for later use or can be used for IF immediately. For Immunofluorescence, cells are first
incubated with PBTx blocking and permeabilization solution (1% BSA, 0.1% Triton-X in PBS)
for 1 hour at room temperature. Primary antibodies are diluted with PBTx in the range of 1:200-
1:500 and incubated with cells at 4°C overnight. The 1°Ab wash is done thrice with PBTx 10
mins each, followed by the addition of the secondary antibody (1:1000), left for 45 ins in dark
at room temperature. Cells were washed again with PBS and then DAPI (0.1 mg/mL in PBS)
was added for 5-10 minutes. Coverslips are mounted on slides using 15-20% glycerol and
imaged using either Leica Dmi8 inverted fluorescence microscope or Leica SP8 confocal
microscope. Images in figures are representative.
Antibodies used: NG2 (sc- 9.2.27), Angpt1 (abcam-),γ-Catenin(sc-514115) , VE-Cadherin
( sc-9989), ki-67
Cell Counting
Immunofluorescence assays quantification was done by taking at least 5 random fields per
sample. All the Ifs for different cell lines were performed simultaneously in identical
conditions, and all images were collected at the same settings of exposure collection and
threshold.
Tube assay
12x matrigel was thawed and 50μl was added per well of a 96-well plate. Approximately 9000
EC in 50μl ECM were added to each well and incubated at 37°C for 20 minutes, and then 3000
PCs in 50μl ECM were added to each well. Cultures were grown for 4 days and were imaged
daily using a Leica Dmi8 inverted fluorescent microscope
Scratch Assay
Cells were grown to about 75% confluency and using a 1ml pipette tip, a uniform scratch was
done and marked on the outside of the plate using a marker. The cells were imaged every 24
hrs until the scratch is filled.
Harmine and FINDY treatment
Primary brain pericytes were split and cultured in pericyte media for ~16h and then treated
with Harmine (Sigma Aldrich) and Findy (Sigma Aldrich) at different concentrations. The
inhibitors were dissolved in DMSO and cells were treated with DMSO in parallel experiments
19
as negative controls.
RNA-Seq data analysis
RNA was isolated from primary brain pericytes of 3 different sAD patients and 2 wild types.
RNA libraries were generated using the KAPA Stranded mRNA-Seq kits and sequenced
using Illumina Nextseq500. The raw sequencing data was aligned using STAR with hg19
reference genome which gave sam files as output. Using samtools, the files were converted to
the bam format and the readcounts were generated using “Featurecounts” in the Rsubread
package in R Bioconductor. Differential analysis of gene expression was done using Deseq2
package which uses an algorithm that does normalization of the counts with a median-of-ratio
method, taking into account sequencing depth and RNA composition. Input for DESEQ2 is a
DESeqDataSetFromMatrix which contains information about the counts matrix and the
information about the samples.

20
RESULTS
APP is expressed in many cell types and is substantially higher in neurons and astrocytes [54]
but is also expressed by brain pericytes and endothelial cells [55, 56]. APP is one of the
targets of PSEN1, and ADAD pericytes showed structural and functional defects, however
whether it depended on APP processing and Aβ production was still unknown. Excessive Aβ
from APP produced by astrocytes and neurons are toxic, but it was still unclear if loss of PC
observed in AD patients was a consequence of aggregation of Aβ. Addition of the toxic form
of Aβ in micromolar quantity exogenously to pericytes and pericytes cultured with
endothelial cells, showed no detrimental effects in the pericyte properties as well as PC-EC
crosstalk. While this did not kill pericytes and they continued proliferating, there is still a
possibility that long term accumulation of Aβ could be detrimental. This implied that
phenotypes associated with PSEN1 mutations were not a consequence of excessive Aβ
produced by pericytes and also that APP may have some other role in pericyte.
Section I: Knocking down APP and DYRK1A did not affect ANGPT1
expression and NG2 localization

1.1 Treatment with DYRK1A inhibitors:

One of the ways to elucidate the role of a particular protein in a cell, is to knock it down or
inhibit it and see what the loss of function does to the cell type. Certain compounds such as
Leucettine L41, carboline compunds like Harmine and FINDY are known to inhibit
DYRK1A function. FINDY inhibits the autophosphorylation of Ser97 in DYRK1A by
targeting its folding process, leading to its degradation, while L41 inhibits its proteolytic
cleavage, and harmine acts on DYRK1A by inhibiting substrate phosphorylation as well as
autophosphorylation of the mature kinase [57-60].

Harmine induces apoptosis in Neuroblastoma cells by activating caspase‐3/7 and caspase‐9
[58] and affected neurite formation in hippocampal neurons [57]. L41 treatment decreases the
accumulation of truncated DYRK1A in astrocytes and promotes microglial cells recruitment
leading to a decrease in aggregated Aβ load in an APP/PS1 transgenic mouse model [59]. This
shows that DYRK1A activity and role differs between cell types.
Viability assays done on HeLa and HEK293T cells showed that Harmine had no effect on the
cells in culture at concentrations that caused near‐complete inhibition of DYRK1A (upto 4μM)
21
[57]. So 1°Pericytes were treated with different concentrations of Harmine and FINDY and
observed them over the course of three days. Using higher dosages that were comparable to
what was done in multiple studies, was detrimental to pericytes, while less than 1 μM showed
little effect. Which implies that DYRK1A may have an important role in cell proliferation and
pericyte survival. Studies have shown that using DYRK1A inhibitors improves the tau
pathology of AD [48, 51]. But perhaps using lower concentrations for a prolonged time could
be harmful to pericytes. Additionally, it was difficult to follow up with assessment of PC
function in mediating EC contacts etc, using the inhibitors as Harmine was also shown to
directly affect endothelial cells, by inhibiting it migratory potential and tube formation [61].

Fig7: Pericytes treated with DYRK1A inhibitors FINDY and HARMINE for 3 days.

22
1.2 Strategy for knocking down candidate genes:

293 T cells were transfected with the validated shRNA construct as mentioned in the methods.
1°Pericytes were infected with the virus using polybrene (1μg/ml) and grown for 14 days in
dox (1μg/ml) and selected with puromycin (1μg/ml) to get a pure population of shRNA infected
(Red) cells. All cells were grown in identical conditions. The shRNA is flanked by miRNA
sequences so that the knockdown will be more efficient as it will be regulated by the
transcriptional and translational mechanism. Tet-inducible shRNA expression permits
reversible gene knockdown, and puromycin resistance gene allows selection of the infected
cells. The shRNA is present 3’ to the RFP reporter which allows the visualization of the
infected cells and also gives a direct read out of the level of shRNA in the cells.

Fig8: Schematic of the method used to knock-down the candidate genes. (1) Co-transfection
of lentiviral assembly plasmids into HEK-293t cells. (2) Lentivirus is released into the medium,
and then the medium is collected from which the virus is concentrated. (3) Infection of Primary
brain pericytes with the virus. (4) Turning on the shRNA by dox and selecting the pure RFP
population with Puromycin
Confirmation of knockdown:
The shRNA for DYRK1A has been used in another study where successful knockdown was
achieved [62]. However actual verification of the knockdown in pericytes for my study was
not done. Overall transcriptome analysis by total RNA sequencing is underway, which will
also independently determine the level of knockdown. So there exists a caveat in my
experiments since the knockdown level wasn’t assessed.
23
1.3 Effect of PSEN1 knockdown in primary pericytes:
Pericytes are elongated cells with processes extending on two sides of the nucleus and
stretching across 2 or more endothelial cells. This elongated structure is essential in stabilizing
blood vessels as well as maintaining the integrity of the barrier. The ADAD patient pericytes
carrying a PSEN1 mutation showed structural defects: where the pericytes are shorter in length
and with multiple processes.
A validated shRNA for PSEN1 that is known to cause about 70% reduction in the γ-secretase
activity was tested out first and it recapitulated some of the defects seen in ADAD pericytes
(Fig). The shPSEN1 1°PC appear flat and with multiple projections as compared to control
pericytes and there is a reduction in the levels of ANGPT1 compared to the controls and NGT
is symmetrically localized in some of the cells (Fig8).

Fig9: Knocking down PSEN1 affects PC structure, protein expression and localization (a)
(c)
(a) (b)
24
Representative confocal images of shPSEN1 1°PC at maximum projection. (b) Violin plots for
size of shPSEN1 1°PC compared to control (c)Representative images of IF of ANGPT1(green)
and NG2(Red) done on shPSEN1 infected cells (Grey)

25
1.4 Knocking down APP and DYRK1A does not affect PC structure and migration:
Pericytes expressing shAPP, shAPOE (figure not shown) and shDYRK1A cultured in similar
conditions as the control and measured their length across different fields and found that there
were no structural differences. Pericytes are important in the regulation of EC proliferation and
angiogenesis, and they migrate to the site of blood vessel formation in response to signals from
endothelial cells [29]. Factors such as NG2, PDGFRb and Angpt1 contribute to the migratory
abilities of pericytes [63]. But it was not known if DYRK1A or APP would have an effect on
the migratory potential of pericytes. When a semi-confluent plate of pericytes was scratched
with a uniform width and allowed to grow, the gap was filled by the pericytes showing that
there is no defect in cell migration

Fig10: Pericyte structure is not affected when APP, APOE or DYRK1A is knocked down. Violin
Plots showing no variation in PC size. Length of pericytes was measured using ImageJ. At least
N>100 cells were measured. (Below) Representative confocal images of pericytes at maximum
projection.
(b)
(a)
26

Fig11: APP and DYRK1A have no effect on the migratory potential of pericytes. Scratch assay
showing migration of cPC into the scratch area at two time points

1.5 Knocking down APP, APOE and DYRK1A show no alterations in PC specific protein
expression/ protein localization:
To begin understanding the possible effects of knocking down these proteins certain pericyte
specific markers were tested for the level of expression and localization. 1°PC expressing the
shRNA for APP, APOE and DYRK1A was cultured in similar conditions with doxycycline for
2 weeks and fixed in a coverslip to perform an IF. The proteins we looked at were NG2 and
ANGPT1. NG2 is a Pericyte specific proteoglycan, which is usually polarized and
asymmetrically localized in pericytes. ANGPT1 ligand is produced by Pericytes and it binds
to the TIE2 receptor in endothelial cells, enabling EC-PC crosstalk. Previous data from our lab
has shown that iPSEN1*-cPC, as well as PC expressing shRNA for PSEN1 and PSEN2 showed
mislocalized NG2 and downregulation of ANGPT1, implicating that it is a feature of defective
γ-Secretase activity. But when Pericytes expressing shAPP and shDYRK1A were assessed,
there was no defect in the localization of NG2 AND there was no change in the levels of
ANGPT1 in shAPP and shDYRK1A pericytes.

27

Fig12: APP, APOE and DYRK1A have no effect on PC protein expression or protein
localization. (a) Representative confocal images Pericytes stained with ANGPT1 and NG2 (b)
Quantification showing that the number of cells expressing asymmetric NG2 is constant in all
the knockdowns and there is no mislocalization of NG2. (c) Quantification of the number of
cells expressing ANGPT1 showing no change in levels of ANGPT1 Scale bars represents 100
um. Counts were done of high-resolution maximum projection confocal images of N > 100
cells from at least 2 experiments
Section II: APP and DYRK1A affects PC-EC Crosstalk independent of
ANGPT1
2.1 APP and DYRK1A knockdown affects PC mediated endothelial junction plakoglobin
expression:
Dysfunctional crosstalk or interplay between endothelial cells and pericytes are the considered
to be the cause of many pathologies. To test whether APP and DYRK1A may be involved in
vascular functions like PC-EC crosstalk, Pericytes and endothelial cells were co-cultured for
three days. The PC mediated EC contacts are formed by maturation of EC that takes place by
first inhibiting proliferation and then establishing contacts between cells through TJs and AJs.
It was found that shAPP and shDYRK1A 1°PC when cultured with endothelial cells were
effectively able to block their proliferation but were not able to induce EC contacts to the same
extent as the shCTRL pericytes due to downregulation of one of the junction proteins required
(a)
(b) (c)
28
for EC contact – γ-catenin. γ-catenin and β-catenin are structurally similar and interact with
VE cadherin to maintain the junctions between endothelial cells. We wanted to know if other
components in the endothelial junctions were affected . However, upon immunofluorescence
analysis and quantitation of VE cadherin stained regions there, there was no significant change
in the levels of VE cadherin in EC junctions.

Fig13: APP and DYRK1A affect EC interaction but not proliferation. Representative confocal
images pericytes cultured with endothelial cells. Cells are co-cultured for 3 days in similar conditions
and stained with the antibodies of interest. (a) DAPI (Blue) stains the nucleus and ki67 (red) is the
proliferation marker. The top row shows that there is a reduction in the number of cells proliferating
when EC is cultured with PC but there is no variation when APP and DYRK1A are knocked down.
Scale bars represent 50 um. (b) Pericytes (red) enable endothelial cells to form contacts with each other
through the expression of Junction Plakoglobin or catenin (Green) but the pericytes expressing
shRNA for APP and DYRK1A show a significantly lower level of plakoglobin. Scale bars represent
100 um. (c) γ-catenin closely interacts with another junction protein VE-Cadherin (Green). Shown here
is a representative image of both the control and knockdown pericytes (red) are able to mediate VE
cadherin expression to a similar extent. Scale bars represent 50 um.

(a)
(b)
(c)
29

Fig14: APP and DYRK1A affect EC interaction but not proliferation. All quantifications were
done in at least 5 different fields/experiment fields in at least 2 biological replicates and
calculating its average. The images were taken in similar conditions and the ratio of pericytes
to endothelial cells was on average consistent. (a)Quantification of the percentage of ki67
positive cells. No significant difference in the number of ki67 positive cells when shAPP and
shDYRK1A pericytes are cultured with EC. (b) Quantification of the total γ-catenin
fluorescence signal. (c) Quantification of the total VE-Cadherin fluorescence signal in at least
5 different fields/experiment fields and calculating its average. No significant difference was
seen in the levels of VE-Cadherin when APP and DYRK1A were knocked down in primary
pericytes. Significance was determined using ANOVA-Tukey’s multiple comparison

Fig15: Western Blot of γ catenin and GAPDH. Quantitation of the signal intensity using
ImageJ.

2.2 APP and DYRK1A knockdown affects PC mediated endothelial tube formation
One of the important functions of pericytes is the regulation of angiogenesis and pericytes are
in close contact with endothelial cells as they extend their processes across several EC to
encircle the EC derived vessels or “tubes”. When endothelial cells and pericytes are co-cultured
in matrigel endothelial cells form tube like structures which are not formed when they are
cultured by themselves. Endothelial tube formation is a density dependent phenomenon. At
low density they do not form branching tubes, and these structures are mediated by the presence
(c) (b) (a)
30
of pericyte, but higher density of EC will result in tube like structure independent of the
presence of pericytes. However, to test how pericytes mediate endothelial tube formation, the
number of cells per 96 well plate was optimised (≃10000 EC and ≃ 3000 PC). Our lab has
shown that PSEN1 mutant pericytes show reduced tube lengths and tube branching and that it
is partially due to downregulated ANGPT1. Since γ-catenin was not induced to the same extent
by pericytes knocked down with APP and DYRK1A , we predicted that it might affect its tube
formation capabilities too.
The control PC, shAPP PC and shDYRK1A PC when cultured with endothelial cells form
tubes with branching in about 3 days after plating them. But the tube lengths of the endothelial
cells when cultured with the shAPP and shDYRK1A pericytes showed reduction in the length
of the tubes.

31

Fig16: APP and DYRK1A affect EC tube formation (a) Representative images of Pericytes
cultured with endothelial cells in matrigel showing tube-like structure formation.
(b,c) Violin plots showing average unbranched tube length in two replicates. Tube area in
pixels was measured in multiple fields/experiment using ImageJ . There is a reduction in tube
length of endothelial cells when cultured with PC expressing shAPP and shDYRK1A. (d)
Quantification of Average number of branching points (e) Representative images showing tube
branching points.

(a)
(b)
(c)
(d) (e)
32
Section III: RNA seq analysis of primary human pericytes

In order to understand the transcriptomic profile of the knockdown pericytes and check how
they differed from other sporadic gene mutations, RNA sequencing data from 3 sporadic AD
pericytes was analysed. It is essential to understand what the common defects in the sAD
pericytes are first. Our lab studied pericyte defects from three sporadic AD patients (78 yrs.,
84 yrs., and 89 yrs.) and an ADAD (PSEN1 mutant) pericyte. We found that even though the
two types of AD are technically different manifestations of the disease, there are many pericyte
specific defects that overlap: such as NG2 Mislocalization, PC-EC cross talk defects, tube
formation defects and structural defects. To understand the possible mechanisms involved in
these pericyte defects and the genetic variations underlying sporadic AD patient pericytes. R
subread and DESEQ2 was used to perform the gene expression analysis of RNAseq data [64,
65]. Using a P value cut off as 0.05 and Log2FoldChange cut off as -1.5 and 1.5 for
downregulated and upregulated genes respectively, we identified that the three patients show
only 4-5% differentially expressed genes with this cut off. This includes 342 genes commonly
downregulated and 74 genes commonly upregulated in the 3 sAD patients.

GO term and Pathway analysis: The 342 genes that were commonly downregulated in the
three sAD patient pericytes were used as the input for enrichment analysis for GO term and
PANTHER pathways [66]. For the GO analysis, only terms that FDR less than 0.1 and p value
less than 1E-06 were considered as significantly enriched. Amongst the terms were Tube
development, Locomotion, blood vessel development and Cell adhesion, all of which are
pericyte related functions. Our lab has also shown these defects invitro using the same sAD
pericytes [40]. This suggests that there are common defects in AD pericytes.

PANTHER(V.14.0) [66] was used to find out what were the major pathways these genes were
involved in. Majority of the genes that are commonly upregulated and downregulated are
involved in the Wnt pathway and the Cadherin pathway, while other pathways like FGF
signaling and TGFb signaling have much fewer genes involved. The Wnt signaling pathway is
involved in many essential cell processes including cell proliferation and differentiation. The
Cadherin and Wnt pathway are linked by β-Catenin [67]. Presenilin1 is also involved in Wnt/b-
catenin signaling, acting as a negative modulator of b-catenin/Tcf-4-mediated transcription
[68, 69] Some of the genes that are misregulated in the Cadherin pathway are Protocadherins.
Protocadherins are the largest mammalian subgroup of cadherins and are organized as α, β, γ.
α- and γ-protocadherin (Pcdhs) are present predominantly in the central nervous system and
33
undergo Presenilin-dependent intramembrane proteolysis, and the cleavage product α C-
terminal fragment 2 (α-CTF-2) and γ-CTF-2 are important in signaling pathways
that determine cell and synapse adhesive properties [70-72]

Other genes that are downregulated include PTPRB (also known as VE-PTP-(vascular
endothelial protein tyrosine phosphatase)) and TEK which is known to be essential for VE
cadherin function and hence EC-EC interactions. Many other cell adhesion proteins are
downregulated such as ICAM, NCAM2, ITGB3 showing that cell-cell contacts and the barrier
integrity could be compromised in AD pericytes. TEK or Tie2 receptor is present in both
endothelial cells and pericytes. It is a tyrosine kinase receptor that is important for ANPT1
signaling. PTPRB also binds to Tie2 receptor and negatively regulates Tie2 activation. In
mouse models, inhibition of PTPRB has been shown to activate ANGPT1/Tie2 signaling, but
also increase vascular permeability and supressed neovascularization in ocular vasculature[73].
γ-catenin is essential for VE-PTP to enhance VE cadherin function of EC adhesion and
permeability[74]. Thus in these sAD patients the pericytes having lower PTPRB may be
attributed to (a) having lesser angiogenesis (tube formation) as well as weaker EC contacts (b)
the pericytes have downregulated PTPRB so that the ANGPT1/Tie2 signaling can be enhanced
to compensate for the lower levels of ANGPT1.

ID Condition Sex Age
ScienCell
1oPC
WT M 20
086 1oPC WT M 52
# 049 AD M 78
# 131 AD F 84
# 044 AD M NA

Table1: Primary pericytes samples (Controls and sAD) used for RNA seq analysis
34

Fig17: Differential expression analysis of sporadic AD pericytes (a) Using a cut off as P < 0.05
and Log2FC > 1.5 and Log2FC < -1.5 for upregulated (b) and downregulated (b) genes
respectively. Number of genes that are upregulated and downregulated in the three sAD patient
pericytes Using a cut off as P < 0.05 and Log2FC > 1.5 and Log2FC < -1.5 for upregulated and
downregulated genes respectively. (Venn diagram created using Biovenn [75])
(c) GO terms and (d)PANTHER pathways for the 342 genes that are commonly downregulated
in the three sAD patients.
(a) (b)
(d)
(c)
35

Fig18: Volcano plots showing the differential
expression pattern in the three sporadic AD
patient pericyte. The volcano plots were
generated using enhancedvolcano package [1]
and it is seen that the number of statistically
significant and differentially expressed genes
varies between patients.
Volcano plots with cut offs as P value = 10e-8 ;
Log2FoldChange = -1.5 and 1.5, comparing (a)
044 vs WT, (b) 049 vs WT, (c) 131 vs WT.
ANGPT1 is downregulated in all the three
patients but more severely in one of the patients
(049)
Genes annotated include: ANGPT1, ICAM,
NCAM2, ITGB3. CDH4, TEK, PTPRB.

(a)
(b)
(c)
36

Table2: Gene ontology terms for the commonly downregulated genes

PANTHER pathways No: of genes Genes involved
Angiogenesis (P00005) 6 TEK KDR F3 AL137145 PTPRB NOS3
Alzheimer disease-presenilin
pathway (P00004)
5 MME AC006483 PCSK6 ACTBL2 AL137145
Wnt signaling pathway (P00057) 10
PCDH12 AC006483 CDH5 CDH4 DCHS1
KREMEN1 CDHR1 PCDHGB7 PCDH7 AL137145
Cadherin signaling pathway
(P00012)
9
PCDH12 AC006483 CDH5 ACTBL2 CDH4 DCHS1
CDHR1 PCDHGB7 PCDH7

Table3: PANTHER pathways for the commonly downregulated genes

Future directions:

RNA has been isolated from the pericytes expressing shRNA for DYRK1A and APP as well
as controls, and libraries are made. The sequencing for these libraries is underway and
analysis for those will be done soon using the same pipeline used for the above analysis.
Further analysis will be done to compare the differences between the knockdowns and
sporadic AD pericytes.

37
DISCUSSION

Brain vasculature defects and loss of pericytes is a phenomenon observed in AD patients.
Based on our lab’s characterization of cranial pericytes and ADAD (PSEN1 mutant) pericytes,
we wanted to know if other AD risk associated genes had any effect on brain pericyte functions.
Four of the five genes that we tested out showed PC specific defects. We started by knocking
down APOE, APP and DYRK1A. While data from PSEN1 mutants and sAD pericytes showed
that ANGPT1 was downregulated and NG2 was mislocalized, knocking down APP, APOE and
DYRK1A in pericytes showed no difference in the levels of ANGPT1 and NG2. Since APOE
did not affect the ability of pericytes to mediate EC contact formation as well, further assays
were not performed on the APOE knockdown lines. However, it is possible that loss of function
of APOE is not detrimental to pericytes, but the mutation resulting in APOE4 allele is what
causes PC defects. Our lab has characterized pericytes that have heterozygous APOE4 and
found that only the transcytosis activity of these pericytes are slightly altered. So perhaps
APOE does not govern the functions of PC that have been tested out. Interestingly, knocking
down DYRK1A and APP had no effect on the levels of ANGPT1 but affected endothelial
contact formation and tube formation. It is possible that APP and DYRK1A are involved in
maintaining the EC contact only but not the EC maturation process (because the proliferation
is not affected). γ-Catenin levels are lower in PC mediated EC junctions, but proteins that
directly interact with γ-Catenin are not affected. Clustering of molecules at a junction, such as
plakoglobin could enable other interactions and further direct signaling cascades. In addition
to γ-Catenin functioning as an adhesion protein it can also modulate Wnt/β-catenin signaling
by competing with β-catenin for binding to the transcriptional coactivator TCF/LEF [76-78].
A recent study reported that knocking down plakoglobin decreased tube formation ability and
increases the permeability of HUVECs. While plakoglobin knockdown mildly reduced the
levels of VE cadherin, it is not required for a connection between VE-cadherin and cytoskeletal
proteins like vimentin or phalloidin in ECs [79]. And clearly because of some downstream
effect of knocking down APP and DYRK1A, there is a reduction of γ-Catenin at the junctions
which is affecting tube formation ability in endothelial cells. There is a caveat to this
conclusion, since the level of knockdown was not verified.
A subset of the defects seen in APP knockdown was seen in PSEN1 mutant pericytes which
could mean that those defects in the ADAD pericytes were governed by APP processing. Based
on this initial characterization, we think that DYRK1A and APP perhaps functioned through a
signaling pathway independent of ANGPT1 regulation, and some downstream effectors of the
38
knockdown were involved in EC-PC crosstalk. The downstream effect of knocking down these
genes in pericytes is potentially affecting a diffusible molecule that mediates PC-EC crosstalk
however, experiments with pericyte conditioned media was not reproducible. This will be
addressed using RNA seq analysis to identify differentially expressed genes in the knockdown
cells. Since the loss of function of single genes like PSEN1, APP or DYRK1A affects different
aspects of pericyte biology, we predict that the RNA seq analysis of the knockdown pericytes
will reveal some of the GO terms and pathway that has been seen in the sporadic AD pericytes,
because only a subset of the defects is shared.
Harmine and other -carboline compounds inhibit the kinase activity of DYRK1A and Tau
phosphorylation in neurons and the hippocampal regions [51]. In transgenic AD mouse models,
inhibition of DYRK1A has shown to improve AD like pathology by altering APP processing
and reduction in tau phosphorylation. The study focused on hippocampal regions of the mouse
brain and found that DYRK1A inhibition reduces APP phosphorylation, thereby modifying
APP turnover [48]. More recently a study done by Samumed in 2018 further supported
previous studies and presented positive results for improved cognitive abilities and protection
against amyloid and tau pathology by a novel DYRK1A inhibitor. And as of April 2019, phase
I clinical trials are ongoing for this drug [80]. Since animal models of DYRK1A inhibition
showed improved AD pathology, our prediction was that BBB is also improved, but contrary
to that hypothesis, inhibiting DYRK1A results in a compromised barrier, which suggests that
pericytes in mice do not express DYRK1A at high levels or they don’t play a role in many
functions. While it may be true that DYRK1A inhibition ameliorates AD pathology, it is
important to consider the effect of an inhibitor on all human cell types in the brain. The
experiments done in our lab show that DYRK1A inhibition by kinase inhibitors like harmine
affect pericyte viability at higher dosages, therefore low dosage over a long period could be
detrimental. Additionally, knocking down DYRK1A results in a compromised barrier. Putative
therapeutic targets for BBB alterations still need time to emerge, it is important to look at the
whole picture including the BBB when it comes to developing a treatment for a disease. The
BBB should be considered as a therapeutic target to ameliorate the pathology of the disease.

39
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44
Acknowledgments:

I am incredibly grateful to Dr.Ruchi Bajpai for her constant support and guidance throughout
this project. I am also very thankful for the support from all our lab members in teaching me
new techniques and helping me out with lab work. I would also like to thank the Biochemistry
and Molecular Medicine department which gave me the opportunity to pursue this research. I
also extend my humble gratitude to my Thesis committee members Dr.Judd Rice and Dr. Axel
Montagne.

And most importantly, my family and friends for encouraging me and supporting throughout
the two years of this program.

Abstract (if available)

Abstract Cerebrovascular dysfunction and leaky blood-brain barrier have emerged as early indicators of Alzheimer's disease (AD). Animal studies have shown that defective pericytes (PC) accelerate Alzheimer’s-like pathology in models of AD. Our lab has shown that Alzheimer’s disease pericytes show structural and functional defects and impaired pericyte-endothelial cell crosstalk. What is not known is, how are PC functions regulated and what are the gene mutations that could cause pericyte specific defects. Various studies including sporadic AD cases, analysis of patients with Down’s syndrome or GWAS, have revealed a cohort of gene variants associated with AD. However, their role, if any in pericyte biology or vascular health is not known. Here we test the function of two genes Amyloid Precursor Protein (APP) and Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) in primary human pericytes using shRNA mediated knockdown. While this did not affect pericyte viability, proliferation, migration or expression of common PC specific proteins, they resulted in a consistent and reproducible defect in PC-EC crosstalk. Understanding what loss of function of these genes does to pericytes will give insights on PC specific defects as well as serve as potential targets for treating AD.

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Asset Metadata

Creator Neelakantan, Varsha (author)

Core Title Understanding the role of APP and DYRK1A in human brain pericytes

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 07/29/2021

Defense Date 06/18/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Alzheimer's disease,blood-brain barrier,neurodegeneration,OAI-PMH Harvest,pericytes

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Bajpai, Ruchi (committee chair), Montagne, Axel (committee member), Rice, Judd (committee member)

Creator Email 96varsha@gmail.com,vneelaka@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-199750

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Rights Neelakantan, Varsha

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