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Lipid Regulation as a Critical Factor in the Development of Alzheimer's Disease

PhD Thesis Defense presented by Michael A. Castello on Friday, September 5, 2014.
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Michael Castello

on 24 February 2015

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Transcript of Lipid Regulation as a Critical Factor in the Development of Alzheimer's Disease


Lipid Regulation as a Critical Factor in the Development of Alzheimer’s Disease
Soriano Neurodegenerative Diseases Laboratory
Friday, September 5, 2014
}
AD
Piece by piece, Alzheimer's disease fatally destroys both
mind and body
Thesis Defense by Michael A. Castello
Alzheimer's Disease (AD) is the most common form of
dementia
Progressive decline in memory and cognitive ability
AD affects
5-6 million
North Americans
1 in 9
by retirement age (over 65)
1 in 3
by age 85
In the US, Alzheimer's disease is the
6th leading cause of death
It has
no effective treatment
Overview
Introduction to Alzheimer's Disease
Adaptive Response Hypothesis
Experimental Testing
Summary and the Future
Amyloid cascade hypothesis
Problems with the amyloid cascade
Stress, lipids, and Alzheimer's
APP as a stress response
Genes
Mechanisms
Practical applications
AD Features
Begins with
difficulty remembering
names and events
Clinical
Progresses to symptoms including
impaired judgement
,
confusion
, and
difficulty speaking
,
walking
, and
swallowing
Ultimately fatal
Pathological
Senile plaques
Neurofibrillary tangles
Aggregations of
amyloid beta (Aβ)
Formed through cleavage of
amyloid precursor protein (APP)
Aggregations of
hyperphophorylated tau protein
Microtubule-associated protein, involved in
axonal transport
Familial Alzheimer's
Amyloid Cascade Hypothesis
Down Syndrome
Rare mutations in
APP
or its
cleavage enzymes
Causes
Aβ accumulation
and
early-onset AD
APP is often
duplicated
, leading to
overexpression
These patients have
Aβ plaques
and dementia

l
oligomers
plaques
Tau
Neuronal Loss


l
oligomers
plaques
Amyloid Hypothesis
AD
plaques
Supported by countless
animal model
and
cell culture
studies [2]
Targeting Aβ has
failed to produce
effective AD therapies
Perhaps due to prior strong evidence, this has
not prompted critical evaluation
of the hypothesis
Instead, failures are attributed to
treatment timing
,
patient selection
, and
undetectable

l
oligomers
-
-
Aβ+
Available Aβ imaging techniques can only visualize Aβ plaques, NOT
l
oligomers!
Medical community
Aβ is an unreliable marker in early stages of AD
Greater emphasis on neurological function in diagnosis
Alzheimer's researchers
Use imaging to identify AD patients with and without Aβ
Eliminate patients without Aβ from clinical trial
Identify AD patients based on neurological function
Tautological Definition of AD
Aβ causes Alzheimer's disease because Alzheimer's disease is a disease of Aβ

=
AD
Understanding Aβ function is critical to understanding AD
STRESS
Aβ±
Aβ±
In this model, Aβ is produced as part of the
brain's response to stress
This process could be both
necessary
and
harmful
Cell division
Inflammation
We would
expect to see
evidence of
Aβ production
whenever the brain is under stress
Evidence for stress-induced Aβ production
Injury
Activity
Metabolic Regulation
In traumatic brain injury, Aβ
increases inversely
with
tau damage

increases after ischemia
, perhaps due to oxidative stress
Regular
neuronal activity increases Aβ
, which promotes synaptic plasticity
Aβ levels
decrease during sleep
Diabetes
increases risk
of developing AD and accelerates accumulation of both
Aβ and tau
in mice [3,4]
Lipid metabolism in the brain
The brain is only 2% of body mass...
...but contains 25% of body cholesterol
2
25
Brain cholesterol is
relatively isolated
by the blood-brain barrier
...form
lipid rafts
and contribute to
membrane fluidity
Cholesterol and other lipids...
...make up
myelin
, become
neurosteroids
, and aid in
synaptic function
DHEA
Complete turnover takes
five years
Interaction with the body occurs via
oxysterols
24-hydroxycholesterol
27-hydroxycholesterol
Lipid regulation is disrupted in AD
Cholesterol synthesis
fails to decline
with age...
...but
myelin lipids
are
decreased
E4 form of lipid transporter
ApoE = 18-fold risk
of AD
27-hydroxycholesterol is
increased
Other genetic risks:
metabolism
,
endocytosis
,
inflammation
APP processing (Aβ production) occurs in
lipid rafts
...
...which can be
altered
in composition
via diet
Insights from Niemann-Pick type C
NPC
begins with lipid dysregulation
via a non-functional cholesterol transporter
In later stages,
Aβ production
and
tau hyperphosphorylation
occur along with
dementia
NPC is a devastating
neurodegenerative disease
affecting
movement
In NPC mice,
loss of APP exacerbates the disease
phenotype
Without APP
symptoms worsen
and
tau is hyperphosphorylated
in the absence of Aβ
[5] Nunes et al, 2011
Linking lipid regulation and APP
Loss of APP negatively affects NPC phenotype
Lipid raft composition affects APP cleavage into Aβ
Becomes AICD, a transcription factor affecting lipid transporters
Binds cell surface lipids; could function as a signaling molecule
STRESS
LIPIDS
APP


AD
Adaptive Response Hypothesis
Lipid dysregulation is a key stressor in the early pathogenesis of AD
APP metabolism–including Aβ production–is part of an adaptive response to stress in the brain
Heritable mutations cause early-onset AD by dysregulating the adaptive response
Adaptive Response Hypothesis
Testing the hypothesis
Specific Aim 1: Identification of genes
involved in brain cholesterol regulation that are associated with APP
Specific Aim 2: Identification of mechanisms
through which APP influences cholesterol homeostasis in the brain
Specific Aim 3: Practical application
of my findings to clinical AD by prediction of AD risk
Specific Aim 1
To identify genes involved in brain cholesterol homeostasis that are regulated by APP
Rationale
SA1 Results
Removing APP from healthy mice results in age-dependent
cognitive
and
locomotor deficits
,
reactive gliosis
, and
synaptic degeneration
These findings suggest that APP has a
specific
,
adaptive
role in cholesterol regulation
Knockout mice enable a
systematic search
for molecular targets of APP function in the
absence and presence of cholesterol dysregulation
Experimental Design
Microarray Analysis of Gene Expression
Analysis and interpretation of data obtained through
genetic microarray
Functional pathways with differentially expressed genes in cortex and cerebellum include
cholesterol biosynthesis
,
membrane trafficking
, and
myelination
Ingenuity Pathway Analysis revealed functional networks of genes
Reelin signaling: Kinases downstream of Very Low-Density Lipoprotein Receptor (VLDLR) are downregulated
only in double knockout mice
VLDLR, originally grouped as trafficking,
interacts with the cholesterol metabolism
pathway via HMG CoA Reductase (HMGCR)
Unexpectedly,
CD59
, an inhibitor of complement activation, is
downregulated
This suggests an
interaction
between the two knockouts that is
more significant
than either alone
In the double knockouts,
C1q
, a complement activator, is
upregulated
, providing evidence for
inflammatory activation
CD59 also relates to
cholesterol synthesis
via HSD17B7, and
phospholipid metabolism
via PEMT
Ermin (ERMN)
, a protein related to myelination, appears to have a
direct regulatory link
to APP
This connection was previously unsuspected
Proteomic Validation
Use
mass spectrometry
to compare
protein expression levels
across animals
Correlate changes with microarray results
Results were
highly variable
Could reduce in the future by
increasing total protein concentration
and
number of fractions
Decreasing trend (0.06 > p > 0.05) in GAPDH
This change has been associated with
oxidative stress
, and could negatively affect
axonal transport
in neurons [6,7]
Specific Aim 1: Summary
Validates that
cholesterol systems are disrupted
in APPko mice despite their lack of obvious phenotype
My work on SA1...
Identified
connections between genes and pathways
for further study
Collected
microarray data
for establishing a simulation model
To identify specific mechanisms through which APP influences cholesterol homeostasis
Specific Aim 2
Rationale
Specific Aim 1
identified functional pathways
and networks suggesting that
APP is capable of regulating lipid homeostasis
in the brain
Specific Aim 2 focuses on
identifying potential mechanisms
through which APP might act
In the process, I discovered the significance of testing a link between filipin fluorescence and cellular damage
Original: Direct manipulation of gene expression
Experimental Design
Modified: In vitro characterization of the link between filipin staining and cellular damage, and
Generation of a biosimulation model to identify metabolic pathway-wide mechanisms based on transcription data
SA2 Results
Filipin fluorescence (blue) is
more intense
in fibroblasts from
young patients
with NPC symptoms (1 year)...
...compared to fibroblasts from
older patients
with later onset of symptoms (23 years)
Control 73 years
AD 73 years
Similar staining occurs in AD cells...
Filipin fluorescence is
quantifiable with flow cytometry (FACS)
and
consistently increased in AD
fibroblasts compared to control cells (p = 0.01)
In NPC, cholesterol is known to be dysregulated, causing
increased
filipin staining
It appears that filipin also correlates with the
damage
caused by the disease
Filipin is
increased
in AD cells compared to controls
Could filipin be used to indicate
cellular damage due to AD?
Revised Experimental Design
Testing a possible link between filipin and AD cellular damage
SH-SY5Y "sushi" cells, differentiated into neuron-like cells
Based on work by Ghribi et al [8]
27-hydroxycholesterol (27-OHC), known to be increased in AD and capable of crossing the blood-brain barrier
Model system
Treatment
U18666A, an inhibitor of cholesterol synthesis that creates an NPC phenotype
Positive control
Vehicle
24-hydroxycholesterol (24OHC), normally found in the brain, to eliminate possible binding of filipin to oxysterols
Negative controls
Cell death, measured by LDH assay,
increases with 27-OHC
but
not with 24-OHC
(p < 0.05)
During analysis, a gate is set to include
all cells to the immediate right
of the peak fluorescence from control (shaded) cells; both control and experimental cells contained by this gate are considered
filipin-positive
.
Filipin fluorescence also
increases with 27-OHC
but
not with 24-OHC
(p = 0.01)
Filipin
correlates with cellular damage
caused by 27-OHC
In AD,
27-OHC may be damaging neurons
in a similar manner
Damage via this mechanism
can be detected
with filipin
SA2: Cell Culture
SA2: Biosimulation
Uses the
Transcriptome-to-Metabolome (TTM)
process developed by Phelix et al and initially
validated in AD
using the
glycolysis pathway
[9]
Models
entire metabolic pathways
beginning with gene transcription data
TTM can re-create
in silico
the
in vivo
effects of APP and NPC1 manipulation and offers a
pathway-wide perspective
Generates
predictions in parallel
that can form the basis of a hypothesis for
subsequent experiments
SA2: Biosimulation Results
Assign constants
to each reaction in a metabolic pathway
based on gene expression
values from control mice
Subsequently
modify constants based on the percent change
in gene expression, relative to control, in knockout mice
Cholesterol synthesis changes in APP knockout mice
Simplified cholesterol synthesis changes in APP knockout mice
SA2: Biosimulation
Despite the lack of disease phenotype in APP knockout mice,
loss of APP results in significant metabolic changes
Continues to suggest that
APP

is part of a mechanism that
protects against stress
to lipid regulation
Animals lacking APP are
more vulnerable to stress
from NPC mutation
In AD,
chronic stress
over years
could prevent APP from successfully responding
, allowing cellular damage to accumulate
Specific Aim 2: Summary
TTM biosimulation model has
significant potential
Filipin fluorescence
correlates with disease severity
27-OHC treatment produces
AD-like filipin fluorescence
in differentiated SH-SY5Y cells
Biosimulation
validates impact of APP loss
on
cholesterol
metabolism
CYP51A1
has been targeted as part of a unique approach to
reducing systemic cholesterol
levels [10]
TTM biosimulation highlights possible experimental approaches
Approaches like this one could be
used experimentally
to characterize APP function, or even become
new AD therapies
Specific Aim 3
To determine if filipin levels can be used as potential predictors of Alzheimer’s disease risk
Rationale
Results from SA1 and SA2
tested the adaptive response hypothesis
of AD on a basic science level
Demonstrating that these findings reflect human AD
validates their significance
as a contribution to the
scientific understanding
of the disease
Experimental Design
Adaptive response hypothesis predicts that
lipid dysregulation will occur
, and become apparent, in the AD process
well before dementia symptoms
Filipin fluorescence is
increased in AD cells
, and
correlates with damage
caused to neuron-like cells by 27-OHC
A test for
early lipid abnormalities
would allow patients to seek further advice from a neurologist or be placed into trials for experimental interventions
If usable with filipin,
peripheral blood mononuclear cells (PBMCs)
can be
easily obtained from patients
during blood collection
Filipin test
Myo armband
Recently,
movement abnormalities
have been found to be another early symptom of AD
Unlike other detectors, Myo is capable of detecting
both movement
and
electrical impulses
SA3 Results
Using
U18666A
, I attempted to
induce cholesterol dysregulation
in PBMCs from healthy patients
Filipin fluorescence
increases with U18666A concentration
, demonstrating a
measurable range
of cholesterol dysregulation (p = 0.03)
Returning to
AD patients
, I tested whether filipin could be used to identify cholesterol abnormalities in
B-lymphocytes
, one type of PBMC
Cells from AD patients have both an
increased percentage of filipin positive cells
(p = 0.01; scope?) and
increased intensity
(p = 0.02; severity?)
Filipin test
Myo armband
This
device is unique
(for now) in that it can detect both
movement
(accelerometer) and
electrical impulses
(skin EMG)
Using the Myo,
movement data is collected
from patients and
analyzed for signal patterns
that correspond to abnormalities
Once identified, the system could also
filter those movements
, allowing impaired patients to
interact with a computer
Specific Aim 3: Summary
Previously observed (in SA2)
cholesterol changes persist in human PBMCs
Strengthens argument of
AD as a systemic disease
A
clinical trial of the filipin test
is underway, with a
second clinical experiment
involving the Myo in process
These trials can be
combined with TTM
biosimulation analysis to look for novel
metabolic abnormalities
Overall Findings
Evidence that APP is indeed involved with cholesterol regulation
Application of these findings to clinical practice
Support of the larger adaptive response hypothesis
Like my predecessors, I have produced a solid body of work for others to continue to expand
I have also made a significant intellectual contribution to the field of Alzheimer's disease research
Acknowledgments
Salvador Soriano-Castell
Ana Nunes
Clyde F. Phelix
Kristy D. Howard
Acknowledgments
Thesis Committee
Jerome Badaut
Othman Ghribi
Wolff Kirsch
William Pearce
Salvador Soriano
Soriano Lab
Arthur J. Castaneda
Brendan Gongol
John D. Jeppson
Oberg Lab
Kerby Oberg
Jennifer Feenstra
Charmaine Pira
Billy Watson
Amber Brown
Kirsch Lab
Andrew Crofton
Matthew Zabel
Badaut Lab

Hughes Lab

Longo Lab
Payne Lab
Kimberly Payne
Terry-Ann Milford
Michael A. Castello
Presentation CC-BY-SA 2014 Michael A. Castello. Literature references follow and are also available upon request. Branding by Caitlyn Mayers Design. This has been a modulate-free presentation.
castello.me
Significance of

l
oligomers
hyper-phosphorylation
tangles
synaptic disconnection
dementia
Aβ+
?
-
-
Aβ+
Aβ+
?
Remember:
Brain tissue from largely
pre-symptomatic mice

with and without APP
, with and without
added

cholesterol dysregulation
Validation by
proteomic analysis
using a shotgun approach
Trafficking
Trafficking + cholesterol synthesis
...across multiple AD patients
New Experimental Question
Variability
in control population
is expected
as these patients are on a
continuum of possible states
, while
AD patients are not
[1]
References
1. 2014 Alzheimer’s disease facts and figures. Alzheimers Dement. J. Alzheimers Assoc. 10, e47–e92 (2014).
2. Castello, M. A., Jeppson, J. D. & Soriano, S. Moving beyond anti-amyloid therapy for the prevention and treatment of Alzheimer’s disease. BMC Neurol. 14, 169 (2014).
3. Castello, M. A. & Soriano, S. On the origin of Alzheimer’s disease. Trials and tribulations of the amyloid hypothesis. Ageing Res. Rev. 13C, 10–12 (2014).
4. Currais, A. et al. Diabetes exacerbates amyloid and neurovascular pathology in aging-accelerated mice. Aging Cell 11, 1017–1026 (2012).
5. Nunes, A., Pressey, S. N. R., Cooper, J. D. & Soriano, S. Loss of amyloid precursor protein in a mouse model of Niemann-Pick type C disease exacerbates its phenotype and disrupts tau homeostasis. Neurobiol. Dis. 42, 349–359 (2011).
6. Ralser, M. et al. Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. J. Biol. 6, 10 (2007).
7. Agarwal, A. R. et al. Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs. Am. J. Physiol. - Lung Cell. Mol. Physiol. 303, L889–L898 (2012).
8. Prasanthi, J. R. P. et al. Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on beta-amyloid precursor protein levels and processing in human neuroblastoma SH-SY5Y cells. Mol. Neurodegener. 4, 1 (2009).
9. Valdez, C. M., Phelix, C. F., Smith, M. A., Perry, G. & Santamaria, F. Modeling cholesterol metabolism by gene expression profiling in the hippocampus. Mol. Biosyst. 7, 1891–1901 (2011).
10. Zhang, L. et al. An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes. Int. J. Biol. Sci. 8, 310–327 (2012).
X
APP loss in NPC1 mice
exacerbates
the dysregulated lipid phenotype
These double mutant mice show
decreased myelination
and
impaired lipid trafficking
[5]
Summary and the Future
Full transcript