Loading presentation...

Present Remotely

Send the link below via email or IM

Copy

Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.

DeleteCancel

Make your likes visible on Facebook?

Connect your Facebook account to Prezi and let your likes appear on your timeline.
You can change this under Settings & Account at any time.

No, thanks

Testing an Adaptive Response Hypothesis of Alzheimer's Disease

LLU Monday seminar presentation 02-11-2013
by

Michael Castello

on 8 November 2013

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of Testing an Adaptive Response Hypothesis of Alzheimer's Disease

Alzheimer's Disease
An overview
Dementia: progressive loss of cognitive ability
Alzheimer's disease (AD) is the most common form of
dementia
, affecting 5.4 million Americans and 747,000 Canadians last year
Testing an Adaptive Response Hypothesis of Alzheimer's Disease
Michael A. Castello
February 11, 2013
65+
1 in 8 Americans
1 in 7 Canadians
Nearly half
85+
Despite years of research, Alzheimer's disease has
no effective treatment
AD research is an opportunity to
make a difference
in a growing number of lives
x3
Outline
Foundation
AD characteristics
Amyloid cascade and its shortcomings
Relationship between cholesterol and AD
The Adaptive Response Hypothesis
Testing the Hypothesis
Conceptual rationale
Experimental design
Results and interpretation
Wrapping Up
Experimental trajectory
Conclusions
What is Alzheimer's Disease?
Clinical
Pathological
Begins with
difficulty remembering
names and events
Progresses to symptoms including
impaired

judgement
,
confusion
, and
difficulty speaking
,
walking
, and
swallowing
Ends in death
Senile plaques
Neurofibrillary tangles
Aggregations of
amyloid beta (Aβ)
Formed through cleavage of
amyloid precursor protein (APP)
Aggregations of
phosphorylated tau protein
Cytoskeletal, involved in
axonal transport
Indictment: Amyloid
Lack of correlation
between presence of Aβ plaques and cognitive decline
Nagging Inconsistencies
Plaques have been identified in at least 30% of
healthy individuals
No conceptual framework
for understanding why Aβ is physiologically produced
Familial Alzheimer's
Supporting Information
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 show
AD-like pathology
If Aβ
l
oligomers start the cascade,
preventing aggregation
or
removing Aβ
entirely will stop it.
Aβ Aggregates
Tau Aggregates
Neuronal Loss
causing tau phosphorylation
disrupting axonal transport
dementia and death
l
oligomers
plaques
tangles
filaments
synaptic disconnection
cell death
=
Prediction
tramiprosate
tarenflurbil
semagacestat
ponezumab
solanezumab
ACC-001
CAD-106
?
This strategy has been a failure
Amyloid Cascade
Evidence suggests that
Aβ is not causing
Alzheimer's disease
Cholesterol dysregulation
is a more likely candidate
Why Cholesterol?
Cholesterol is heavily involved in cellular processes critical to brain function
myelin sheaths
synapses
membrane fluidity
neurosteroid synthesis
Cholesterol dysregulation leads to tau phosphorylation
Phosphorylated tau forms neurofibrillary tangles
Widespread cholesterol dysregulation found in AD
Associations between AD and genes involved in
cholesterol metabolism
,
transport
, and
endocytosis
Aβ Aggregates
Tau Aggregates
Neuronal Loss
causing tau phosphorylation
disrupting axonal transport
dementia and death
l
oligomers
plaques
tangles
filaments
synaptic disconnection
cell death
Cholesterol Dysregulation
Adaptive Response Hypothesis
Cholesterol dysregulation
is a key early pathogenic trigger in Alzheimer's disease
Aβ generation occurs as part of an
adaptive response
to cholesterol-associated cellular distress
Can cholesterol distress cause an adaptive response from APP?
Can cholesterol distress cause cellular dysfunction?
Testing the Hypothesis
Elements of cholesterol distress
will
provoke a response from APP
Cholesterol distress
will
cause signs of cellular dysfunction
Foundation
Predictions
Questions
Can cholesterol distress cause an adaptive response from APP?
Can cholesterol distress cause cellular dysfunction?
Experimental Design
Testing the Hypothesis
Elements of cholesterol distress
will
provoke a response from APP
Cholesterol distress
will
cause signs of cellular dysfunction
Predictions
Questions
Can cholesterol distress cause an adaptive response from APP?
Can cholesterol distress cause cellular dysfunction?
Can cholesterol distress cause an adaptive response from APP?
Relevant Model
Induce Stress
Observe Changes
SHSY-5Y cells
Derived from
human neuroblastoma
cells
Can be differentiated into
neuron-like cells
with
retinoic acid
"Shishy" cells?
"
Sushi
" cells
Oxidized cholesterol
27-hydroxycholesterol (
27OHC
)
Capable of
crossing
blood-brain barrier
Capable of being
neurotoxic
Shows signs of
dysregulation in AD
APP Alterations
Differences in
protein expression
or
cleavage
Changes in
localization patterns
Western blot
Lyse cells in Triton X-100 buffer
Run on gradient tris-glycine gels
Data
APP C-terminal fragment (CTF) is
significantly reduced
after 12h 27OHC treatment
So?
Interpretation
Current results in neuron-like SHSY-5Y cells suggests that
27OHC
, an oxidized form of cholesterol,
affects APP cleavage
Can cholesterol distress cause cellular dysfunction?
Relevant Model
Induce Stress
Observe Changes
BV2 cells
Derived from
mouse microglia
Non-neuronal cell type responsible for
brain inflammatory response
Oxidized cholesterol
27-hydroxycholesterol (
27OHC
)
Crosses
blood-brain barrier
Capable of being
neurotoxic
Cholesterol regulation
Differences in
amount or intensity of filipin
staining
Immunofluorescence
Paraformaldehyde-fix cells on glass coverslips
Expose to
filipin
, a fluorescent cholesterol stain
Data
Filipin-positive cells
significantly increase
after 12h 27OHC treatment
So?
Interpretation
Work in BV2 cells suggests that
oxidized cholesterol has an effect
on intracellular cholesterol regulation
Manipulation
Wrapping Up
SHSY-5Y Cells
BV2 Cells
Structural similarity between RA-treated SHSY-5Y cells and neurons allows for testing changes in
key neuronal proteins
, such as
tau
How does 27OHC treatment affect
cell death
?
ELISA on cell culture media to detect changes in
secreted APP fragments
, such as

Demonstrated response of microglia to 27OHC must be
characterized
Test for release of
inflammatory

cytokines
Repeat experiments in
human-derived microglia
(collaboration with Kirsch lab)
Measurement
Hypothesis
In the Future
Conclusions
Sound experimental foundation
for testing the Adaptive Response hypothesis
Encouraging early results
create opportunities for further investigation and discovery
Understanding
of APP function and the molecular pathways surrounding the inception of Alzheimer's disease

Validation
of a hypothesis that may be applicable to multiple neurodegenerative diseases

Conversion
of knowledge into novel therapeutic interventions

Salvador Soriano
Kristy Howard
Thesis Committee
Acknowledgments
Jerome Badaut
Othman Ghribi
Wolff Kirsch
William Pearce
Salvador Soriano
Kirsch Lab
Andrew Crofton
Matthew Zabel
Oberg Lab
Charmaine Pira
Michael A. Castello
February 11, 2013
Presentation CC-BY-SA 2013 Michael A. Castello. Literature references available upon request. This has been a modulate-free presentation.
mcastello@llu.edu
Adaptive response hypothesis claims that APP responds to cholesterol stress
Examples of cholesterol stress provided by the hypothesis include
oxidation
,
location
, and
amount
Measurements of responses include
changes in APP
,
cholesterol dysregulation
,
tau phosphorylation
, and
cell death
Full transcript