Transcript Probing sporadic and familial Alzheimer*s disease using induced

Mason A. Israel et. al.
Nature 2012
Presentation by Airan Jansen
Program Administrator
CIRM Bridges to Stem Cell Research
California Polytechnic University, Pomona
California State University, Los Angeles
Alzheimer’s disease
• Common neurodegenerative disorder
• 6th leading cause of death in the US
• More than 5 million Americans are
living with the disease
• 1 in 3 seniors dies with Alzheimer’s or
another dementia
• Alzheimer’s is the only cause of
death among the top 10 in America
without a way to prevent it, cure it
or even slow its progression.
• Today, there are no survivors of
Alzheimer’s. If you do not die from it,
you die with it.
Alzheimer’s is defined post mortem by the increased
presence of amyloid plaques and neurofibrillary tangles in
the brain.
• Amyloid plaques:
extracellular deposits
consisting primarily of
amyloid-β peptides
• Neurofibrillary tangles:
intraneuronal aggregations
of hyperphosphorylated tau
• Tau: a microtubuleassociated protein involved
in microtubule stabilization
Sporadic (sAD) vs. Familial Alzheimer’s Disease (FAD)
• FAD only involve 3 genes (APP, amyloid precursor protein; PSEN1 and 2,
presenilin 1 and 2).
• sAD involves many genes and affects many pathways.
• Vast majority of Alzheimer’s Disease is sporadic and not familial
• Studying the known mechanisms of FAD can lead to the development
of appropriate directions for sAD research
• The focus of this study is on developing an in vitro model using iPSCs to
understand the differences between sAD and FAD.
Limits to understanding Alzheimer's
Disease Pathogenesis
1. Difficulties in obtaining live neurons from Alzheimer's
patients
2. Inability to model the sAD form of the disease
To overcome these difficulties,
the investigators reprogrammed
Alzheimer’s Disease patient
fibroblast cells to form induced
pluripotent stem cells (iPSCs)
which could be differentiated
into neurons.
Induced Pluripotent Stem Cells (iPSCs)
ADULT CELL
iPSC Reprogramming Factors:
Inserted into the nucleus
(DNA) of the cell to reverse
development of the cell
DEDIFFERENTIATION
iPSCs
Cardiac Muscle
Kidney Tubule
Smooth Muscle
Cell
Red Blood
BloodCells
Cells
Skeletal Muscle Cells
Pancreatic Cell
Lung Cell
Thyroid Cell
Skin Cell
Pigment Cell
Neuron
Questions to be addressed in this study
1. Can iPSC technology be used to produce neuronal cell
phenotypes of patients with Alzheimer’s Disease?
2. Can iPSC technology be used to predict Alzheimer’s disease
before a patient manifests the disease?
3. Is there a causative relationship between amyloid-β
precursor protein (APP) processing and tau phosphorylation
in the neurons?
4. Can neurons with the genome of an sAD patient exhibit
phenotypes seen in an FAD patient?
Experimental Approach
NDC1
Fibroblasts NDC2
Reprogramming
with OSKM vectors
iPSCs
Directed neuronal
differentiation and
FACs purification
Purified Neurons
sAD1
sAD2
APPDp1
APPDp2
Characterization of patient fibroblasts
Familial Alzheimer’s disease
fibroblasts (APP) expressed
higher levels of APP mRNA
relative to NDC and sAD
samples.
APP Dp1 and APP Dp2
fibroblasts secrete increased
levels of amyloid-ß(1-40)
compared to NDC cells
Dedifferentiation
(Patient fibroblasts)
OSKM
(OCT4, SOX2, KLF4, c-MYC)
• Maintain embryonic stem cell like
morphology
• Express pluripotent-associated
proteins (NANOG and TRA1-81)
• Can differentiate into cells of
ectodermal, mesodermal and
endodermal lineages under in vitro
conditions
• Form teratomas when injected into
nude rats
Teratoma formation shows pluripotency of iPSCs
H&E stained horizontal section
of a whole spinal cord showing
the formation of multiple
teratomas.
Scale bars, 50 µm
Scale bar, 2 mm
One iPSC line per
individual plus an ESC
line (HUES-9) was
tested for pluripo-tency
in vivo by ten bilateral
injections into lumbar
spinal cords of nude
rats.
Higher magnication images showing
the presence of ectodermal,
mesodermal and endoder-mal lineages
for each iPSC line.
Fluorescence-activated cell sorting (FACS) for
purification of neurons derived from iPSCs
Fibroblast culture
iPSC culture showing
human Embryonic stem
cell (hESC)-like
morphology
Fluorescence-activated cell sorting (FACS) for
purification of neurons derived from iPSCs
Neural progenitor
cells (NPCs)
differentiated NPCs
Nucleated FACS-purified neurons express MAP2 and βIII-tubulin
Almost all neurons tested generated voltage-dependent
action potentials and currents indicating true neuronal
phenotype
Purified neurons from
sAD2, APPDp1 and APPDp2
patients secrete increased
amyloid-β(1–40)
(Aβ(1–
40)) compared to NDC
patient samples.
Background: Tau forms neurofibrillary tangles
(NFTs) and adds to Alzheimer’s Disease severity
Kinase GSK-3β phosphorylates tau at Thr231 (p-tau(Thr231). P-tau(Thr231)
regulates microtubule stability and correlates with:
1. neurofibrillary tangle number
2. degree of cognitive decline
Neurons from sAD2, APPDp1 and APPDp2 patients had
significantly higher p-tau/total tau (p-tau/t-tau)
compared to NDC patient samples
Neurons from sAD2, APPDp1 and APPDp2 patients had
significantly higher active GSK-3β compared to NDC
patient samples
Two additional iPSC lines from the sAD2 patient were
analyzed to confirm elevated levels of amyloid-β,
aGSK-3β and p-tau/t-tau compared to NDC controls
There are strong positive correlations between
amyloid-β(1–40), aGSK-3β and p-tau/total tau in
purified neurons from FAD and sAD patients
Twenty-four hour treatment with β- and γ-secretase inhibitors
reduced secreted amyloid-β(1–40) compared to control DMSO
treatment. β-secretase inhibitors partially rescued aGSK-3β and ptau/total tau in sAD2 and APPDp2 neurons
Neurons from both sAD2 and APPDp2 patients frequently had
Rab5+ early endosomes similar in volume, morphology and
localization to that observed in neurons from
Alzheimer’s Disease patient autopsy samples (not shown)
The neurons from both sAD2 and APPDp2 patients had
significantly increased numbers of both large and very large early
endosomes relative to NDC controls
No significant difference in the number of synapsin I+ puncta per
μm MAP2+dendrite was observed between NDC and either sAD2
or APPDp2 patients
Summary of Results
NDC1
Fibroblasts NDC2
Reprogramming
with OSKM
vectors
iPSCs
Directed neuronal
differentiation and
FACs purification
Purified Neurons
sAD1
sAD2
APPDp1
APPDp2
iPSC technology can be used to study early pathogenesis
and drug response in both Sporadic and Familial
Alzheimer’s disease
SUMMARY
• There were significantly increased levels of three major
biochemical markers of Alzheimer’s disease ( amyloid-β(1–40),
aGSK-3β and p-tau/total tau) in neurons from one Sporadic
Alzheimer’s disease and two Familial Alzheimer’s disease
patients.
• These studies suggest that the APP processing pathway has a
causative role in tau Thr 231 phosphorylation in human neurons.
• Products of APP processing other than amyloid-β may have a role
in induction of GSK-3β activity and p-tau.
• Early endosome phenotypes have been found in neurons from
sAD2 (Sporadic Alzheimer’s) and APPDp2 (Familial Alzheimer’s)
patients.