Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges...
-
Upload
richard-neal-hodge -
Category
Documents
-
view
218 -
download
0
Transcript of Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges...
![Page 1: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/1.jpg)
Mason A. Israel et. al. Nature 2012
Probing sporadic and familial Alzheimer’s
disease using induced pluripotent stem cells
Presentation by Airan JansenProgram Administrator CIRM Bridges to Stem Cell ResearchCalifornia Polytechnic University, PomonaCalifornia State University, Los Angeles
![Page 2: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/2.jpg)
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.
![Page 3: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/3.jpg)
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 microtubule-associated protein involved in microtubule stabilization
![Page 4: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/4.jpg)
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.
![Page 5: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/5.jpg)
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.
![Page 6: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/6.jpg)
DEDIFFERENTIATION
iPSC Reprogramming Factors: Inserted into the nucleus (DNA) of the cell to reverse development of the cell
iPSCs
ADULT CELL
Cardiac Muscle
Skeletal Muscle Cells
Kidney Tubule Cell
Red Blood Cells
Smooth Muscle
Red Blood Cells
Lung Cell
Thyroid Cell
Pancreatic Cell
NeuronSkin Cell Pigment Cell
Induced Pluripotent Stem Cells (iPSCs)
![Page 7: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/7.jpg)
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?
![Page 8: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/8.jpg)
Experimental Approach
Fibroblasts
iPSCs
Purified Neurons
Reprogramming with OSKM vectors
Directed neuronal differentiation and FACs purification
NDC1
NDC2
sAD1 sAD2APPDp1
APPDp2
![Page 9: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/9.jpg)
![Page 10: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/10.jpg)
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
![Page 11: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/11.jpg)
(Patient fibroblasts)
(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
Dedifferentiation
OSKM
![Page 12: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/12.jpg)
Teratoma formation shows pluripotency of iPSCs
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.
H&E stained horizontal section of a whole spinal cord showing the formation of multiple teratomas.
Higher magnication images showing the presence of ectodermal, mesodermal and endoder-mal lineages for each iPSC line.
Sca
le b
ars, 5
0 µ
m
Sca
le b
ar, 2
mm
![Page 13: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/13.jpg)
Fluorescence-activated cell sorting (FACS) for purification of neurons derived from iPSCs
Fibroblast culture
iPSC culture showing human Embryonic stem
cell (hESC)-like morphology
![Page 14: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/14.jpg)
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
![Page 15: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/15.jpg)
Almost all neurons tested generated voltage-dependent action potentials and currents indicating true neuronal
phenotype
![Page 16: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/16.jpg)
Purified neurons from sAD2, APPDp1 and APPDp2 patients secrete increased amyloid-β(1–40) (Aβ(1–40)) compared to NDC patient samples.
![Page 17: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/17.jpg)
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
![Page 18: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/18.jpg)
Neurons from sAD2, APPDp1 and APPDp2 patients had significantly higher p-tau/total tau (p-tau/t-tau)
compared to NDC patient samples
![Page 19: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/19.jpg)
Neurons from sAD2, APPDp1 and APPDp2 patients had significantly higher active GSK-3β compared to NDC
patient samples
![Page 20: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/20.jpg)
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
![Page 21: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/21.jpg)
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
![Page 22: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/22.jpg)
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 p-tau/total tau in sAD2 and APPDp2 neurons
![Page 23: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/23.jpg)
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)
![Page 24: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/24.jpg)
The neurons from both sAD2 and APPDp2 patients had significantly increased numbers of both large and very large early endosomes
relative to NDC controls
![Page 25: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/25.jpg)
No significant difference in the number of synapsin I+ puncta per μm MAP2+dendrite was observed between NDC and either sAD2
or APPDp2 patients
![Page 26: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/26.jpg)
Summary of Results
Fibroblasts
iPSCs
Purified Neurons
Reprogramming with OSKM vectors
Directed neuronal differentiation and FACs purification
NDC1
NDC2
sAD1 sAD2APPDp1
APPDp2
![Page 27: Mason A. Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bridges to Stem Cell Research California Polytechnic University,](https://reader036.fdocuments.us/reader036/viewer/2022062315/5697bff01a28abf838cbae3d/html5/thumbnails/27.jpg)
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.