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

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 microtubule-associated 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.

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)

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

Fibroblasts

iPSCs

Purified Neurons

Reprogramming with OSKM vectors

Directed neuronal differentiation and FACs purification

NDC1

NDC2

sAD1 sAD2APPDp1

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

(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

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

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 p-tau/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

Fibroblasts

iPSCs

Purified Neurons

Reprogramming with OSKM vectors

Directed neuronal differentiation and FACs purification

NDC1

NDC2

sAD1 sAD2APPDp1

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.