Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells

1. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells Mason A.Israel et. al. Nature 2012 Presentation by Airan Jansen Program Administrator CIRM Bidgesto Stem Cell Research California Polytechnic University, Pomona California State University, Los Angeles

2. 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.

3. 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

4. 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.

5. Limits to understanding Alzheimer's Disease Pathogenesis • Difficulties in obtaining live neurons from Alzheimer's patients • 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.

6. 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 Cell Pancreatic Cell Smooth Muscle Lung Cell Pigment Cell Skin Cell Neuron Thyroid Cell Red Blood Cells Red Blood Cells Skeletal Muscle Cells

7. Questions to be addressed in this study Can iPSC technology be used to produce neuronal cell phenotypes of patients with Alzheimer’s Disease? Can iPSC technology be used to predict Alzheimer’s disease before a patient manifests the disease? Is there a causative relationship between amyloid-β precursor protein (APP) processing and tau phosphorylation in the neurons? Can neurons with the genome of an sAD patient exhibit phenotypes seen in an FAD patient?

8. Experimental Approach sAD2 sAD1 NDC1 APPDp1 Fibroblasts NDC2 APPDp2 Reprogramming with OSKM vectors iPSCs Directed neuronal differentiation and FACs purification Purified Neurons

10. 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

11. 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

12. Teratoma formation shows pluripotency of iPSCs One iPSC line per individual plus an ESC line (HUES-9) was tested for pluripo-tencyin vivo by ten bilateral injections into lumbar spinal cords of nude rats. Scale bars, 50 µm Scale bar, 2 mm 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.

13.  Fluorescence-activated cell sorting (FACS) for purification of neurons derived from iPSCs Fibroblast culture iPSC culture showing human Embryonic stem cell (hESC)-like morphology

14. 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

15. Almost all neurons tested generated voltage-dependent action potentials and currents indicating true neuronal phenotype

16. Purified neurons from sAD2, APPDp1 and APPDp2 patients secrete increased amyloid-β(1–40)(Aβ(1–40)) compared to NDC patient samples.

17. 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

18. Neurons from sAD2, APPDp1 and APPDp2 patients had significantly higher p-tau/total tau (p-tau/t-tau) compared to NDC patient samples

19. Neurons from sAD2, APPDp1 and APPDp2 patients had significantly higher active GSK-3β compared to NDC patient samples

20. 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

21. 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 

22. 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 

23. 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)

24. The neurons from both sAD2 and APPDp2 patients had significantly increased numbers of both large and very large early endosomes relative to NDC controls 

25. No significant difference in the number of synapsin I+ puncta per μm MAP2+dendrite was observed between NDC and either sAD2 or APPDp2 patients

26. Summary of Results sAD2 sAD1 NDC1 APPDp1 Fibroblasts NDC2 APPDp2 Reprogramming with OSKM vectors iPSCs Directed neuronal differentiation and FACs purification Purified Neurons

27. 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.