George Perry presented on developing a stem cell model of Alzheimer's disease (AD) as a platform to better understand the human disease and develop drugs. Traditional models like human tissue, animal models, and cell cultures have major limitations. Perry's team aims to generate induced pluripotent stem cells from AD patients to create "disease-in-a-dish" models that exhibit key AD features. Their preliminary studies show AD cells have mitochondrial abnormalities and amyloid-beta overexpression can disrupt mitochondria, supporting the potential of this human stem cell approach.

1. George Perry
Semmes Foundation Distinguished University Chair in Neurobiology
The University of Texas at San Antonio
RegenMed SA
Session III – Clinical Progress & Applicaitons
Alzheimer ‘Disease-in-a-Dish’ and
Development of Novel Therapeutics
San Antonio, Texas 12 February 2016
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2. 2

3. 3
Alzheimer disease is a complex
biological and social condition that
requires development of new technical
and conceptual insights to be solved.
While we can see the disease in
humans, it is difficult to dissect and
while we can study the components in
animals and their cells, it only models
portions of the disease.

4. 4
Poor models are the major reason 99.6% of AD drugs
have failed. Our goal is to develop a stem cell model of
human AD as a platform to understand human AD and
drug development.
Alzheimer disease patients live for years with every
feature of normal physiology, cell biology, molecular, and
social interaction altered.
Traditionally we have used:
1) Human tissue and proteins
2) Animal models, either normal or genetically modified
3) Cell culture models
a. Animal
b. Human
c. Stem cell technology

5. CaMKII promotor tTA
tetO MYCp
Dox
Activation
Inactivation
Establishment of an animal model CaMKII-MYC mice
MYC is specifically induced in cerebral cortical and
hippocampal neurons by depletion of doxycyline
C-MYC (now referred to as MYC) is a member of a family of proto-oncogenes comprising C-
MYC, N-MYC, and L-MYC. MYC encodes a transcription factor that, as part of a heterodimeric
complex with MAX, regulates the expression of a multitude of genes involved in regulating
cellular proliferation and growth. Overexpression of MYC is commonly associated with
tumorigenesis. Where MYC exerts its neoplastic function by inducing autonomous cellular
proliferation and cellular growth, blocking differentiation, and inducing genomic
destabilization. 5

6. Selective Neurodegeneration in CaMKII-MYC mice
MYC-ON MYC-OFF
6

7. Oxidative stress is an early event in
AD.
.
..
..
.
t
Glycation
Normal
Neuron
? Pre-NFT I-NFT E-NFT
FREE CARBONYLS
80HG
HNE
Potential Mechanisms of
Reactive Oxygen Species
Generation in apparently normal
neurons in Alzheimer Disease
Active microglia
Redox active
metals
Amyloid-b
Advanced
glycation
endproducts
Mitochondria??
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Human Tissue

8. 8

9. Alzheimer Control
In situ hybridization of mtDNA
Immunolocalization of Cytochrome oxidase 1 is increased in AD
Alzheimer disease Control
Mitochondrial components are
increased in Alzheimer disease
Wild type probe
Chimera probe
4977 bp deletion
Deleted mtDNANormal mtDNA
8482 10897
8454 10941
13475
13475
8454
8482/13460
13460
ACACAAACTACCACCTACCTCCCTCACCA
TTGGCAGCCTA GCATT
CAACAACCTATTTAGCTGTTCCCCAACCTT
TTCCTCCGACCCCCT
16,569 bp 11,592 bp
OHOH
4977 bp deletion contains ATPase subunit 8, subunit 6, cytochrome-c oxidase subunit III, and
NADH-coenzyme Q oxidoreductase subunit 3, 4 and 5.
9

10. Examination of
neurons in biopsy
specimens shows
normal mitochondria
(A), mitochondria with
broken cristae (B), and
lipofuscin with
vacuoles (C).
0
0.2
0.4
0.6
0.8
1
Numberperm
2
AD
Control
0
0.2
0.4
0.6
0.8
1
Sizeinm
2
0
2
4
6
8
10
Percentageofarea
Total
mitochondria
Normal
mitochondria
Brokencristae
mitochondria
Vacuolar
lipofuscin
Electrondense
lipofuscin
*
Mitochondria
0
0.2
0.4
0.6
0.8
1
Numberperm
2
AD
Control
0.6
0.8
1
m
2
Percentage area of intact
mitochondria is significantly
decreased in cases of AD (n=8)
as compared to age-matched
controls (n=5). p=0.012
While mtDNA is increased, mitochondria are not.
10

11. D
Disbursed mt
Collapsed mtNumberofMitochondria
ADNormal
MeanofMitochondria
Length(micrometers) ADNormal
AD Fibroblasts
ADNormal
%ofcollapsed
mitochondria
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12. Fission Fusion
A Delicate Balance between Mitochondrial
Fission and Fusion
12

13. Normal AD
Decreased DLP1 levels are found in AD fibroblasts
13

14. Reduction of DLP1
levels in normal cell
fibroblasts by iRNA
affects mitochondria
14

15. Mitochondrial morphology and distribution in rat E18 primary neurons
(DIV 7) either overexpressing or knocking down mitochondrial
fission/fusion proteins
15

16. Tubulin DsRed-Mono Mito-AcGFP DAPi Merged
DsRed-Mono vector
WT AβPP- DsRed-Mono
AβPPswe-DsRed-Mono
AβPPswe-DsRed-Mono
+ BACE inhibitor IV
A
0
20
40
60
80
100
120
Vector Vector+inhibitor WT APP WT APP+inhibitor APPswe APPswe+inhibitor
%ofco-transfectedCellswithvarious
mitochondrialmorphology
Normal Fragmented Elongated
**
*
*
*
*
*
*
*
0
10
20
30
40
50
60
70
80
Vector Vector+inhibitor WT APP WT APP+inhibitor APPswe APPswe+inhibitor
%ofCellswithabnormal
mitochondrialdistribution
*
*
*
B
C D
R² = 0.938
0
20
40
60
80
100
R² = 0.9589
0
20
40
60
80
100
R² = 0.9463
0
20
40
60
80
100
0 1000 2000 3000 4000 5000 6000
Ab
%cellswithabnormal
mitochondrialdistribution
%cellswithfragmented
mitochondria
%cellswithnormal
mitochondria
Aβ Overproduction Underlies AβPP-Induced Mitochondrial Abnormalities
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17. Amyloid plaque cores purified from
brain demonstrate characteristic
Congo red birefringence (A), are
strongly reactive with antisera to Ab
1-42 (B), but not with irrelevant
antisera (C).
A
B
C
Spectra of plaque cores (B) are virtually
identical to synthetic peptide (A). Intensity at
1604 is indicative of Zn binding and at 1278 for
Cu binding, both increased in purified cores.
B sheet
Zn binding
Cu binding
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18. 18

19. 19
Imaging Mass Spectrometry

20. 20
Alzheimer’s Patient (APOE3/4)
Green = bIII tubulin
Blue = DNA
Green = MAP2
Blue = DNA
Control Patient
Human Neuronal Cells Derived from Fibroblasts

21. Conclusion
Development of human-based AD cell
models will allow us to understand the
uniquely human disease with greater
clarity than prior approaches, where
parallels were compared with rodents.
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