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George PerrySemmes Foundation Distinguished University Chair in Neurobiology
The University of Texas at San Antonio
RegenMed SASession III – Clinical Progress & Applicaitons
Alzheimer ‘Disease-in-a-Dish’ and Development of Novel Therapeutics
San Antonio, Texas 12 February 2016
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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.
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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 proteins2) Animal models, either normal or genetically modified3) Cell culture models
a. Animalb. Humanc. Stem cell technology
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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.
Cere
bral
Cor
tex
Brai
n st
emCe
rebe
llum
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Selective Neurodegeneration in CaMKII-MYC mice
MYC-ON MYC-OFF
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Oxidative stress is an early event in AD.
......
t
GlycationNormalNeuron
? Pre-NFT I-NFT E-NFT
FREE CARBONYLS
80HGHNE
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??
Human Tissue
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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
Wi l d type probe
Chi mera probe
4977 bp del et i on
Deleted mtDNANormal mtDNA
8482 108978454 10941
1347513475
8454
8482/ 1346013460
ACACAAACTACCACCTACCTCCCTCACCATTGGCAGCCTA GCATT
CAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCT
16, 569 bp 11, 592 bpOHOH
4977 bp del et i on cont ai ns ATPase subuni t 8, subuni t 6, cyt ochrome-c oxi dase subuni t I I I , and NADH-coenzyme Q oxi dor educt ase subuni t 3, 4 and 5.
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Examination of neurons in biopsy specimens shows normal mitochondria (A), mitochondria with broken cristae (B), and lipofuscin with vacuoles (C).
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F i g u r e 7
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Mitochondria0
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F i g u r e 7
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.
DDisbursed mt
Collapsed mtN
umbe
r of M
itoch
ondr
ia
ADNormalM
ean
of M
itoch
ondr
ia
Leng
th (m
icro
met
ers)
ADNormal
AD Fibroblasts
ADNormal
% o
f col
laps
ed
mito
chon
dria
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Fission Fusion
A Delicate Balance between Mitochondrial Fission and Fusion
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Normal AD
Normal
Normal
AD AD
Decreased DLP1 levels are found in AD fibroblasts
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Reduction of DLP1 levels in normal cell fibroblasts by iRNA affects mitochondria
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Mitochondrial morphology and distribution in rat E18 primary neurons (DIV 7) either overexpressing or knocking down mitochondrial
fission/fusion proteins
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Tubulin DsRed-Mono Mito-AcGFP DAPi Merged
DsRed-Mono vector
WT AβPP- DsRed-Mono
AβPPswe-DsRed-Mono
AβPPswe-DsRed-Mono+ BACE inhibitor IV
A
Vector Vector+inhibitor WT APP WT APP+inhibitor APPswe APPswe+inhibitor0
102030405060708090
100
Normal Fragmented Elongated
% o
f co-
tran
sfec
ted
Cells
wit
h va
riou
s mi-
toch
ondr
ial m
orph
olog
y
* *
*
*
*
*
**
*
Vector Vector+inhibitor WT APP WT APP+inhibitor APPswe APPswe+inhibitor0
10
20
30
40
50
60
70%
of C
ells
with
abn
orm
al m
i-to
chon
dria
l dis
trib
utio
n
*
*
*
B
C D
0
20
40
60
80
100R² = 0.938039966911449
0
20
40
60
80
100R² = 0.958907420307873
0 1000 2000 3000 4000 5000 60000
20
40
60
80
100
R² = 0.946331381866869
Ab
% c
ells
with
abn
orm
al
mito
chon
dria
l dis
tribu
tion
% c
ells
with
frag
men
ted
mito
chon
dria
% c
ells
with
nor
mal
m
itoch
ondr
ia
Aβ Overproduction Underlies AβPP-Induced Mitochondrial Abnormalities
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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 bindingCu binding
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Imaging Mass Spectrometry
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Alzheimer’s Patient (APOE3/4)
Green = bIII tubulinBlue = DNA
Green = MAP2Blue = DNA
Control Patient
Human Neuronal Cells Derived from Fibroblasts
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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.
