1. Molecular Mechanisms
Involved in
Alzheimer’s Disease Pathology
Atul Deshpande, Ph.D
P.I.: Dr. Jorge Busciglio
Dept. of Neurobiology and Behavior
University of California, Irvine

2. Background: Prevalence
 Alzheimer’s Disease is the leading cause of
dementia among elderly people.
 An estimate of 12% Americans over the age
of 65 and almost half above the age of 85
have AD (~5 million).
 Alzheimer’s Disease is the 3rd most
expensive disease to treat, costing the society
close to $100 billion annually.
 AD is a progressive disorder beginning with
memory deficits and leading to dementia.

3. Background: Etiology
 The disease is characterized by two main lesions:
Amyloid Plaques Neurofibrillary Tangles
 Synaptic loss precedes neuronal loss
 The neurodegeneration occurs in several brain
areas including cortex, hippocampus and basal
forebrain.

4. This Talk...

5. This Talk...
 Aß -
• Brief Background: Different conformations
• Results
• Effect of different conformations of Aß on HCN
• Role in AD pathogenesis
• Conclusions with possible model system
for mechanism(s) of neurotoxicity

6. Beta Amyloid (Aß): main component of plaques
 Two types of Aß deposits are found, a) the dense core neuritic plaques
consisting of fibrillar insoluble Aß, and b) the diffuse plaques
 A 40-42 amino acid peptide product of ß and γ secretase mediated
cleavage of amyloid precursor protein (APP)
 Fibrilization of Aß is
preceded by multiple
conformational changes but
the pathways to
oligomerization and
fibrilization can be
independent (Necula et. al.,
2007)

7. Different Conformations of ß-amyloid
Aβ42 Monomers
Aß Derived Diffusible Ligands (ADDLs) Aβ42 Oligomer (AβO)
AFM EM
O
L
EM
Aß*56 M
O
G
I R
E S
(Lambert et. al., 1998) Demuro et. al. 2004
Annular Protofibrils
Protofibrils
EM
Aβ42 Fibrils (AβF)

8. Different Conformations of ß-amyloid
Aβ42 Monomers
Aß Derived Diffusible Ligands (ADDLs) Aβ42 Oligomer (AβO)
AFM EM
O
L
EM
Aß*56 M
O
G
I R
Soluble
E S
(Lambert et. al., 1998) forms Demuro et. al. 2004
of Aß Annular Protofibrils
Protofibrils
EM
Aβ42 Fibrils (AβF)

9. Alzheimer’s Disease:
The Different Faces of Amyloid-ß…
Amyloid-ß
Ravana Amyloid Precursor
Protein

10. Alzheimer’s Disease:
The Different Faces of Amyloid-ß…
Amyloid-ß
…and all of them are Evil!!!

11. Soluble Aß…
M.W.
AßO and ADDLs are found
100
AßO (~90kd) in the brains of AD patients
75 and have been found to…
50 a) Induce acute
electrophysiological changes,
25 b) Inhibit long-term potentiation
20
ADDLs (~17-24kd)
and impair memory function
15
c) Permeabilize cell membrane
10 and lead to influx of calcium,
Monomer (~4.5kd)
d) Affect neuronal viability

12. Objective of this study…
Characterization of soluble oligomeric amyloid
has raised the possibility that different Aß
conformations may contribute to AD pathology
via different mechanism(s)
To characterize the toxicity of different Aß species:
We tested side by side the effect of well
characterized AßO, ADDLs and Aß fibrils in
Human Cortical Neurons.

13. Experimental Model: Human
Cortical Neurons (HCN)
 Target Cells in Alzheimer’s Disease
 Similar profiles of protein expression to the adult human brain
(e.g. tau, etc.).
HCN (0 DIV) HCN (20 DIV)

14. The most Sophisticated system ever designed…

15. The most Sophisticated system ever designed…
By us!!!
Control
0 4 8 12
hr hrs hrs hrs
β-amyloid treated

16. The most Sophisticated system ever designed…
By Zeiss!!!

17. Aß42f induce neuronal dystrophy, synaptic
loss and modest cell death over an
extended period of time
Control AβF (20μM; 10 Days)
Tau
Aβ
(Grace et.al;2002; Grace and Busciglio; 2003)

18. Soluble Aß colocalizes with synaptic markers in HCN
60
% colocolization
40
20
0
5 30 60 min
AßO are rapidly targeted to synaptic terminals

19. AßO induces rapid and extensive neuronal death
AßO (5µM) - 24 hours

20. AßO induces rapid and extensive neuronal death
AßO (5µM) - 24 hours

21. AßO induce rapid mitochondrial alterations
AßO at 5µM very rapidly activate an apoptotic cascade in HCN

22. Soluble AßOs induce translocation of cyt. c and AIF
from mitochondria to the cytoplasmic compartment

23. AßO neurotoxicity under normal and low extracellular calcium
After 12 hr with 5µM AßO…
Regular Low Ca+2
medium
↓ mitochondrial oxido- No Change
reductase activity
↑ activated caspases No Change
3/7
↑ LDH in supernatant No Change
After 24 hr with 5µM AßO…
Regular Low Ca+2
medium
↓ mitochondrial oxido- ↓ mitochondrial oxido-
reductase activity reductase activity
↑ activated caspases ↑ activated caspases
3/7 3/7
↑ LDH in supernatant ↑ LDH in supernatant
AßO neurotoxicity is accelerated by calcium influx

24. Incubation with AßO at lower concentrations
Lower concentrations of AßO showed chronic mitochondrial
alterations but no cell death

25. Summary…
MMP: Mitochondrial Membrane Potential
ATP: Energy currency of the Neurons
MTS: Measure of efficiency of some of the key enzymes of
the electron transport chain
Cyt.C/AIF: Cytochrome C/Apoptosis Inducing Factor
LDH: Lactate Dehydrogenase released/leaked into culture media

26. Mechanisms of AßO synaptic targeting
a) Activity dependent AßO targeting?
b) Role of metal ions?
c) Endocytosis?
d) Binding to specific receptor(s)?

27. Methods:
 Two different culture systems were used:
1. Rat hippocampal organotypic cultures: allows the study of well
characterized structures and synapses
2. HCN: allows the tracking of individual endocytic vesicles
 Colocalization studies were carried out using three different antibodies
(A11, NU2 and M69)
 Z-sections were reconstructed using a rendering software to visualize
the colocalization of AßO and synaptophysin

28. Neuronal activity modulates localization of
AßO to synaptic terminals
 Stimulation: 5min
 20 mM KCl
 100µM glutamate
 Inhibition: 15 min
2µM TTX

29. Neuronal activity modulates localization of
AßO to synaptic terminals
 Stimulation: 5min
 20 mM KCl
 100µM glutamate
 Inhibition: 15 min
2µM TTX
min

30. Role of metal-ions:
✓ Metal ions, especially Cu+2 and Zn+2, are released at
synaptic terminals during synaptic activity
✓ Aß binds to Cu+2 and Zn+2 with very high affinity (K1(Zn):
7; K1(Cu): 8.9)
✓ Thus, released metal-ions in the synaptic cleft could
“attract” AßO to the synaptic terminals

31. Clioquinol:
Effects of Oral Treatment of 15-Month-Old APP2576
Transgenic Mice with Clioquinol (from Cherny et. al., 2001)
5-chloro-7-iodo-quinolin-8-ol
 Clioquinol, iodochlorhydroxyquin, is a metal-protein- attenuating compound (MPAC)
 Clioquinol topical is an antifungal and antibacterial medication
 Clioquinol is a chelator that allows the sequesstration of Zn/Cu-binding compounds,
including Aß
 A phase II trial reports that AD patients on the drug had a slower cognitive decline
than patients on placebo (Ritchie CW, et. al., 2004)
 Also effective to reduce htt accumulation in a Huntington’s mouse model

32. Effect of metal-ion chelation by clioquinol
on synaptic localization of AßO
Metal ions appear to be involved in synaptic
localization of AßO

33. Analysis of AßO endocytosis at
synaptic terminals
AßO are not
endocytosed
at synaptic
terminals

34. NMDAR activation appears to mediate
synaptic localization of AßO
• Recent studies have suggested role of specific receptors in AßO
mediated neurotoxicity
• NMDA receptor activity was inhibited using APV, memantine and
ifenprodil.
• AMPA receptor activity was inhibited using CNQX

35. NMDAR activation appears to mediate
synaptic localization of AßO
• Recent studies have suggested role of specific receptors in AßO
mediated neurotoxicity
• NMDA receptor activity was inhibited using APV, memantine and
ifenprodil.
• AMPA receptor activity was inhibited using CNQX
AßO neurotoxicity appears to be mediated via
NMDA receptor

36. Oligomeric specific antibody labeling
colocalizes with synaptic markers in the
AD Brain
8 NL and 10 AD cases were
analyzed:
 AßO labeling
colocalized in 8 of 10
AD cases
 Weak AßO labeling observed
in 2 of 8 NL cases

37. Oligomeric specific antibody labeling
colocalizes with synaptic markers in the
AD Brain
8 NL and 10 AD cases were
analyzed:
 AßO labeling
colocalized in 8 of 10
AD cases
 Weak AßO labeling observed
in 2 of 8 NL cases

38. Mechanisms of AßO synaptic targeting

39. Mechanisms of AßO synaptic targeting
Activity dependent AßO targeting ✓

40. Mechanisms of AßO synaptic targeting
Activity dependent AßO targeting ✓
Role of metal ions ✓

41. Mechanisms of AßO synaptic targeting
Activity dependent AßO targeting ✓
Role of metal ions ✓
Endocytosis X

42. Mechanisms of AßO synaptic targeting
Activity dependent AßO targeting ✓
Role of metal ions ✓
Endocytosis X
Binding to specific receptor(s) ?

43. Working Model for AßO mediated molecular
mechanism(s) in AD pathogenesis

44. Conclusion:
 AßO affect synaptic plasticity and neuronal function
 Multiple Aß species capable of deleterious effects at
multiple levels coexist in the AD brain.
 Therapeutic strategies to address Aß-mediated
neurotoxicity will require a refined multimodal approach.

45. Alzheimer’s Disease Lesions:
Amyloid Plaques Neurofibrillary Tangles

46. Alzheimer’s Disease Lesions:
Amyloid Plaques Neurofibrillary Tangles
Multimeric and oligomeric tau and Aß species appear to have a
significant role in AD pathology
Establishing how the load of Aß-soluble species changes during
disease progression
Exploring the role of specific receptors in AßO mediated
neurotoxicity and relationship of AßO with post synaptic
receptors in the AD brain

47. Acknowledgements
P.I: Dr. Jorge Busciglio
Supported by funding from NIH and Alzheimer’s Association, Hilblom Foundation and ADRC, UCI

48. Acknowledgements
P.I: Dr. Jorge Busciglio
Lab Members
Dr. Gustavo Pigino
Ale Pelsman
Pablo Helguera
Khin Win
Michael Hanna
Rosa Resende
Jackie Selgie
Octavio Garcia
Ardy Rahman
Supported by funding from NIH and Alzheimer’s Association, Hilblom Foundation and ADRC, UCI

49. Acknowledgements
P.I: Dr. Jorge Busciglio
Lab Members Collaborators:
Dr. Gustavo Pigino Dr. Charlie Glabe
Ale Pelsman Glabe Lab
Special Thanks
Pablo Helguera Dr. Frank LaFerla
Dr. Raju Metherate
Khin Win LaFerla Lab Dr. Hideki Kawai
Michael Hanna Dr. Katumi Sumikawa Dr. John Marshal
Rosa Resende Dr. Ian Parker Dr. Amit Deshpande
Jackie Selgie Dr. Angelo Demuro
Octavio Garcia Dr. Marcelo Wood
Ardy Rahman Dr. Gustavo Pigino, UIC
Dr. Scott Brady, UIC
Supported by funding from NIH and Alzheimer’s Association, Hilblom Foundation and ADRC, UCI