1. and Hippocampal Cells in Murine and Human AD Brains
Amyloid ß1-42-Associated p75NTR Expression in Human Neuroblastoma Cells
and Hippocampal Cells in Murine and Human AD Brains
ADI Conference-Toronto March 26-29, 2011
Balu Chakravarthy
Molecular Signalling Laboratory
Institute for Biological Sciences
National Research Council, Canada

2. Conflict of Interest Disclosure Balu Chakravarthy, Ph.D.
Has no real or apparent
conflicts of interest to report. 2

3. p75 neurotrophin receptor (p75NTR)
p75NTR is a member of TNF super family of receptors
p75NTR promiscuously binds, and is activated by, NGF, BDNF, NT3 and NT4 neurotrophins, has higher affinity towards pro-neurotrophins
implicated in cell survival and differentiation, neurite growth, and cell death (apoptosis)
it is known to bind Aß peptides and may mediate Aß-induced apoptotic cell death

4. Aß1-42 induces apoptotic death of human neuroblastoma cells (SH-SY5Y)
Apoptotic death (% Total)
n=4
CON µM Aß 10 20 30 40 50
Control (0 µM)
5µM
10µM
20µM 4

5. Time- and dose dependent increase in membrane p75NTR levels
in Aß1-42 treated human SH-SY5Y neuroblastoma cells
p75/actin
Mol Wt
(KDa) µM
250
150
100 75 50 37
Actin
p75
NTR h
p75/actin 5

6. Increase in the level of membrane-associated p75NTR in ß-amyloid treated human neuroblastoma cells
Con Aß Con Aß Con Aß Con Aß 1 2 3 4
P < .005
Control Aß1-42
Ratio of p75NTR to actin
p75NTR
actin
Antigen peptide
p75NTR
Con Aß1-42
SH-SY SK-N
Con Aß25-35 Aß35-25 Aß1-42 Aß42-1 rAß1-42 rAß42-1
p75NTR
SH-SY5Y
p75NTR 6

7. Protein synthesis inhibitor cycloheximide (CHX) blocks ß-amyloid-induced increase in membrane p75NTR level
p75NTR
Aß25-35
Control Aß
CHX-Aß
actin
Aß1-42
Control Aß CHX+Aß1-42
0.0
0.5
1.0
1.5
2.0
2.5
p< 0.01,
Ratio of p75NTR to actin
p< 0.01, 7

8. ß-amyloid treatment does not affect trk A and trk B levels
120kD
150kD
actin
Con Aß Con Aß Con Aß Con Aß
Trk A
TrkA
Aß25-35
Aß1-42
85kD
100kD
Trk B
Con Aß Con Aß Con Aß Con Aß
TrkB 8

9. Hippocampal Membrane p75NTR level is increased inAD transgenic mice
Wt Tg
p75
NTR
Actin
250
150
100 75 50
Mol Wt
KDa
Wt Tg
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Ratio of p75NTR to actin
p< 0.05,
3xTg
(PS1, APP
Tau)
Wt Tg
p75
NTR
Actin
250
150
100 75 50
Mol Wt
KDa
Wt Tg
0.0
0.2
0.4
0.6
0.8
Ratio of p75NTR to actin
2xTg
(PS1,APP) 9

10. ß-Amyloid load in 3xTg mice hippocampus
Immunostaining
Quantification by ELISA
month old
Aß1-42 ng/mg Wt
Tg- 9m
Tg- 12m 10

11. compared to cognitively normal (CN) patients
Membrane p75NTR level is significantly higher in the hippocampii of AD patients
compared to cognitively normal (CN) patients
p75NTR
ß-actin CN AD
Antigen peptide
CN AD 1 2 3 4
CN AD
Ratio of p75NTR to actin
Lower Band
CN AD
Upper Band
p< 0.05,
p< 0.02,
All female, Average age- CN = 75.1 ± 9.4; AD = 79.4 ± 5.7 (Douglas Hospital, Montreal) 11

12. Amyloid precursor protein APP expression and ß-amyloid load in cognitively normal (CN) and AD patients
APP expression (6E10 IR)
ß-amyloid load (ELISA)
AßPP
actin 1 3 2 4 6 5
CN AD
0.0
1.0
2.0
3.0
4.0
Ratio of AßPP to actin CN AD
CN AD 12

13. In conclusion, we have shown that:
Aβ peptides stimulate the expression of p75NTR in the membranes of human neuroblastoma cells and this induction is dependent on new protein synthesis.
The level of membrane p75NTR is increased in the Aβ1-42 accumulating hippocampi of AD transgenic mice
There is an increased p75NTR expression that accompanies Aβ accumulation in the hippocampi of human AD patients
An increased p75NTR expression may be particularly lethal to hippocampal neurons as the accumulating Aß and pro-neurotrophins, whose levels are elevated in AD, may activate the ‘death domain’ of p75NTR triggering a cytocidal apoptogenic cascade. 13

14. Acknowledgement
Trevor Atkinson Leslie Brown Michel Ménard Chantal Gaudet (ASC Grant)
Dr. Shingo Ito Dr. Jim Whitfield
Dr. Ubaldo Armato Dr. Anna Chiarini Dr. Ilaria Dal Prà (University of Verona)
Alzheimer’s Society of Canada Grant (ASC# 09-14) 14