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 ChakravarthyMolecular Signalling LaboratoryInstitute for Biological Sciences

National Research Council, Canada

Conflict of Interest Disclosure

Balu Chakravarthy, Ph.D.

Has no real or apparent

conflicts of interest to report.

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

Aß1-42 induces apoptotic death of human neuroblastoma cells (SH-SY5Y)A

po

pto

tic

de

ath

(%

To

tal)

n=4

CON 5 10 20 µM Aß0

10

20

30

40

50

Control (0 µM) 5µM

10µM 20µM

Time- and dose dependent increase in membrane p75NTR levels in Aß1-42 treated human SH-SY5Y neuroblastoma cells

p75/actin 0.50 0.80 1. 2 1.4 1.0

Mol Wt (KDa)

0 5 10 15 20 µM

250150

100

75

50

37

Actin

p75 NTR

p75 NTR

Actin

250150

100

75

50

37

0 2 6 24 h

Mol Wt (KDa)

p75/actin 0.25 0.25 0.35 0.50

0

1

2

3

4 P < .005

Control Aß1-42

Ra

tio

of

p7

5N

TR t

o a

cti

n

Con Aß Con Aß Con Aß Con Aß

p75NTR

actin

Increase in the level of membrane-associated p75NTR in ß-amyloid treated human neuroblastoma cells

Con Aß25-35 Aß35-25 Aß1-42 Aß42-1 rAß1-42 rAß42-1

p75NTR

SH-SY5Y

p75NTR

- +Antigen peptide

p75NTR

Con Aß1-42 Con Aß1-42 Con Aß1-42 Con Aß1-42

SH-SY SK-N SH-SY SK-N

Control Aß1-42 CHX+Aß1-42

0.0

0.5

1.0

1.5

2.0

2.5

Ra

tio

of

p7

5N

TR t

o a

cti

n

p75NTR

Aß25-35

Control Aß CHX-Aß

actin

Aß1-42

Control Aß CHX-Aß

p75NTR

Protein synthesis inhibitor cycloheximide (CHX) blocks ß-amyloid-induced increase in membrane p75NTR level

p< 0.01,

p< 0.01,

120kD

150kD

actin

Con Aß Con Aß Con Aß Con Aß

Trk A

TrkA

Aß25-35 Aß1-42

85kD 100kD

150kD

actin

Trk B

Con Aß Con Aß Con Aß Con Aß

TrkB

Aß25-35 Aß1-42

ß-amyloid treatment does not affect trk A and trk B levels

Wt Tg

p75NTR

Actin

25015010075

50

Mol Wt KDa

1 2 3 1 2 3

Wt Tg

1 2 3 1 2 3

p75NTR

Actin

250150

10075

50

Mol Wt KDa

Wt Tg

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Rat

io o

f p

75N

TR t

o a

ctin

Wt Tg

0.0

0.2

0.4

0.6

0.8

Rat

io o

f p

75N

TR t

o a

ctin

Hippocampal Membrane p75NTR level is increased inAD transgenic mice

3xTg(PS1, APP Tau)

2xTg(PS1,APP)

p< 0.05,

0

20

40

60

80

100

120

140

160

9 12 15 month old

Aß

1-42

ng

/mg

Wt

Tg- 9m

Tg- 12m

Immunostaining Quantification by ELISA

ß-Amyloid load in 3xTg mice hippocampus

Membrane p75NTR level is significantly higher in the hippocampii of AD patients compared to cognitively normal (CN) patients

0

1

2

3

4

CN AD

Ra

tio

of

p7

5N

TR t

o a

cti

n

Lower Band

0

1

2

3

4

CN AD

Ra

tio

of

p7

5N

TR t

o a

cti

n

Upper Band

p75NTR

ß-actin

CN AD

Antigen peptide - +

CN AD CN AD

p75NTR

1 2 3 4 5 6 7 1 2 3 4 5 6 7

All female, Average age- CN = 75.1 ± 9.4; AD = 79.4 ± 5.7 (Douglas Hospital, Montreal)

p< 0.05, p< 0.02,

0.0

1.0

2.0

3.0

4.0

CN AD

Ra

tio

of

Aß

PP

to

ac

tin

Amyloid precursor protein APP expression and ß-amyloid load in cognitively normal (CN) and AD patients

CN AD

1 2 3 4 5 6

AßPP

actin

APP expression (6E10 IR)

CN AD

1

3

2

4

6

5

ß-amyloid load (ELISA)

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 Aβ1-42

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.

Acknowledgement

Trevor AtkinsonLeslie BrownMichel MénardChantal Gaudet (ASC Grant)

Dr. Shingo ItoDr. Jim Whitfield

Dr. Ubaldo Armato Dr. Anna ChiariniDr. Ilaria Dal Prà(University of Verona)

Alzheimer’s Society of Canada Grant (ASC# 09-14)