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

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

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) Apoptotic death (% Total) n=4 CON 5 10 20 µM Aß 10 20 30 40 50 Control (0 µM) 5µM 10µM 20µM 4

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 250 150 100 75 50 37 Actin p75 NTR 0 2 6 24 h p75/actin 0.25 0.25 0.35 0.50 5

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

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 Aß1-42 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

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

Hippocampal Membrane p75NTR level is increased inAD transgenic mice Wt Tg 1 2 3 1 2 3 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 1 2 3 1 2 3 Wt Tg 0.0 0.2 0.4 0.6 0.8 Ratio of p75NTR to actin 2xTg (PS1,APP) 9

ß-Amyloid load in 3xTg mice hippocampus Immunostaining Quantification by ELISA 9 12 15 month old Aß1-42 ng/mg Wt Tg- 9m Tg- 12m 10

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 5 6 7 1 2 3 4 5 6 7 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

Amyloid precursor protein APP expression and ß-amyloid load in cognitively normal (CN) and AD patients APP expression (6E10 IR) ß-amyloid load (ELISA) 1 2 3 4 5 6 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

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. 13

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