1. Qilu International Neuroscience Symposium
Non-catalytic role of acetylcholinesterease (AChE) in the regulation of glutamatergic synaptic stability
Wei-Yang Lu, MD, PhD
Robarts Research Institute
Department of Physiology and Pharmacology
University of Western Ontario
Qilu International Neuroscience Symposium
October 12, 2011

2. Glutamatergic and cholinergic synapses, and two isoforms of AChE in the brain
Presynaptic
cholinergic
Glutamatergic
synapse
AChE-S
AChE-R
Postsynaptic
dendritic spine

3. Excess AChE is associated with neuronal cell apoptosis
■ Colocalization with β-amyloid protein in aged and Alzheimer's brains
─ Acta Neuropathol.1993;85(4):362-9.
■ Accelerating assembly of amyloid-β (Aβ) peptides into Alzheimer's fibrils, and increasing the toxicity of Aβ.
─ Neuron 1996 Apr;16(4):881-91
■ A major component of Senile plaques
Acta Neuropathol.1990;80(6):624-8
Interaction with
other proteins - +

4. The active site & the surface anionic site (SAS) of AChE- -
physostigmine
BW584c51 - -

5. Blockade of the “SAS” of AChE results in an increased expression of AChE-
BW584c51 -
physostigmine

6. Increased expression of AChE decreases glutamate receptor mediated currents

7. Excessive AChE reduces dendritic spines and surface glutamate receptors

8. Excess AChE decreases the number of excitatory synapses
Excessive
AChE
“Normal”

9. Increased expression of AChE alters the expression levels of neurexin and neuroliginNB
WT mice AChE-mice
5Wk
mRNA of Nrxn-1β

10. The molecular sequence and dimensional structure of the ectodomain of Nlgn are highly homologous to that of AChE
Nlgn
PSD95
NR1A
GluR2
esterase
side
Nrxn
Pre-
Post-
AChE
Hypothesis:
Excessive AChE interacts with neurexin, competitively interrupting Nrxn-Nlgn association and consequently destabilizing synapses.

11. AChE co-localizes with neurexin, and can be co-precipitated with neurexin from neurons
Nrxn AChE Overlay
A-1
Control
BW284c51
40μm
A-2
A-3
Ctrl BW
Nrxn
AChE B C
Blot: AChE
IgG
α-Nrxn
IP (antibody):
Lysates: 
AChE
input
Control
Blot: Nrxn
IP (antibody): 
Control BW
Lysates:
α-AChE

12. Expressing neuronal isoforms of AChE and neurexin-1β in non-neuronal cells
AChE-S
(AChE-R)
cDNA
Nrxn-1b G2 G1
175 83
kDa 62
Medium
Lysate
Vector
AChE-R
AChE-S
Cell lysate + ─
Membrane fraction
AChE-S + + ─
Nrxn1-1’ ─ + +
Nrxn
kDa
Blot: His
150
105 75 60 55
100 75 50

13. Neuronal AChE physically interacts with neurexins
IP: His
Blot: AChE
IP: AChE
Blot: His + ─
Co-transfection
Nrxn1-1’
AChE-S 50 75
100
150
kDa
AChE 70 67
105 60 55
Nrxn B + ─
Nrxn1-1’
AChE-R
IP: AChE
Blot: His 60 55
Nrxn
Co-transfection C + _
IP:
IgG
AChE
Nrxn1-1’
AChE-S
Blot: His 60 55
Nrxn
Co-transfection

14. AChE interacts with neurexins in situ
lyset
lyset
Mixed
B-1
A-2
Co-culture
47.5
kDa 62 ─
Nrxn1-ß
AChE-S +
Physo
55kD
Nrxn
Separated culture ─ +
47.5
kDa 62
B-2

15. Excess AChE reduces NRXN-NLGN association and inhibits NLGN-induced synaptogenesis
GFP Synapsin Overlay
Nlgn Nlgn Ctrl
AChE-S A ─ +
Nrxn1-1’
Nlgn
AChE-S
IgG
IP: -His
Blot: -Nlgn
Blot: -His
Nrxn B’ C
Synapsin
clusters

16. Excess AChE decreases glutamatergic synapses
Ctrl AChE+ Physo.
Physo. * D A
Perm Non-perm Overlay
Physo AChE+Physo Control
20 m
Control AChE + Physo Physo C B
Ctrl AChE+ Physo. C’
Control
AChE+

17. Increased expression of AChE reduces glutamate-induced current in neurons
Light Fluorescent
40 m B E * C
Control
AChE-R
AChE-S * D
Control
AChE-R
AChE-S F

18. Excess AChE decreases glutamatergic synaptic activities
Ctrl+physo
AChE+physo
A-2
B-1
B-2
C-1
C-2 *

19. Acknowledgments Dr. Yanna Xiang Dr. Haiheng Dong Dr. Burton B. Yang
Dr. John F. Macdonald