Wei-Yang Lu , MD, PhD Robarts Research Institute Department of Physiology and Pharmacology

1. 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 SAS BW584c51 physostigmine

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

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. mRNA of Nrxn-1β WT mice AChE-mice E-17 NB 5Wk Increased expression of AChE alters the expression levels of neurexin and neuroligin

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

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

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

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

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

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

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

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

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

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