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

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

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 - +

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

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

Increased expression of AChE decreases glutamate receptor mediated currents

Excessive AChE reduces dendritic spines and surface glutamate receptors

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

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

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.

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

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

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

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

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

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+

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

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

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