Acetylcholinesterase (AChE) accumulates on axonal varicosities and is primarily found as tetramers associated with a proline-rich membrane anchor (PRiMA). PRiMA is a small transmembrane protein that efficiently transforms secreted AChE to an enzyme anchored on the outer cell surface. Surprisingly, in the striatum of the PRiMA knock-out mouse, despite a normal level of AChE mRNA, we find only 2-3% of wild type AChE activity, with the residual AChE localized in the endoplasmic reticulum, demonstrating that PRiMA in vivo is necessary for intracellular processing of AChE in neurons. Moreover, deletion of the retention signal of the AChE catalytic subunit in mice, which is the domain of interaction with PRiMA, does not restore AChE activity in the striatum, establishing that PRiMA is necessary to target and/or to stabilize nascent AChE in neurons. These unexpected findings open new avenues to modulating AChE activity and its distribution in CNS disorders.

Cholinesterase inhibitors are commonly used to improve cognition and treat psychosis and other behavioral symptoms in Alzheimer's disease, Parkinson's disease, and other neuropsychiatric conditions. However, mechanisms may exist that down-regulate the synaptic response to altered cholinergic transmission, thus limiting the efficacy of cholinomimetics in treating disease. Acetylcholinesterase knockout (AChEϪ/Ϫ) mice were used to investigate the neuronal adaptations to diminished synaptic acetylcholine (ACh) metabolism. The striatum of AChEϪ/Ϫ mice showed no changes in choline acetyltransferase activity or levels of the vesicular ACh transporter but showed striking 60% increases in the levels of the highaffinity choline transporter. This transporter takes choline from the synapse into the neuron for resynthesis of ACh. In addition, the striata of AChEϪ/Ϫ mice showed dramatic reductions in levels of the M 1 , M 2 , and M 4 muscarinic ACh receptors (mAChRs), but no alterations in dopamine receptors or the ␤2 subunit of nicotinic receptors. M 1 , M 2 , and M 4 also showed decreased dendritic and cell surface distributions and enhanced intracellular localizations in striatal neurons of AChEϪ/Ϫ mice. mAChR antagonist treatment reversed the shifts in mAChR distribution, indicating that internalized receptors in AChEϪ/Ϫ mice can recover to basal distributions. Finally, AChEϪ/Ϫ mice showed increased sensitivity to mAChR antagonist-induced increases in locomotor activity, demonstrating functional mAChR down-regulation. mAChR downregulation in AChEϪ/Ϫ mice has important implications for the long-term use of cholinesterase inhibitors and other cholinomimetics in treating disorders characterized by perturbed cholinergic function.

Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic post-synaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the post-synaptic surface, but is instead clustered along the presynaptic membrane and deep in the post-synaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus, the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extra-junctional regions.