Endocytic trafficking of muscarinic acetylcholine receptors in cultured neuronal cells and mouse models of hypercholinergic activity.

摘要

Muscarinic acetylcholine receptors (mAChRs), which belong to the family of G protein-coupled receptors, play a crucial role in CNS functions including memory, motor behavior, pain, and sleep. This dissertation examined the intracellular trafficking of mAChRs following stimulation. Intracellular trafficking provides a crucial component of GPCR regulation and includes: internalization, subsequent return to the cell surface, or degradation.; Initially following stimulation, M₄ endogenously expressed in PC12 cells internalizes from the cell surface and traffics to early endosomes. M₄ early endosomal trafficking and localization within multivesicular bodies depend on the activity of the small GTPase, Rab5a. Upon prolonged stimulation, M₄ traffics to perinuclear endosomes and returns to the cell surface following agonist washout. Expression of inactive forms of Rab11a and the unconventional myosin Vb dramatically alter M₄ trafficking to perinuclear endosomes and impair M₄ recycling. Thus, M₄ traffics through specific endocytic and recycling pathways through mechanisms dependent on Rab5a, Rab11a and myosin Vb.; Having identified the intracellular mechanisms involved in mAChR internalization and recycling, it was determined if mAChRs following similar patterns of trafficking in the brain. Furthermore, the fate of mAChRs in response to chronic diminished ACh metabolism was also examined. The cholinergic system provides a primary target for the treatment of disorders such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, and schizophrenia. However, mechanisms may exist that control the synaptic response to altered cholinergic transmission. AChE knockout (AChE −/−) mice were utilized to investigate the neuronal adaptations to diminished ACh metabolism. M₁, M₂ and M₄ mAChRs showed dramatically reduced immunoreactivity in the striatum, hippocampus and cortex of AChE −/− mice. In addition, mAChRs showed enhanced intracellular localization and increased internalization from dendrites, presynaptic terminals and the cell surface. However, these alterations in mAChR subcellular distribution were reversible. AChE −/− mice also showed diminished mAChR function. The profound mAChR downregulation following chronic stimulation found in this study provides important implications regarding the chronic use of cholinesterase inhibitors and other cholinomimetics to treat CNS disorders. Understanding the factors regulating expression and localization of molecules involved in cholinergic transmission is crucial to improve the efficacy of chronic cholinergic drug treatments.