Doctor of Philosophy
Pharmacology and Toxicology
Cholinergic neurons play a key role in cognitive processes through the action of the neurotransmitter acetylcholine (ACh). Dysfunction of these neurons occurs in several neurodegenerative disorders, including Alzheimer’s disease (AD). The high-affinity choline transporter CHT recycles choline back into synaptic terminals, which is the rate-limiting step to ACh production. CHT proteins traffic between the cell surface and subcellular organelles in a constitutive manner, which maintains plasma membrane transporter levels, thereby regulating CHT activity and maintaining cholinergic transmission. Pathological conditions associated with AD may alter CHT function in a manner that reduces choline uptake activity and impairs cholinergic neurotransmission. Thus, my experiments focused on understanding the mechanisms regulating the subcellular distribution and activity of CHT in neural cells stably expressing the transporter exposed to AD-related pathological conditions. Important AD risk factors that can lead to pathological changes in the cholinergic nerve terminal microenvironment include increased generation of the reactive oxygen species (ROS) peroxynitrite (ONOO-), high serum cholesterol level at midlife and mutations in genes encoding the amyloid precursor protein (APP). My investigation revealed that ONOO- alters CHT function through changes to CHT subcellular trafficking, as opposed to nitrosative or oxidative modification to CHT itself. Moreover, CHT movement through subcellular endosomes and lysosomes was not altered by ONOO-, but blocking proteasome function attenuated ONOO- mediated inhibition of CHT function. Second, I evaluated how CHT activity and trafficking is regulated by plasma membrane cholesterol and its association with cholesterol-rich lipid rafts. The results of these experiments showed that membrane cholesterol and lipid rafts play an important role in regulating CHT trafficking and activity by retaining functional CHT at the cell surface. Finally, my studies revealed that CHT function is inhibited by both wild-type and Swedish mutant APP but the underlying mechanisms differ. Wild-type APP facilitates CHT endocytosis, whereas Swedish mutant APP mediated inhibition of CHT function is caused by the susceptibility of CHT to Aβ released into the extracellular environment. My studies help define the molecular regulation of CHT proteins under pathological conditions. Information from studies such as these will aid the design of therapeutic strategies to treat pathologies involving cholinergic dysfunction.
Cuddy, Leah K., "Regulation of the High-Affinity Choline Transporter Activity and Trafficking in Alzheimer’s Disease-Related Pathological Conditions" (2014). Electronic Thesis and Dissertation Repository. 2513.