Roles of the Presynaptic Choline Transporter in Sustaining Cholinergic Signaling as Revealed Using Genetically Altered Mice

Abstract

The presynaptic choline transporter (CHT, SLC5A7) supplies choline for acetylcholine (ACh) synthesis in cholinergic neurons. CHT is particularly important as the demands of cholinergic signaling increase and ACh stores are depleted is vital for sustaining normal autonomic and cognitive function. Despite its role at the plasma membrane in importing choline, CHT is predominately localized at neuromuscular and brain synapses on intracellular synaptic vesicles. Whether augmentation of CHT through genetic or pharmacological approaches will enhance cholinergic tone is presently unknown. This dissertation examines models providing for genetically-instated reduced and elevated CHT expression generated to evaluate more clearly how such changes impact cholinergic signaling. Brain slice studies in CHT heterozygous mice are used to evaluate the contribution of loss of transporters on CNS ACh release and to evaluate whether alterations observed derive from compromised ACh synthesis or altered vesicular CHT support for release. A second model produces specific overexpression of CHT in motor neurons using the motor neuron specific promoter Hb9. I provide evidence that motor neuron overexpression of CHT can modestly extend life of CHT knockout mice and when combined with expression of native genomic expression of CHT results in enhanced endurance in a forced exercise paradigm. Surprisingly, analysis of stimulated muscle action potentials in these mice reveals a change of muscle responses from the unimodal wild-type distribution to a bimodal distribution where each population of the transgenic distribution exhibits different recovery kinetics following high frequency stimulation. A third model uses multiple copies of the genomic region encoded by a Bacterial Artificial Chromosome (BAC) for CHT to produce constitutive CHT overexpression. Although finding that these mice express substantially more CHT, such expression does not result in a commensurate increase in uptake. An initial behavioral battery has suggested that these mice have more anxiety and reduced working memory. Together, the results of this dissertation support both a classical role of CHT in sustaining ACh synthesis but also a novel role in contributing to the composition or mobilization of cholinergic vesicle pools.