Date of Award

Winter 1-2017

Embargo Period

1-5-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Marcel P. Bruchez

Abstract

Numerous neurological disorders and defects, including seizures and neurodegenerative diseases, stem from alterations in neuronal firing. Neuronal activity is dictated by its complement of cell surface proteins, including ion channels. Furthermore transmission of messages from one neuron to the next requires careful control over the secretion and replenishment of neurotransmitters. Changes in the way these involved proteins are trafficked could underlie neuronal plasticity in learning and memory formation. When these mechanisms go awry, neurological disease results. Studying protein trafficking and delivery to the neuronal cell surface is challenging. Electrophysiology has classically been used to measure ion flux and cellular activity, which can indirectly measure trafficking on a per-cell basis, but is hampered by its low throughput. Current optical methods enable visualization of larger brain regions; typically, pH-sensitive fluorescent tags or antibodies directed against extracellular portions of the proteins of interest are used, but have steep limitations in brain tissue. To tackle these issues, we have developed fluorogen-activated peptides (FAPs), which involve a non- fluorescent protein tag that binds a normally non-fluorescent dye (fluorogen); this combination induces bright fluorescence. By modifying the fluorogen, we can confer special properties such as selective labeling of surface proteins or changing the color of fluorescence. We have employed FAPs in a series of studies aimed at elucidating the mechanisms of synaptic vesicle reuse and trafficking of a critical potassium channel involved in seizures and epilepsy. The large conductance voltage and calcium activated potassium (BK) channel has been implicated in sensitization to seizures, possibly by increasing the complement of surface channels and enhancing intrinsic cellular excitability. To screen for trafficking e ffectors, we established the green-inside, red-outside (GIRO) approach which enables the simultaneous labeling of surface proteins with a red fluorogen, and the internal protein with a green fluorogen. Using this method, we identified adenylyl cyclase as a regulator of BK channel surface expression. We next generated a genetically engineered mouse model in which the endogenous BK channels are tagged with FAP, with the goal of enabling visualization of BK in the mammalian brain under intrinsic regulatory conditions. Unlike mice lacking BK, we characterized the FAP-BK mouse as having normal locomotor behavior. We also observed the assembly of surface channels into well-defined puncta in Purkinje Cells of the cerebellum, corroborating electron microscopy studies demonstrating the presence of clustered ion channels. We anticipate that future work with this mouse line will provide insight into the regulation of BK channel trafficking in seizure disorders, learning, and memory. The unique approach afforded us by FAPs also enabled us to characterize the recycling of synaptic vesicles (SVs) carrying dopamine and acetylcholine, mediators of mood, motivation, and of altered availability in neurodegenerative diseases. Previous studies characterized bulk SV fusion and retrieval; however, our method enabled tagging of a mobile subset of SVs and characterizing reuse of the same SVs. We found that FAP tagging does not disrupt protein function, and we found unique reuse properties of VMAT2 and VAchT in a neuronal-like cell line.

Available for download on Friday, January 05, 2018

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