Publication Date

2013-12-13

Availability

Embargoed

Embargo Period

2015-12-13

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2013-11-15

First Committee Member

Rong Grace Zhai

Second Committee Member

Sandra Lemmon

Third Committee Member

Michael Kim

Fourth Committee Member

Pedro Salas

Fifth Committee Member

George R. Jackson

Abstract

Synapses are specialized cell-cell contacts where signals are reliably transduced from a neuron to its target cell in a regulated manner. Structurally, synapses are comprised of the pre- and post-synaptic terminals, and the synaptic cleft. Within each of these compartments is a complex network of molecular machinery, the components of which require constant turnover and “maintenance” to ensure a consistent relay of information. In neurodegenerative diseases, such as Alzheimer’s (AD), Parkinson’s (PD), and prion-related diseases, synaptic degeneration is often an early event associated with disease severity, which is evinced by the degree of cognitive decline. A dysfunction of synaptic maintenance may therefore represent a novel mechanism contributing to the pathogenesis of neurological disease as well as a new potential target for therapeutic design. However, the precise mechanisms underlying synaptic maintenance to date remain largely unexplored. In my thesis, I explore two distinct mechanisms underlying synaptic maintenance: (1) the maintenance of active zone structure through protein-protein interaction of a structural active zone protein, Bruchpilot (BRP), with the neuronal maintenance factor, Nicotinamide mononucleotide adenylyltransferase (NMNAT). (2) Synaptic degeneration induced by misregulated glucose metabolism owing to mutations in degeneration induced by misregulated glucose metabolism owing to mutations in the glycolytic enzyme, enolase. NMNAT is an essential housekeeping enzyme required for the synthesis of NAD+. Previous work has shown that NMNAT is a neuroprotective factor required for maintaining neuronal integrity, as loss of NMNAT causes severe neurodegeneration in Drosophila and mice, while overexpression of NMNAT offers potent neuroprotection in activity- or protein misfolding-induced neurodegeneration. While the mechanism of NMNAT-mediated neuroprotection remains controversial, it is clear that this can be partly attributed to its role as a molecular chaperone independent of its enzymatic function. In addition to its general neuroprotective ability, I have shown that NMNAT is also necessary for active zone maintenance and is present in the synapse. In my thesis, I explore the detailed mechanism governing NMNAT maintainance of the active zone structure. I demonstrate that NMNAT is localized to the peri-active zone, colocalizing and interacting biochemically with the protein, Bruchpilot (BRP), an essential structural protein of the active zone and necessary for efficient synaptic transmission. NMNAT is required for maintaining the amount of BRP at the active zone by stabilizing BRP. Importantly, the requirement for NMNAT in maintaining synaptic structure and the interaction between NMNAT and BRP is activity-dependent. Glycolysis is the metabolic pathway that converts glucose to pyruvate in the cytosol. Enolase, also known as phosphopyruvate hydratase, catalyzes the dehydratation of 2-phosphoglycerate to phosphoenolpyruvate, in the last steps of the catabolic glycolytic pathway. Mutations in enolase have been implicated in human glycogen storage disease (GSD) and it has been shown recently that glycogen formation can directly induce neuronal death. It has also been reported that glycogen accumulates in neurons of diabetic rats, possibly contributing to diabetic neuropathy. Glycogen in the granules may be cross-linked abnormally and thus harder to dissolve. Consistent with this, mutations in Drosophila enolase not only impair visual neurotransmission, but also introduce glycogen-like granules in postsynaptic terminals. The association between neuronal glycogen accumulation and neuropathology has been established; however, the underlying mechanism remains unclear. Here I have characterized mutations in enolase that cause the loss of enolase enzymatic activity, disrupt glycolysis, reduce glycogen synthase kinase-3β (GSK-3β) activity, and promote glycogen synthesis and accumulation. Abnormal glycogen accumulation induces synaptic degeneration by activating the autophagy-lysosome pathway. My work demonstrates critical roles of enolase and glycolysis in maintaining synaptic structural and functional integrity, and reveal a detailed mechanism of glucose metabolic dysregulation-induced synaptic degeneration.

Keywords

Synaptic maintenance, NMNAT, enolase, glia

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