Publication Date

2016-01-28

Availability

Embargoed

Embargo Period

2018-01-27

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2016-01-20

First Committee Member

Rong Grace Zhai

Second Committee Member

Abigail Hackam

Third Committee Member

Mary Lou King

Fourth Committee Member

Miguel Perez-Pinzon

Fifth Committee Member

Fangliang Zhang

Sixth Committee Member

Bing Zhang

Abstract

Neurodegenerative diseases triggered by a variety of genetic, epigenetic, and environmental factors are increasingly prevalent worldwide. Maintaining neuronal homeostasis is a prerequisite for proper neurological activity. However, details of mechanisms of neuronal maintenance are largely unknown, and in particular it is unclear whether and how healthy neurons regulate their self-protective activities. Previous work in our laboratory and others has found housekeeping NAD synthetic enzyme NMNAT proteins to be among the most effective and versatile neuroprotective factors in Drosophila and mammals. It has been shown that a stem-loop secondary RNA structure within the Drosophila Nmnat (DmNmnat) intron can modulate its alternative splicing. Alternative splicing is a common cellular post-translational regulation mechanism to produce structurally and functionally distinct mRNA and protein variants from a single gene, which can be regulated by environmental stress. However, the post-transcriptional regulation of DmNmnat under stress and the functional properties of alternatively spliced protein isoforms are still not understood. I hypothesize that DmNmnat is differentially regulated under stress, and the functional properties of each variant may be distinct. Complete dissection of the mechanisms of the post-transcriptional regulation of the DmNmnat gene under stress and the neuronal functions of all potential NMNAT isoforms will provide important insights into some of the fundamental mechanisms of neural degeneration as well as the neuroprotective ability of NMNAT. To test this hypothesis, I investigated the following aims: (A1) Determine whether DmNMNAT isoforms have different neuroprotective capacity in multiple neurodegenerative disease models; (A2) Determine whether alternative splicing functions as a switch that regulates the expressions of DmNmnat variants; (A3) Identify other potential regulatory mechanisms involved in post-transcriptional regulation of DmNmnat. In these studies I report that the DmNmnat gene is alternatively spliced into two mRNA variants, RA and RB, which leads to two sets of functionally distinct proteins: PA/PC from RA is nuclear localized with minimal neuroprotective ability; PB/PD from RB is cytoplasmic and has robust neuroprotective capacity to protect against multiple neurodegenerative diseases. Furthermore, under stress conditions, the RB variant is preferably spliced in neurons to produce the neuroprotective PB/PD protein isoform. All these data indicate that alternative splicing acts as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the switch to produce the neuroprotective variants, and therefore achieve self-protection. This study reveals a key mechanism of how neurons achieve and regulate self-protection against adverse conditions. The obtained information will provide significant insights for neural protective therapy in neurodegenerative disorders under stress.

Keywords

Drosophila; Neurodegenerative disease; Neuroprotection; Alternative splicing; Nmnat

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