ORCID ID

https://orcid.org/0000-0002-4509-7913

Publication Date

2021-01-21

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

Biochemistry and Molecular Pharmacology

First Thesis Advisor

Paul Thompson

Second Thesis Advisor

Celia Schiffer

Keywords

SARM1, neurodegeneration, NAD, therapeutics

Abstract

Traumatic brain injury, peripheral neuropathies, and other neurodegenerative diseases exhibit diverse clinical manifestations but are connected by their underlying trigger, axonal degeneration. These diseases cause extensive morbidity and mortality worldwide, as treatments are palliative and no curative treatments exist. SARM1 has recently emerged as a therapeutic target for these diseases as knockdown prevents axonal degeneration and ameliorates disease prognosis. Later, it was shown that SARM1 hydrolyzes NAD+ in response to degenerative stressors. Given that NAD+ supplementation delays axonal degeneration, we expect therapeutically targeting SARM1 will be efficacious for neurodegenerative diseases. However, the design of SARM1 therapeutics is limited by the dearth of knowledge surrounding its NAD+ hydrolase activity and active structural state.

Illuminating this black box has been hindered by technical difficulties in obtaining pure active protein. To circumvent these issues, I began by studying SARM1 in lysates. I synthesized truncated constructs and developed three different assays, which enabled me to characterize the kinetic activity. I also established a high–throughput screening pipeline to identify inhibitors and screened >4,000 compounds. Recently, I identified additives (i.e., PEG and citrate) that activate SARM1 by ~2,000–fold, making it feasible to study the purified protein. I found that the additives enhance activity by inducing SARM1 to form a multimeric precipitate. To further interrogate the role multimerization plays in activity, I performed detailed mutagenesis and cell culture studies. The insights from this thesis have aided in our understanding of this elusive enzyme and provided strategic direction for future SARM1 investigation and drug development.

DOI

10.13028/b5rq-w097

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Thursday, February 03, 2022

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