Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Pathology and Laboratory Medicine

Supervisor

Dr. Martin Duennwald

Abstract

Protein misfolding characterizes most neurodegenerative diseases. Protein misfolding is the conversion of specific proteins from their normal, often soluble, and native three-dimensional conformation into an aberrant, often insoluble, non-functional conformation. Protein inclusions and aggregates are among the major pathological hallmarks of protein misfolding associated with many neurodegenerative diseases. Yet, the role of aggregates and inclusions is not clearly defined and heavily debated. This study utilizes powerful genetic approaches in yeast and verification in mammalian neuronal cell lines to address the misfolding and toxicity of three proteins, the Rho Guanine Nucleotide Exchange Factor (RGNEF), Matrin3, which are involved in amyotrophic lateral sclerosis (ALS) and polyglutamine (polyQ) expanded huntingtin, which causes Huntington’s disease (HD).

Genetic, biochemical, and pathological findings implicate RGNEF and Matrin3 in Amyotrophic Lateral Sclerosis (ALS). In this thesis we establish two novel humanized yeast models to study RGNEF and Matrin3. We find that RGNEF is toxic in yeast and can misfold and form inclusions. We also identify a potential new role for RGNEF as a microtubule regulator. Similarly, Matrin3 is also toxic and forms inclusions in yeast. We identify members of the Hsp90 and Hsp70 cytosolic chaperoning machinery as potent determinants of Matrin3 associated toxicity and misfolding and verify our findings in neuronal cell lines. Also, polyQ expanded repeats of the huntingtin protein are the sole known cause of Huntington’s disease (HD). We address the nexus of aging and aggregation, and toxicity of polyQ expanded repeats in a yeast model of aging. Aging is the most significant risk factor for all neurodegeneration, and we find that polyQ toxicity is exacerbated in aged cells while aggregates are lost. We also demonstrate that treating cells expressing polyQ with the steroidal lactone Withaferin A (WA) reduces toxicity while increasing aggregation. In essence, this study contributes to a deeper understanding of three misfolded proteins. Also, our results challenge the long-held postulation that aggregates cause neurodegeneration. Our findings provide further insight into the role of aggregation and inclusions that are crucial for developing effective therapeutic strategies that are not currently available for ALS and HD.

Summary for Lay Audience

Proteins are functional units in the cell that carry out specialized tasks that sustain life. The ability of a protein to perform a given function is heavily dependent upon folding into the correct three-dimensional structure. In neurodegenerative diseases, such as ALS or HD, proteins take on an aberrant structure that can lead to a loss of function or damage cells. This can be caused by genetic mutations and aging and changes in the cellular environment, such as temperature and pH. Misfolded proteins are packaged together in the cell and form aggregates or inclusions. It is heavily debated if protein aggregation is beneficial in removing potentially harmful proteins or if these aggregates directly damage neurons and cause neurodegeneration. My thesis aims to address this problem by modeling protein misfolding in yeast, a simple yet powerful living test tube as well as in cultured neuronal cells. My thesis explores three different proteins that misfold in neurodegenerative diseases.

RGNEF and Matrin3 are implicated in the development of ALS, a devastating motor-neuron disease. In order to study the basic function of these proteins as well as their misfolding, we have expressed them in yeast. We observe that both RGNEF and Matrin3 are toxic to yeast cells and form inclusions. Using this model, we have identified a novel role for RGNEF as a regulator of cell scaffolding. In addition, we demonstrate that molecular chaperones are potent modifiers of Matrin3 toxicity and misfolding. Finally, we show that polyQ expanded repeats are more toxic in aged yeast cells and that aggregates are lost. Further, we show that treatment of neurological cells with a plant-derived small molecule reduces polyQ toxicity and induces aggregation.

My thesis discovered new processes that affect the misfolded proteins studied. Enhancing our understanding of these proteins and their misfolding can inform novel therapeutic strategies to treat ALD and HD. Our results find that protein aggregates may be protective instead of drivers of disease. Thus, drugs designed to breakdown aggregates should be reconsidered.

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