Dissertations, Theses, and Capstone Projects

Date of Degree

9-2023

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Patrizia Casaccia

Committee Members

Alejandra Alonso

Luke E. Berchowitz

Mariana Torrente

Sebastien Poget

Subject Categories

Biochemistry | Molecular and Cellular Neuroscience | Molecular Genetics | Structural Biology

Keywords

Tau, Phase Separation, Alzheimer's Disease, Neurodegeneration, SecretionTau is a protein expressed exclusively in glia and neurons in the central nervous system and implicated in several neurogenerative diseases called “tauopathies”. Among all the tauopathies, one third is characterized by the presence of genetic mutations leading to the synthesis of tau proteins with single amino acid substitutions at specific locations and affecting protein function. While most of the initial studies have emphasize the functional role of tau as modulator of the axonal cytoskeleton, it has recently been well accepted that tau is also an intrinsically disordered protein that tends to form membraneless organelles called coacervates, due to a process called phase separation. My Thesis tested the hypothesis that tau mutations, as those identified in primary tauopathies (e.g. P301L and G389R), might impair the phase separation properties of the protein and impact its pathological role in cells. I first tested the effect of single amino acid substitutions in vitro, using recombinant tau and the results revealed that the tau pathogenic mutations P301L and G389R increased the propensity of tau to phase separate and form coacervates with higher viscosity than wild type. I then hypothesized that tau mutants and wild type tau may also differentially impact cell function. This was studied by transfecting tau and its two mutants P301L and G389R, into either HEK293T, which do not express endogenous tau, or in SHSY-5Y neuroblastoma cells, which endogenously express tau. For the experiments, we used a bimolecular fluorescence complementation assay based on the formation of tau dimers fused with either the N or C terminus of the reporter VENUS fluorescent protein. Using live imaging, we determined that tau P301L and G389R, both enhanced vesicular release of tau in the extracellular space, via calcium-dependent membrane blebbing, in both HEK293T and SHSY-5Y cells compared to wild type. However, the expression of tau mutants did not affect cell viability in response to oxidative stress. While the relationship between the biophysical properties of tau and its function in cells remain to be elucidated, this study suggest potential mechanisms by which specific mutations of tau may induce its release in the extracellular space and suggest a potential role in spreading of the pathology.

Abstract

Tau is a protein expressed exclusively in glia and neurons in the central nervous system and implicated in several neurogenerative diseases called “tauopathies”. Among all the tauopathies, one third is characterized by the presence of genetic mutations leading to the synthesis of tau proteins with single amino acid substitutions at specific locations and affecting protein function. While most of the initial studies have emphasize the functional role of tau as modulator of the axonal cytoskeleton, it has recently been well accepted that tau is also an intrinsically disordered protein that tends to form membraneless organelles called coacervates, due to a process called phase separation. My Thesis tested the hypothesis that tau mutations, as those identified in primary tauopathies (e.g. P301L and G389R), might impair the phase separation properties of the protein and impact its pathological role in cells. I first tested the effect of single amino acid substitutions in vitro, using recombinant tau and the results revealed that the tau pathogenic mutations P301L and G389R increased the propensity of tau to phase separate and form coacervates with higher viscosity than wild type. I then hypothesized that tau mutants and wild type tau may also differentially impact cell function. This was studied by transfecting tau and its two mutants P301L and G389R, into either HEK293T, which do not express endogenous tau, or in SHSY-5Y neuroblastoma cells, which endogenously express tau. For the experiments, we used a bimolecular fluorescence complementation assay based on the formation of tau dimers fused with either the N or C terminus of the reporter VENUS fluorescent protein. Using live imaging, we determined that tau P301L and G389R, both enhanced vesicular release of tau in the extracellular space, via calcium-dependent membrane blebbing, in both HEK293T and SHSY-5Y cells compared to wild type. However, the expression of tau mutants did not affect cell viability in response to oxidative stress.

While the relationship between the biophysical properties of tau and its function in cells remain to be elucidated, this study suggest potential mechanisms by which specific mutations of tau may induce its release in the extracellular space and suggest a potential role in spreading of the pathology.

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