Date of Award

2021

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Paula Traktman

Second Advisor

Robin C. Muise-Helmericks

Third Advisor

C. Amanda LaRue

Fourth Advisor

Denis Guttridge

Fifth Advisor

W. Dave Hill

Abstract

The nuclear envelope is a dynamic structure and organizing hub for cell structure and function. The underlying nuclear lamina is composed of a thick network of intermediate filaments, Lamin A/C and B, and inner nuclear membrane-associated proteins such as Lap2, Emerin and Man1. BAF, a central organizing protein, is a small, dimeric, essential protein that binds to dsDNA, proteins of the inner nuclear membrane and Lamin A. Cells with perturbations of the nuclear lamina display dysmorphic nuclei which have the potential to lead to altered gene expression, increased DNA damage events, and premature cellular senescence. In this study, we address a single amino acid substitution from Alanine 12 to Threonine (A12T) in BAF that gives rise to a nuclear morphology defect. At the clinical level, patients who inherit a homozygous BAF A12T mutation have a rare premature aging syndrome called Nestor Guillermo Progeria Syndrome (NGPS) with symptoms such as osteolysis, adipolysis and craniofacial defects that present at approximately two years of age and progress rapidly, ultimately shortening patient lifespan significantly. Tissues of mesenchymal origin appear to be most severely impacted in this disease, leading to the hypothesis that mesenchymal stem cells are preferentially affected. To address the cellular and organismal pathology of NGPS, we used CRISPR/Cas9 technology to establish BAFA12T/BAFA12T human induced pluripotent stem cells (hiPSCs). A12T BAF iPSCs appeared normal, however when differentiated to induced mesenchymal stem cells (iMSCs), A12T BAF cells exhibited a marked increase in cellular senescence and an elevated cellular stress response. The mechanism of A12T BAF’s contribution of this cell-type specific pathology was not well understood. Our data indicate that A12T BAF ‘s binding to Lamin A is greatly diminished, thereby weakening the Lamin A network of the nuclear lamina and the structural integrity of the nuclear envelope. Mechanosensitive iMSCs are unable to withstand this weakened network and the nuclei appear to rupture, thereby causing cellular stress and ultimately, senescence. Our results indicate that the A12T BAF mutation is necessary but not sufficient to cause premature cellular aging. All cell types expressing BAFA12T/BAFA12T have dysmorphic nuclei, however cells derived from BAFA12T/BAFA12T mice are viable and primary dermal fibroblasts are not senescent, nor do they demonstrate elevated levels of DNA damage. Furthermore, A12T BAF mice do not have shortened lifespans nor do they have defects in mesenchymal derived tissue. These data highlight the nuances of this mutation’s effect both at the cellular and organismal level and elucidates a new role for BAF at the nuclear envelope as a key player in cell mechanosensitivity and the network of the nucleoskeleton and cytoskeleton.

Rights

All rights reserved. Copyright is held by the author.

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