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The role of karyopherin-alpha in the pathogenesis of TDP-43 proteinopathy Armstrong, Bryson Walter


Aberrant cellular processing and targeting of TDP-43 has been implicated in a wide variety of neurological diseases such as frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). These diseases are characterized by the sequestration of TDP-43 into the cytoplasm of afflicted neurons, leading to the formation of ubiquitinated, cytoplasmic inclusions and an increased susceptibility to cellular insults. While the underlying causes of TDP-43 proteinopathy are unknown, we are investigating the role of a protein family known as the karyopherins in the nuclear targeting of TDP-43. Using co-immunoprecipitation in SH-SY5Y cells we determined that a major binding partner of TDP-43 is karyopherin-alpha 2 (KPNA2). Next, utilizing a high-density peptide array comprised of overlapping peptide sequences derived from TDP-43 we identified six regions where KPNA2 may directly interact with TDP-43. From these regions we developed six small, cell-penetrating peptides designed to specifically inhibit the interaction between KPNA2 and TDP-43. Through the use of these synthetic peptides, we were able to interfere with the binding of KPNA2 to TDP-43 in vitro. We found that the disruption of this specific protein-protein interaction was not sufficient to induce TDP-43 cytoplasmic sequestration, as determined by co-immunoprecipitation and subcellular fractionation assays. As our research focused primarily on healthy SH-SY5Y cells, future studies will focus on investigating the effects of peptide-mediated TDP-43 nuclear import impairment paired alongside oxidative insult. We will also investigate whether compensatory mechanisms within SH-SY5Y cells are responsible for the nuclear localization of TDP-43 in the absence of KPNA2-mediated nuclear import.

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