Electronic Theses and Dissertations for Graduate School
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Author Last Name
Adaptive Defense Mechanism Associated with HFE Gene Variant
Restricted (Penn State Only)
Doctor of Philosophy
Date of Defense:
July 10, 2020
James Robert Connor, Dissertation Advisor/Co-Advisor
James Robert Connor, Committee Chair/Co-Chair
Xuemei Huang, Committee Member
Nadine Hempel, Committee Member
Elizabeth Anne Proctor, Outside Member
Alistair J Barber, Program Head/Chair
There is a gap in knowledge regarding the connection between neurodegenerative diseases and the homeostatic iron regulator (HFE) gene variant, which is found in over 15% of the U.S. population. Previously, our group found that the Hfe gene variant mouse model (H67D-Hfe) has limited vulnerability to paraquat-induced motor impairment and α-synuclein, but accelerated death in a model of amyotrophic lateral sclerosis. The purpose of this thesis is to elucidate the cellular and molecular mechanisms underlying the modification that the HFE gene variant induces that enables it to function as a genetic modifier of neurodegenerative disease. The scientific premise is that the adaptive responses to the presence of the Hfe gene variant establishes hormesis, which provides a protective milieu that limits vulnerability to oxidative stress. In chapter 2, we interrogated the cellular mechanism underlying the protection from paraquat toxicity, as a model of oxidative stress, using primary astrocytes cultured from WT-Hfe and H67D-Hfe mouse pups. We found that the H67D-Hfe astrocytes had a higher Nuclear factor erythroid 2-related factor 2 (Nrf2) accumulation in the nucleus after paraquat exposure. Furthermore, H67D-Hfe astrocytes exhibited protection from paraquat-induced mitochondrial damage, cell death, and senescence. Decreasing expression of Nrf2 via Nrf2 siRNA treatment completely eliminated the protection afforded by the H67D-Hfe genotype in the astrocytes. Moreover, the downstream antioxidant of Nrf2, NAD(P)H quinone dehydrogenase 1 (NQO1), was elevated in the H67D-Hfe astrocytes as well as in the H63D-HFE carriers in the human population. Applying the results from chapter 2, in chapter 3 we further explored the differences in the functionality of the brain that are driven by the Hfe genotype, which may modulate the susceptibility to oxidative stress. Bulk RNA-sequencing was performed on the ventral midbrain of the WT-Hfe and H67D-Hfe mice to assess the difference in gene expression profile. From transcriptomic analysis, we identified Hfe gene variant-driven differences in the expressions of genes involved in mitochondrial function and neuroprotective response. We further interrogated the Nrf2 pathway activation in the primary astrocytes by examining the difference in the expression of Nrf2 regulator. The data showed an upregulation of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a positive regulator of Nrf2. Furthermore, we have revealed a difference in Glycogen synthase kinase 3 beta (GSK3β) inactivation, which is a downstream target of PGC-1α. GSK3β either directly phosphorylates Nrf2 to induce degradation or phosphorylates tyrosine kinase Fyn to promote Nrf2 export. Thus, inactivating GSK3β facilitates nuclear Nrf2 accumulation to initiate antioxidant gene transcription. To translate this study to a potential therapeutic approach, we pre-treated the cells with a GSK3β inactivation compound, CHIR99021, which resulted in rescue from paraquat-induced cell death. As far as we know, this is the first study showing the impact of the CHIR99021 compound on preventing cell death induced by paraquat. The findings from my thesis project revealed that the stress generated by iron accumulation in the Hfe gene variant model establishes an adaptive defense response that leads to an alteration in the Nrf2 regulators and mitochondrial-related gene, which ultimately provides protection from mitochondrial damage and cell death induced by paraquat toxicity. We have further progressed the findings to a translational study using GSK3β inactivator, CHIR99021 compound. Consequently, this gene variant model offers new insight for how a common gene variant can impact disease susceptibility and further suggests a therapeutic approach to treating Parkinson’s Disease and possibly other neurodegenerative diseases involving oxidative stress. Finally, the data strongly suggests that individuals with PD could have an altered disease course and therapeutic response that differs based on HFE genotype.
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