Defense Date
12-18-2023
Graduation Date
Spring 5-18-2024
Availability
One-year Embargo
Submission Type
dissertation
Degree Name
PhD
Department
Pharmacology
School
School of Pharmacy
Committee Chair
Rehana K. Leak
Committee Member
Lauren A. O’Donnell
Committee Member
Paula A. Witt-Enderby
Committee Member
David A. Johnson
Committee Member
Laurie H. Sanders
Keywords
Dementia with Lewy bodies, APE1, APEX1, Parkinson's disease, Preformed Fibrils, Synuclein
Abstract
Lewy body disorders are a family of neurodegenerative conditions characterized by proteostatic and redox disequilibrium, leading to deposition of the abundant protein α-synuclein in hallmark inclusions and oxidative damage to DNA. Sublethal oxidative DNA damage in neurons is repaired predominantly by the base excision repair pathway, which involves the coordinated activity of several enzymes, including apurinic/apyrimidinic endonuclease 1 (APE1). Despite emerging connections between α-synuclein, oxidative stress, and DNA damage/repair, along with evidence demonstrating the multifunctional nature of APE1, the link between APE1 and α-synuclein has not yet been explored.
Here, we conducted a broad investigation of APE1 in the context of Lewy body disorders. In Aim 1, we tested if loss of APE1 expression or inhibition of APE1 amplifies inclusion formation in primary hippocampal rat cultures challenged with preformed α-synuclein fibrils. In vitro knockdown of APE1 with two independent short hairpin RNA sequences or pharmacological inhibition of APE1 DNA repair activity increased the number of inclusions bearing hyperphosphorylated α-synuclein (pSer129), suggesting a mechanistic link between APE1 and the emergence of Lewy-like pathology that could be explored further in vivo.
In Aim 2, we measured the effects of α-synucleinopathy and biological sex on APE1 expression and DNA excision/repair function in mice and humans. Given findings of dense α-synucleinopathy in the anterior olfactory nucleus and amygdala of humans with Lewy body disease that may contribute towards non-motor symptoms, we chose to model limbic-predominant Lewy-like pathology by infusing preformed α-synuclein fibrils into the mouse olfactory bulb/anterior olfactory nucleus (OB/AON). OB/AON fibril infusions induced a mild decrease in APE1 expression in the brains of male mice but an increase in females, and the loss of APE1 expression observed in males was abolished by dietary administration of the antioxidant N-acetylcysteine. Preformed fibril infusions into the mouse OB/AON also elicited more robust DNA repair activities for oxidative DNA lesions in females than males. Lastly, men with Lewy body disorders displayed lower APE1 expression in the OB and amygdala compared to women. These findings support a sex-biased impact of α-synucleinopathy on APE1 in mice and humans.
In Aim 3, we examined the impact of biological sex on the expression, phosphorylation, and insolubility of α-synuclein within limbic tissues of humans with Lewy body disorders or unaffected controls and outbred mice and rats bilaterally infused into the OB/AON with vehicle or fibrils. We demonstrated, for the first time, that OB/AON fibril infusions can be used to generate limbic ⍺-synucleinopathy in mice and rats. Furthermore, more of the whole-fraction sex differences in pan and phosphorylated α-synuclein translated from rats to humans, suggesting an advantage of the rat model over the mouse model. Sequential extraction of soluble and insoluble ⍺-synuclein in limbic tissues revealed that sex is not a main driver of variation in hyperphosphorylated, insoluble ⍺-synuclein in humans, rats, and mice. Rather, the disease itself (i.e., fibril infusions in mice and rats or a diagnosis of Lewy body disease in humans) was a main source of variability.
In Aim 4, we further tested the role of APE1 in the development of Lewy-like pathology by infusing α-synuclein fibrils or vehicle in the OB/AON of wildtype and transgenic APE1 overexpressing rats. Hemizygous overexpression of the human APEX1 gene prevented several detrimental effects of fibril exposure on neurological outcomes in rats. However, homozygous APEX1 expression exerted CNS toxicity in rats, and hemizygous APEX1 induced some negative outcomes, including on key variables such as bodyweight. Increasing APE1 expression in vivo failed to reduce inclusion counts in the dorsal AON and increased the proportion of insoluble a-synuclein that was hyperphosphorylated in fractionated amygdala extracts. In contrast, morphometric changes in Hoechst+ and NeuN+ structures were ameliorated by the hemizygous transgene. Finally, correlation analyses revealed that a shorter lifespan in men and women is associated with lower APE1 protein expression and fewer APEX1 transcripts.
Collectively, the body of work presented in this dissertation support the presence of a previously unrecognized link between expression of APE1 and α-synuclein in mice, rats, and humans that may guide future studies seeking to improve understanding of DNA damage and repair systems in α-synucleinopathies.
Language
English
Recommended Citation
Miner, K. M. (2024). The Multipurpose DNA Repair/Redox Protein APE1 in Rodent and Human Lewy Body Disease (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/2345