Tuesday, May 7, 2024
5/7/2024
The genome is the foremost determinant of inherent defenses, and loss of its integrity can culminate in pathological conditions. Cellular DNA repair systems have evolved to mitigate this risk, but are poorly understood in neurological disorders. We propose to examine if changes in DNA repair modify behavioral and histological outcomes in limbic-predominant Lewy body disorder, a neurological condition in which insoluble deposits of a-synuclein protein fill areas of the brain involved in olfaction, emotion, and memory. The repair of oxidized, damaged DNA is facilitated by apurinic/apyrimidinic endonuclease I (APE1). The pleiotropic APE1 gene has both a DNA repair function and a redox function—the former is impaired in brain tissue from Parkinson’s patients. It is known that oxidized a-synuclein enters the nucleus and elicits breaks in DNA, but the relationship between APE1 and a-synucleinopathy has not, until now, been scrutinized. We have collected pilot data to support the central hypothesis that APE1 tempers the pathological sequelae of exposure to preformed a-synuclein fibrils in cellular and animal models of limbic-predominant Lewy body disorders. The olfactory bulb/anterior olfactory nucleus (OB/AON) complex is one of the earliest brain regions to display inclusions in Lewy body disorders. Our pilot data reveal that male mice respond to fibril infusions in the OB/AON with loss of smell, dense limbic inclusions, and a decrease in APE1 expression within six months. In contrast, females respond with a global increase in APE1, do not develop smell deficits, and display fewer limbic inclusions. These sex differences parallel the 2 to 3-fold higher risk of Lewy body disorders in men compared to women. Our recent human postmortem work similarly reveals that gender modifies the impact of limbic Lewy body disease on APE1 expression in limbic brain regions. New pilot data also show that shRNA-mediated knockdown of APE1 increases inclusions in primary neurons. Here, we propose further mechanistic studies that will put the causality of the link between APE1 and a-synucleinopathy to the test. AIM 1. We will test the hypothesis that lentivirus-mediated APE1 knockdown in primary neuron/astrocyte and primary neuron cultures treated with a-synuclein fibrils will elicit loss of APE1 expression and endonuclease activity, greater DNA damage, and more protein aggregations and cell loss. We will employ two shRNA sequences to address this hypothesis and determine if overexpressing human WT APE1 prevents the knockdown phenotype, to ensure shRNA specificity. In AIM 2, we will test the hypothesis that APE1 mitigates the behavioral and histological impact of a-synuclein fibril infusions in the male and female rat OB/AON. We will contrast human wildtype APE1-overexpressing rats to transgenic rats that overexpress mutant APE1 with impaired DNA repair (D210A mutation) or loss of redox capacity (C65A mutation). Cognitive, anxiety, olfactory, and motor behavior will be assessed at monthly intervals. APE1 expression and activity, DNA damage markers, proteinopathic inclusions, and cell counts will be measured at 3, 6, and 12 months. If funded, these studies will be the first to examine the relationship between a-synucleinopathy and DNA repair capacity and to characterize sex differences in limbic Lewy pathology. The experiments are designed to either support or reject the notion that DNA reparatory mechanisms should be pursued as a rational target for stymieing a-synucleinopathic disease.
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