The most frequent DNA lesion caused by oxidative stress is 8-oxo-7,8-dihydroguanine (8-oxodG) and it is often associated with neurodegenerative diseases including Parkinson’s disease (PD), Alzheimer’s disease (AD) and aging processes. In terminally differentiated cells like neurons, 8-oxodG DNA lesions in the transcribed strand of an active gene could be bypassed by RNA polymerase II and generate erroneous proteins through a process called transcriptional mutagenesis (TM). Studies have reported selective increase of 8-oxodG in the substantia nigra dopaminergic neurons of PD brain tissue. Decreased activity of the 8-oxodG-specific repair enzyme, 8-oxoguanine-DNA glycosylase (OGG1), was also documented in PD and aging conditions. Coding region of human SNCA contains 43 potential sites for TM. We recently found that oxidative stress or Ogg1 knockdown increase transcriptional mutagenesis of α-synuclein (α-SYN), leading to protein aggregation. We also identified various TM-generated α-SYN mutants including S42Y and A53E from human PD brain samples. Using highly specific anti-S42Y antibody, we have found S42Y-positive Lewy bodies from postmortem brain samples of PD and dementia with Lewy bodies. Together, our preliminary results strongly suggest that transcriptional mutagenesis contributes to generation of novel pathogenic species of α-SYN in 8-oxodG accumulation conditions such as PD and other synucleinopathy.

The objective of this study is to further identify oxidative stress-derived TM mutant species of α-SYN and investigate their contribution to α-SYN aggregation and the pathogenesis of PD. Successful completion of the project will create a paradigm shift in our understanding of the molecular mechanisms underlying oxidative stress-mediated α-SYN pathology in PD. Knowledge of TM events in α-SYN might be equally important to understand other molecules, such as Aβ and tau in other neurodegenerative conditions.

α-SYN and mitochondrial dysfunction are two central components in PD pathogenesis. Mitochondrial dysfunction is a common feature of the many iterations of PD pathogenesis and α-SYN toxicity seems to affect mitochondria most significantly. Complex interplay between α-SYN and mitochondria has been widely observed. While the intricate crosstalk between mitochondria and α-SYN is poorly understood, our preliminary studies suggest that the 3’-untranslated region (3’-UTR) of a-SYN mRNA plays a key role in translational regulation of α-SYN near mitochondria. Our studies demonstrate that 1) α-SYN mRNA is localized to the mitochondrial surface where its translation is initiated by mitochondrial ROS; 2) this translational control is governed by Pum2, a RNA-binding translational repressor, which binds to the 3’-untranslated region (3’-UTR) of α-SYN transcript; 3) interestingly, mitochondrial Pum2 levels in post-mortem PD brain were significantly lower compared to control subjects, while α-SYN levels were opposite, implying Pum2’s repressive role on α-SYN near mitochondria. In addition, recent studies showing the association of single nucleotide polymorphisms in the α-SYN 3’-UTR with PD strongly suggest that 3‘-UTR-mediated regulation of a-SYN could become a critical player in PD pathogenesis. Our central hypothesis is that deregulation of Pum2-mediated α-SYN translational repression on the outer surface of mitochondria contributes to mitochondrial dysfunction observed in PD. The successful completion of this project could create a paradigm shift in our understanding of molecular mechanisms that control α-SYN expression near mitochondria in PD pathogenesis by elucidating the role of Pum2 and the 3’-UTR of α-SYN in translational regulation

To date, midbrain dopaminergic neurons have been the primary focus of Parkinson’s disease (PD) research. However, mounting evidence has suggested that other cell types including microglia, astrocytes, oligodendrocytes, and peripheral lymphocytes also contribute to PD pathogenesis, which highlights the importance of identifying cell types relevant to PD to improve understanding of the causal processes underlying molecular etiology. Moreover, recent advances in single-cell analysis techniques allow further dissection of each cell type into various subpopulations according to epigenomic and transcriptomic profiles. While the nigrostriatal pathway is the primary region affected in PD and the region responsible for motor symptoms, other brain regions are widely affected and responsible for non-motor features. Some non-motor features frequently present before classical motor symptoms, and brain structures beyond the nigrostriatal pathway, including the olfactory bulb, locus coeruleus, and limbic system, are involved in these premotor or prodromal symptoms. To understand these complexities of PD pathogenesis, more comprehensive approaches to investigate different cell types and multiple brain regions over the course of disease progression are necessary. To this end, we will perform combined analysis of single-nuclei RNA-seq and ATAC-seq as well as whole genome sequencing (WGS) of postmortem PD brain and matched control subjects. We will use recently released a Chromium Single-Cell Multiome ATAC plus Gene Expression platform (10XGenomics) which allows for simultaneous profiling of gene expression and open chromatin from the same cell. We are one of the first labs to apply this innovative technique to frozen postmortem brain samples of the SNpc from 14 PD patients, 10 age-matched controls, and 10 young control subjects. This innovative approach opens a new avenue for our understanding of cell-type specific transcriptional as well as epigenetic mechanisms underpinning PD pathogenesis

Dysregulation of α-SYN gene, SNCA, plays a major role in pathogenic expression of the protein in Parkinson’s disease (PD). Molecular mechanisms underlying transcriptional dysregulation remains unknown. Elucidating the precise epigenomic architecture of SNCA is critically important because it could help us to determine how α-SYN is regulated and how the protein contributes to PD pathogenesis. Two epigenetic histone marks are critical in gene regulation: trimethylation of H3 at lysine 4 (H3K4me3), a transcription-initiating histone modification, and trimethylation of H3 at lysine 27 (H3K27me3), a repressive histone modification. We found that H3K4me3 was significantly higher in the substantia nigra (SN) of idiopathic PD (iPD) brains compared to controls, while H3K27me3 levels were low in both conditions, favoring increased expression of a-SYN in PD. We also observed similar changes in differentiated iPSCs from iPD patients and control subjects with higher α-SYN protein levels in iPD-derived iPSC lines. To modify these epigenetic marks in a locus-specific manner, we created an epigenetic editor system by adapting CRISPR/dCas9-SunTag, successfully reversing elevated H3K4me3 in iPD-iPSC with subsequent reduction in α-SYN levels. The goals of the study are to reveal the PD-specific pathogenic epigenomic architecture of the entire SNCA genome using postmortem brain tissue and iPSC lines from controls and PD subjects, and to reverse dysregulated α-SYN expression in PD by genomic locus-specific epigenetic modulation

Braak reported that α-SYN pathology spreads in a topographically predictable sequence: at the early stage of PD, it starts in olfactory and enteric neurons, propagates to the brain stem and eventually to the neocortex toward the later stage of PD. This observation highlights the gut as the first region to develop α-SYN pathology. Mounting evidence has suggested “the gut hypothesis for PD pathogenesis”, which posits that perturbations in the intestinal environment initiate α-SYN aggregation in enteric neurons, propagating trans-synaptically through the vagus nerve into the brain. However, how initial α-SYN aggregation is triggered in the gut is yet to be elucidated. Reactive oxygen species (ROS) in the intestinal lumen is the potential candidate implicated in this pathogenic process. NADPH oxidase 1 (Nox1), a superoxide generating enzyme which is highly expressed in intestinal epithelial cells, especially in the colon, has been implicated in PD. Nox1-derived ROS in the gut lumen is thought to either facilitate intestinal epithelial proliferation or act as a part of host responses to microbiota. Enteroendocrine cells (EECs) embedded in the mucosal lining of the intestine express both α-SYN and Nox1, and they are synaptically connected to enteric neurons, suggesting that they could be an initial cellular component in α-SYN pathology in the gut. Our preliminary results exhibited that intracolonic injection of rotenone increased intestinal α-SYN aggregates and led to motor deficit accompanied by loss of midbrain dopaminergic neurons, which is largely ameliorated in Nox1 knockout mice. The results imply that Nox1 is a key player in initial α-SYN pathology in the colon induced by either environmental toxin or bacterial LPS. Our central hypothesis is that Nox1-derived ROS contributes to early-stage α-SYN aggregation in the gut caused by fecal microbiota of PD patients and Nox1 inhibition effectively blocks this gut-driven PD pathogenesis.

We will pursue the following three specific aims: Aim 1. Investigate the role of Nox1-derived ROS in α-synuclein aggregation and propagation from enteroendocrine cells to enteric neurons. Aim 2. Investigate whether α-synuclein pathology caused by intracolonic injection of environmental toxins is ameliorated by Nox1 KO in EECs. Aim 3. Investigate whether a Nox1-specific inhibitor, ML171, ameliorates gut-driven α-synuclein pathology in the brain.

Successful completion of the project will decipher the molecular mechanism underpinning how α-SYN aggregation is initiated in the gut and to which extent fecal microbiota of PD patients play roles in this process. Moreover, the proposed study could suggest a specific Nox1 inhibitor as a novel treatment for PD.