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 PD 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. 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 transcriptional mutagenesis. We recently found that oxidative stress or Ogg1 knockdown increase transcriptional mutagenesis of ?-SYN, leading to protein ag- gregation. Moreover using a novel technique, RNase H2-dependent PCR, we were able to identify various TM- generated ?-SYN mutants including S42Y and A53E from human PD brain samples. We have also found S42Y- positive Lewy bodies from postmortem brain samples of PD and dementia with Lewy bodies (DLB) using highly specific anti-S42Y antibody. Together, our preliminary results strongly suggest that transcriptional mutagenesis contributes to generation of novel pathogenic species of ?-SYN in 8-oxodG accumulation conditions such as Parkinson's disease and other synucleinopathy. Currently, there are major gaps in knowledge regarding the mechanism by which these mutant species may affect ?-SYN pathology and if ?-SYN aggregates in LBs contain mutant proteins produced by transcriptional mutagenesis. Our central hypothesis is that 8-oxodG-mediated transcriptional mutagenesis event leads to the generation of novel mutant variants of ?-SYN which causes nucleation-dependent aggregation and toxicity as seen in PD. The objective here is to identify oxidative stress-derived TM mutant species of ?-SYN and investigate their contribution to ?-SYN aggregation and the pathogenesis of PD. The following three specific aims will be pursued:
In Aim 1, levels of 8-oxodG and the entire profile of TM- derived mutant variants of ?-SYN mRNA in human postmortem brain samples of PD and control will be meas- ured.
In Aim 2, the role of TM-generated ?-SYN mutants in nucleation-dependent aggregation process will be investigated and ?-SYN TM mutant proteins will be detected in human postmortem brain samples.
In Aim 3, the collective effect of TM-generated mutants on ?-SYN aggregation, toxicity, and neuron-to-neuron transmission will be assessed. Successful completion of the project will create a paradigm shift in our understanding of the molecular mech- anisms underlying oxidative stress-mediated ?-SYN pathology in PD. Knowledge of TM events in ?-SYN might be equally important to understand other molecules, such as A? and tau in other neurodegenerative conditions.
?-synucleinopathy and oxidative insults have long been implicated in the pathogenesis of Parkinson's disease but their pathogenic interaction mechanism is still not clear. The proposed project is to elucidate a novel molecular mechanism in which oxidative damaged DNA lesions generate mutant ?-synuclein species via transcriptional mutagenesis, contributing to ?-synuclein aggregation and toxicity. The present research proposal is particularly relevant to public health since successful accomplishment of the project will help to develop new therapeutic strategies to deal not only with the exponentially increasing population of Parkinson's disease in the United States (60,000 new cases/year) but also other synucleinopathy such as dementia with Lewy bodies and multiple system atrophy. The new knowledge that will be acquired from the project will move the PD into new avenues of research and may lead to novel therapeutic approaches to combat onset and progression of PD and other synucleinopathy.
