Yoon-Seong Kim, Ph.D. - US grants

DESCRIPTION (provided by applicant): Oxidative stress is a major contributing factor in the pathogenesis of Parkinson's disease (PD). Mitochondria have long been implicated as the culprit for oxidative stress in PD. However, molecular sources for reactive oxygen species (ROS) in PD have not been clearly elucidated. A family of NADPH oxidase (NOX) is the first enzyme complex discovered which is specialized to generate superoxide. Here, we first demonstrate that the expression of Nox1, a Nox homologue, is increased in DA cells by oxidative stress such as 6-OHDA both in vivo and in vitro. Rac1, a key component of the Nox1 system, is also activated. Nox1 expression is increased in DA neurons of postmortem human brains from sporadic PD patients. Mutations in Leucine-rich-repeat-kinase 2 (LRRK2), the newly identified causative gene for PD also increase Nox1 expression and ROS generation in DA cells. Interestingly, Nox1 induction is affected by mitochondrial respiratory chain inhibitors, suggesting the interplay between mitochondrial dysfunction and Nox1 activation. Recent studies also suggest a prominent role of mitochondrial dysfunction in Nox1-mediated superoxide generation. Finally, inhibition of Nox1-mediated superoxide generation protects substantia nigra DA neurons from 6-OHDA- induced degeneration. These proposed studies will investigate 1) whether mitochondrial ROS plays a key role in Nox1 induction and activation, 2) the role of the Nox1/Rac1 system in degeneration of the nigrostriatal pathway by inhibition or activation of this system using AAV2-mediated gene transduction, and 3) whether LRRK2 mutations affect the activation of Nox1-mediated ROS production and consequential DA neurodegeneration. PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction and oxidative stress are strongly implicated in the pathogenesis of PD. The overall goal of this proposed study is to define the role of NADPH oxidase 1 and Rac1, the specialized superoxide generation system in degeneration of the dopaminergic nigrostriatal pathway. We will investigate whether mitochondria play a key role in the Nox1 induction and the intervention of the Nox system prevents DA neurodegeneration. Additionally, the study on the interaction between LRRK2 mutations and the Nox1/Rac1 activation may identify common molecular pathways involved in the pathogenesis of PD. Collectively, these results will help us to understand cellular mechanism governing the vulnerability of the DA nigrostriatal pathway to oxidative stress and lead to the development of novel therapeutic target.

DESCRIPTION (provided by applicant): The presence of alpha-synuclein (alpha-SYN) aggregate in Lewy neurites (LNs) and Lewy bodies (LBs), the pathological hallmarks of Parkinson's disease (PD), and mitochondrial dysfunction are two central components in PD pathogenesis. Known physiological roles of alpha-SYN are limited in synaptic terminals, raising the possibility that alpha-SYN mRNA is transported to neurites and its translation is locally controlled. In fact, we have recently found that alpha-SYN mRNA is translocated into neurites of fully differentiated human dopaminergic neuronal cells. We also identified that Pum2, a paralogue of mammalian PUF family RNA binding protein, binds to a PUM binding motif of the alpha-SYN 3'UTR and is responsible for neuritic localization as well as translational repression of alpha-SYN mRNA. Mitochondrial ROS generated by respiratory complex I inhibition led to local translation of alpha-SYN from mRNA associated with mitochondria, suggesting that Pum2 might transmit mitochondrial signal to alpha-SYN translation. Although it is obvious that there is an inter- relationship between mitochondria and alpha-SYN underlying the development of disease, molecular mechanisms governing this cross-talk remain elusive. Our central hypothesis is that Pum2 is responsible for alpha-SYN mRNA transport to the juxta-mitochondrial compartment, alpha-SYN translational regulation and aggregation in neurites. The objectives of this project are to understand the interplay between alpha-SYN and mitochondria in neurites through Pum2-mediated translational regulation of mitochondria-associated alpha-SYN mRNA. To achieve these objectives we propose the following three specific aims: Aim 1. Examine the role of Pum2 in mitochondrial localization of alpha-SYN mRNA. Aim 2. Examine how mitochondrial ROS control alpha-SYN translation. Aim 3. Determine how newly synthesized alpha-SYN near mitochondria affects mitochondrial fragmentation and alpha-SYN aggregation in neurites. The successful completion of the project will bring a paradigm shift in our understanding of molecular mechanisms that control alpha-SYN level in neurites and how mitochondrial ROS contribute to this alpha-SYN regulation. The results expected from the project will open new avenues to understand 1) 3'UTR-dependent posttranscriptional regulation of alpha-SYN in neurites and Pum2's role in this process, 2) intimate crosstalk between alpha-SYN and mitochondria in neurites and 3) its contribution to mitochondrial fragmentation and LN formation.

PROJECT SUMMARY 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.

Alpha-synuclein (?-SYN) and mitochondrial dysfunction are two central components in Parkinson's disease (PD) pathogenesis. Mitochondrial dysfunction is a common feature of the many iterations of PD pathogenesis and ?-SYN toxicity seems to affect mitochondria most significantly. Complex interplay between ?-SYN and mitochondria has been widely observed. While the intricate crosstalk between mitochondria and ?-SYN is poorly understood, our preliminary studies suggest that the 3'-untranslated region (3'-UTR) of ?-SYN mRNA plays a key role in translational regulation of ?-SYN near mitochondria. Our preliminary findings demonstrate that 1) ?-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 ?-SYN transcript; 3) interestingly, mitochondrial Pum2 levels in post-mortem PD brain were significantly lower compared to control subjects, while ?-SYN levels were opposite, implying Pum2?s repressive role on ?-SYN near mitochondria. In addition, recent studies showing the association of single nucleotide polymorphisms in the ?-SYN 3'-UTR with PD strongly suggest that 3`-UTR-mediated regulation of ?-SYN could become a critical player in PD pathogenesis. Our central hypothesis is that deregulation of Pum2-mediated ?-SYN translational repression on the outer surface of mitochondria contributes to mitochondrial dysfunction observed in PD. The following three specific aims will be pursued: In Aim 1, both the cis-regulatory elements and the trans- factors responsible for mitochondrial localization of ?-SYN will be identified. In Aim 2, it will be determined how mitochondrial ROS controls Pum2-mediated translation of ?-SYN mRNA and the roles of newly synthesized ?- SYN. In Aim 3, it will be investigated whether PD-associated SNPs in the 3'-UTR of ?-SYN cause changes in Pum2 binding, translocation of the protein to mitochondria, and mitochondrial functions. The successful completion of this project could create a paradigm shift in our understanding of molecular mechanisms that control ?-SYN expression near mitochondria in PD pathogenesis by elucidating the role of Pum2 and the 3'-UTR of ?-SYN in translational regulation

PROJECT SUMMARY 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.