II. Impact of our study on injury-‐induced ROS signaling in Hydra
5. Conclusions and Perspectives
Work presented in this doctoral thesis can be summarized:
A. Injury in Hydra produces ROS, from which two types were explored.
B. Mitochondrial superoxide acts in the cell-‐autonomous way, orchestrating wound healing possibly through Rho1/ROCK/F-‐Actin pathway. This molecule is produced after any kind of injury, but symmetrically after the bisection in AR and BR.
C. Hydrogen peroxide, a derivative of cellular superoxide is a putative paracrine signal that is responsible for apical regeneration. This molecule is unique for the regeneration process and is produced asymmetrically after the cut.
D. MAPK/ERK/RSK pathway is activated asymmetrically in AR and is dependent on hydrogen peroxide production. Hydrogen peroxide is also crucial for the phosphorylation of CREB and injury-‐induced cell death during the AR.
E. There are several candidates for putative redox sensor proteins in Hydra. For now, Nrf-‐l shows that in vitro can act as a protein that initiates an antioxidative response by binding to ARE.
F. Hydra serves as a good model to study in vivo various biological processes in the adult with the use of genetically encoded biosensors.
In the future of this project, we are going to focus to further characterize the targets of both mitoO2.-‐, and H2O2 during the regeneration. Our best candidate as a target of mitoO2.-‐, is Rho1/ROCK/F-‐Actin pathway. We can dissect this pathway either with the RNAi approach or with the pharmacological inhibitors, such as Y27632, an inhibitor of ROCK phosphorylation.
Confirming that H2O2 is directly inducing the cell death of i-‐cells and then in turn gets amplified is crucial for this work. Biggest flaw of this research is the lack of ways to specifically image H2O2. Even though effort to obtain HyPer transgenic lines was not fruitful, there are another options. One of them is roGFP-‐orp1, a redox sensitive GFP protein that is used for H2O2 quantification. With this tool, first thing to explore would be a spatiotemporal distribution of H2O2 during regeneration. First, does any part of the cell that is committed to regenerate shows higher levels of H O than the other
parts? We could look if H2O2 is accumulating during the earliest time points at the plasma membrane, which would suggest that NOX enzymes are involved in producing the paracrine H2O2, as we hypothesize at the moment. Second, we could specifically pinpoint at what time the amplification occurs, and if the cell death is following it, or it occurs in the same time. Third, we could also examine the cells that are committed to die, i-‐cells and its progenitors. What is the level of H2O2, and if they accumulate it, is it transient or sustained during the early phases of regeneration?
MAPK/ERK activation plays an important role in the apical regeneration in Hydra.
Further way to examine it in the presence/absence of ROS is as well with the existing genetically encoded biosensors. This can be done with the FRET imaging of the reversible ERK reporter mRFP-‐cdc25C-‐eGFP (Harvey et al., 2008) or using the similar variation of this tool (Burack and Shaw, 2005; de la Cova et al., 2017). This could further elucidate the function of H2O2 by following the activity of its putative target:
MAPK/ERK.
We have shown that Hydra Nrf-‐l performs as an oxidative sensor in mammalian cell culture. Our next step is to confirm these preliminary data with the functional assays in vivo. We plan to test this in several contexts. The impact of Nrf-‐l in Hydra can be dissected with monitoring the wound healing, apical regeneration, cell death and ROS production in the Nrf-‐l RNAi animals. Another read-‐out of the Nrf-‐l on the molecular level could be the level of p62 protein. For this, we will utilize our custom made antibody that was developed for the running ageing project of the lb.
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