Edey, Jade
(2023)
Investigating the SUMOylation and transcriptional regulation of Nurr1 and the splice variant Nurr-1a.
PhD thesis, University of Nottingham.
Abstract
Nuclear receptor-related 1 protein (also known as Nurr1 or NR4A2) is an orphan nuclear receptor with multiple roles in many different tissues and pathological settings. It is most commonly known for its vital role in the development, maintenance and survival of dopaminergic (DA) neurons throughout the midbrain, particularly in the substantia nigra (Jankovic et al., 2005). Nurr1 also has a reported role in inflammatory cells such as microglia and macrophages, where it is suggested to dampen down the inflammatory response to prevent excessive inflammation (Lallier et al., 2016, Smith et al., 2015). This reduction in inflammation, thought to be preceded by Nurr1 LBD SUMOylation, is believed to protect surrounding cells such as DA neurons from potentially damaging inflammatory mediators (Saijo et al., 2009). Nurr1 has many reported splice variants found in the human brain. However one of particular interest, termed Nurr-1a, is reported to have truncated LBD whilst retaining an intact AF-1; previously described as the key region for transcriptional regulation in NR42 nuclear receptors (Michelhaugh et al., 2005, Ichinose et al., 1999, Maxwell and Muscat, 2006). Albeit the function of Nurr-1a remains to be investigated. Fully understanding the mechanisms in which Nurr1 regulates the expression of key neuronal and inflammatory genes, and any role Nurr-1a may or may not have in these processes, will help to clarify, or reveal targets for therapeutic manipulation in diseases with a neuroinflammatory component such as Parkinson’s Disease. Therefore, this work aimed to clarify the mechanisms surrounding Nurr1 and Nurr-1a SUMOylation, and the effect this may have on gene transcription. Whilst also focusing on the development of human based neuronal and inflammatory models which can better recapitulate diseases with a neuroinflammatory factor.
To investigate the SUMOylation of Nurr1 and Nurr-1a initial experiments employed a SUMO-pulldown assay to enrich conjugates overexpressed in HeLa cells followed by western blotting. In combination with site directed mutagenesis of a predicted SUMO site, this data revealed Nurr-1a is SUMOylated significantly more than Nurr1, occurring in both receptors at a site dissimilar from the LBD which had been previously described. In addition, the E3 ligase PIASg, previously reported as a key interaction partner of Nurr1, was also found to be the E3 ligase responsible for Nurr-1a SUMOylation and exhibited important activity as a stabilising factor independent of its E3 ligase role. The transcriptional activity of Nurr1 and Nurr-1a was investigated in the neuronal cell line SH-SY5Y using a TH promoter driven luciferase reporter. In accordance with previous reports, Nurr-1a was found to be less transcriptionally active than Nurr1. However, the transcriptional activity of both receptors was significantly enhanced by the presence of SUMO. This was further validated through the use of SUMO site mutants. Furthermore, ligands were unable to majorly affect the SUMOylation of either receptor and had little effect on the transcriptional activity. Highlighting transcriptional activity is largely dependent on cell type (HEK293 vs SH-SY5Y) and whether a natural TH or synthetic 3XNBRE promoter driven reporter was used.
Human based models were developed so that Nurr1 and Nurr-1a transcriptional regulation of key genes could be investigated in setting more representative of human physiology than cell lines and overexpressed rodent genes. Human iPSC’s were differentiated into DA neurons using a polycistronic vector which overexpressed Nurr1 ,in addition to two other key transcriptional factors, and validated through ICC. Blood derived monocytes were differentiated into macrophages and microglial-like cells which were characterised though RT-qPCR, ICC and functional assays. Macrophages were subsequently used to investigate the role of Nurr1 in immune tolerance; the reduced responsiveness of immune response after repeated TLR activation. Identifying Nurr1 is superinduced during tolerance and may therefore have an important function dampening down the production of inflammatory mediators during this state.
Overall, these data reveal Nurr-1a partakes in many of the same mechanisms reported for Nurr1. Albeit a lesser ability to transcriptionally activate genes, it appears Nurr-1a is capable of a greater and more versatile level of SUMOylation. Which may suggest a more prolific role in the inflammatory transrepression mechanism previously reported. Although the SUMOylation in this mechanism was previously reported to occur within the LBD, this study demonstrates SUMOylation occurs within the AF-1 domain of both Nurr1 and Nurr-1a. Highlighting AF-1 as a key region of transcriptional regulation, aligning with other members of the NR4A family. SUMOylation may contribute to the fine tuning of transcriptional regulation by Nurr1 and Nurr-1a, exemplified by its ability to enhance transcriptional activation in neuronal cells whilst being reported to feature in the transrepression of inflammatory genes in microglia and macrophages. The successful generation of DA neurons through the transduction of iPSC’s with Nurr1 amongst two other TF’s again highlights the importance of Nurr1 in DA neuronal development and provides a framework model to identify the role of Nurr-1a, if any, in this process. Lastly the exploration of Nurr1 and inflammatory gene expression during immune tolerance may suggest a novel function for Nurr1 in regulating this protective process. Immune tolerance may have a role in PD and could be abrogated due to increases in IFNy which has been reported in PD patients. Therefore, fully understanding what happens to Nurr1 in this process is important if it is to be considered a therapeutic target.
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