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Defining the potential of ZNHIT1, an SNCA co-expressed gene in the substantia nigra, as a therapeutic target for Parkinson’s disease
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Date
2022
Authors
McCarthy, Erin
Journal Title
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Publisher
University College Cork
Published Version
Abstract
Parkinson’s Disease (PD) is synucleinopathy that is characterised by the formation of toxic α-Synuclein (αSyn)-containing Lewy Bodies (LBs) in the midbrain leading to the progressing death of dopaminergic (DAergic) neurons in the substantia nigra (SN). Toxic aggregation of αSyn results in the dysfunction of important neuronal processes, leading to increased neurotoxicity and neurodegeneration. SNCA and its mutant variants have been linked to several cases of familial PD. Given the lack of effective disease-modifying therapies, there is an increasing focus on examining SNCA-induced changes in epigenetic regulation in the hopes of identifying novel targets for gene therapy in PD.
In the first experimental chapter, we used gene co-expression analysis to identify Synuclein Alpha (SNCA) co-expressed genes in the SN, whose co-expression pattern was lost in PD. We identified nuclear zinc finger HIT-type containing 1 (ZNHIT1) as an important interacting partner of SNCA in the SN, and that this co-expression pattern is lost in PD indicating functional dysregulation.. We went on to investigate the functional role of ZNHIT1, which revealed that overexpression of ZNHIT1 promotes neurite growth and prevents αSyn-induced reductions in neurite growth and cell viability in SH-SY5Y cells. Analysis of ZNHIT1 co-expressed genes in the SN revealed a significant enrichment of genes associated with the regulation of mitochondrial function. Bioenergetic state analysis agreed with these findings and revealed that ZNHIT1 overexpression increases ATP synthesis, and rescues αSyn-induced impairments in oxygen consumption rate (OCR), basal respiration, maximal respiration, and spare respiratory capacity. These findings reveal that ZNHIT1 can protect against αSyn-induced neurotoxicity and mitochondrial dysfunction in ZNHIT1-overexpressing cells, this rationalising further investigation into ZNHIT1 as a potential therapeutic target for PD.
In the second experimental chapter, we investigated the role of ZNHIT1 in αSyn-induced neurotoxicity and mitochondrial dysfunction in PD. PD is characterised by impairments in mitochondrial function and reductions in ATP levels. ZNHIT1 overexpression protects against αSyn-induced deficits in mitochondrial function through an upregulation of genes associated with mitochondrial function. Proteomic and bioinformatic analysis revealed that ZNHIT1 interacts with mitochondrial proteins that are significantly enriched in functional categories important for mitochondrial function such as mitochondrial transport, ATP synthesis, and ATP-dependent activity. We also found that ZNHIT1 upregulates and is co-expressed with hub protein HSP90B1, which is known to deter PD progression, thus indicating a neuroprotective role for ZNHIT1-HSP90BI in the SN. Indeed, we show that ZNHIT1 is also co-expressed with DAergic markers TH and ALDH1A1 in control samples, but that this correlation is lost in PD samples. These results indicate functional dysregulation of ZNHIT1 in PD that may result in the misregulation of its mitochondrial interacting proteins in the cytosol, leading to mitochondrial dysfunction and reductions in ATP synthesis that is characteristic of PD, and thus validates our previous findings that highlight ZNHIT1 as a potential target for PD therapy In the third experimental chapter, we investigated the role of ZNHIT1 in BMP-Smad-dependent transcriptional activation in SH-SY5Y cells overexpressing ZNHIT1. Our analysis revealed that ZNHIT1 activates the BMP-Smad pathway, which has been shown to promote DAergic neurite growth and survival and protects them against αSyn-induced neurotoxicity. However, SNCA overexpression was found to inhibit these ZNHIT1-induced increases in BMP-Smad activation. Further investigation revealed that the neuroprotective effects of ZNHIT1 against αSyn-induced cellular and mitochondrial dysfunction, were inhibited by the BMP receptor (BMPR) inhibitors, Dorsomorphin and K02288, indicating that the neuroprotective effects of ZNHIT1 may be dependent on BMP-Smad signalling. We also show that SMAD4 expression in SH-SY5Y cells overexpressing dominant negative SMAD4 was rescued by ZNHIT1 overexpressing. These results support the hypothesis that αSyn in PD inhibits BMP-Smad signalling, which could lead to the inhibition of the growth promoting effects of ZNHIT1, which appear to be mediated by BMP-Smad signalling. Together, these results further highlight the potential role of ZNHIT1 as a therapeutic target for PD.
In the fourth and final experimental chapter, we sought to examine the gene expression changes associated with important signalling networks induced by αSyn overexpression in an in vivo AAV-αSyn rat model of PD, in order to further our understanding of the role of αSyn in PD pathology. Analysis of gene expression changes of 84 genes known to be associated with PD pathology revealed significant reductions in the expression of genes associated with DA synthesis. Further analysis of gene expression changes in the SN induced by αSyn revealed 2,305 differentially expressed genes. The top ranked gene to be overexpressed in this list was Skor1, a known inhibitor of BMP-Smad signalling. Further investigation revealed a significant reduction in constitutively active BMPR1B-stimulated luciferase activity in HEK293T and SH-SY5Y cells overexpressing αSyn. Gene set enrichment analysis (GSEA) revealed that the overexpression of αSyn causes disruptions to cytoskeletal organisation, DNA repair networks and ATP binding, while analysis of cellular bioenergetic states showed reduced ATP synthesis, oxygen consumption rates and basal rates of respiration. This study highlights the role of αSyn as a regulator of mitochondrial function, ATP synthesis and BMP-Smad signalling.
Collectively, the data presented in this thesis rationalises the future development of strategies focused on ZNHIT1 overexpression as a potential neuroprotective strategy for PD.
In this thesis, we show that overexpression of ZNHIT1 is neuroprotective against αSyn-induced neurotoxicity in PD, including reductions in cell viability and growth, as well as mitochondrial dysfunction, ATP synthesis and BMP-Smad signalling. We also show a functional dysregulation of ZNHIT1-SNCA in PD, suggesting that altered expression patterns of ZNHIT1 may play an important role in PD progression. We hypothesis that overexpression of ZNHIT1 in the SN of PD patients may result in neuroprotection against the progression of PD. Our results highlight a potential role for ZNHIT1 in cellular dysfunction that may underlie PD pathology. Collectively the data in this thesis rationalises the future development of strategies focused on ZNHIT1 overexpression as a potential neuroprotective strategy for PD.
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Keywords
Neurodegeneration , Parkinson's disease , Epigenetics , Axon , Dopamine , Mitochondria , SMAD , BMP , HDAC , Histone , Gene ontology
Citation
McCarthy, E. 2022. Defining the potential of ZNHIT1, an SNCA co-expressed gene in the substantia nigra, as a therapeutic target for Parkinson’s disease. PhD Thesis, University College Cork.