Characterisation of the molecular mechanisms of dopaminergic axonal growth and their impairment in Parkinson's disease

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Anantha, Jayanth
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University College Cork
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Parkinson’s disease (PD) is a neurodegenerative disease that affects 6.1 million people globally as of 2016. Axonal degeneration has been identified as the earliest event in the pathogenesis of the disease. For this reason, the application of neurotrophic growth factors for the protection and/or restoration of dopaminergic neurons and their axons has been the subject of intensive research efforts. Glial derived neurotrophic factor (GDNF) was the first of these growth factors to be tested. Despite an extensive body of pre-clinical evidence, clinical trials of GDNF did not meet their primary end points. Thus, the search for new neurotrophic growth factors that can promote the survival of dopaminergic neurons and restore axonal degeneration has remained an ongoing pursuit. Growth differentiation factor (GDF)5 is one such neurotrophic factor under consideration for PD therapy. Therefore, in this body of work, we evaluate the molecular mediators through which GDF5 promotes neurite growth in cellular models of PD. GDF5 is known to mediate its neurotrophic growth factor effects by regulating the BMP-Smad signalling pathway. Therefore, for GDF5 to be a suitable candidate therapy for PD, it is important that the cellular response to GDF5 is unaffected by the disease process. In Chapter 2, we demonstrate that GDF5 signalling is normal in cellular models of PD. Leucine rich repeat kinase (LRRK)2, both wild type and the PD associated G2019S mutant, had no impact on the BMP-Smad mediated transcriptional response thus, confirming the integrity of this pathway in the LRRK2 models of PD. In contrast, α-synuclein impaired the BMP-Smad transcriptional response in cells overexpressing α-synuclein. α-synuclein impaired both BMPR1B- and Smad1- mediated transcription, however, GDF5 and BMP2 treatment restored α-synuclein-mediated impairments in the BMP-Smad transcriptional response. Histone deacetylase (HDAC)5 also regulates the BMP-Smad pathway, and here we demonstrate that HDAC5 nuclear localization or sequestration is central to the regulation of the BMP-Smad pathway. Finally, we demonstrate that GDNF, unlike GDF5, cannot activate its downstream transcriptional response in the presence of α-synuclein. Taken together, these findings suggest that the GDF5-mediated transcriptional response is unimpaired in cellular models of PD. Despite various studies demonstrating the neurite growth promoting effects of GDF5, in vitro and in vivo, its downstream effectors have remained largely unknown. In Chapter 3, we use a proteomics-based approach to identify the global change in proteome and identify upregulated proteins which play a critical role in mediating the axonal and neurite growth effects of GDF5. We identify two proteins called serine threonine receptor associated protein (STRAP) and nucleoside diphosphate kinase (NME)1 as proteins that play a critical role in GDF5-mediated axonal growth in SH-SY5Y cells. Using siRNA-mediated gene silencing, we demonstrated that the expression of both STRAP and NME1 proteins is crucial for GDF5-mediated neurite growth. Furthermore, we report that the overexpression of STRAP or NME1 was sufficient to promote neurite growth in SH-SY5Y cells. Additionally, we also demonstrate that treatment with recombinant NME1 promoted neurite growth in dopaminergic neurons from the embryonic day (E) 14 rat ventral mesencephalon (VM). Finally, we show that the coexpression pattern of the STRAP and NME1, with midbrain dopaminergic neuronal markers was reduced in the PD brain. Since we establish the role of NME1 in promoting neurite growth in neuronal cells and dopaminergic neurons, we next examine the effects of NME1 in cellular models of PD in Chapter 4. We demonstrate that NME1 protects against 6 hydroxydopamine-induced neurite degeneration in SH-SY5Y cells and E14 rat VM dopaminergic neurons. Additionally, we also demonstrate that NME1 prevented α-synuclein-induced reductions in neurite growth in SH-SY5Y cells. This effect of NME1 was confirmed in cultured primary dopaminergic neurons of E14 VM transduced with AAV-α-synuclein viral vectors. NME1 also rescued neurite growth impairment in cells stably expressing the LRRK2 G2019S mutant. Cumulatively, these findings demonstrated that NME1 treatment can protect against neurite growth impairments in multiple models of PD. We also demonstrated that NME1 exerts its neurite growth-promoting effects through the ROR2 and RORα receptors, and the expression of these receptors is critical for the neurite growth promoting effects of NME1. In the human substantia nigra, we found that NME1 is co-expressed with a distinct set of genes that regulate mitochondrial respiration. Therefore, we finally explore the effects of NME1 on mitochondrial respiration in SH-SY5Y cells. We found that NME1 treatment increases maximal respiration and proton leak in SH-SY5Y cells which confirmed that NME1 regulates mitochondrial respiration and promotes neurite growth. In Chapter 5, we explore the immediate effectors of GDF5 on neuronal cells using a proteomics approach. Our proteomics screen revealed that GDF5 upregulated 14 proteins which had significant coexpression pattern with BMPR2 and multiple dopaminergic neuronal markers. The gene ontology analysis on the proteins upregulated by GDF5 revealed their involvement in oxidative phosphorylation and mitochondrial respiratory processes. We next performed a Seahorse mitochondrial respiration assay to study the effect of GDF5 on mitochondrial function in SH-SY5Y cells. This revealed that GDF5 lowered oxygen consumption rates in SH-SY5Y cells and reduced basal and maximal respiration. Additionally, we demonstrate that in cell lines stably expressing LRRK2 wild type or the G2019S mutant, the expression of BMP-Smad signalling proteins was unaltered. We demonstrated that GDF5 promotes neurite growth in these cells and induces a BMP-Smad dependent transcriptional response. In G2019S-LRRK2 cells, GDF5 lowered basal respiration, maximal respiration, and ATP production, thus lowering bioenergetic demand which is a characteristic of differentiated cells. In summary, the work presented in this thesis identifies new downstream effectors and effects of GDF5. We demonstrate that the GDF5-mediated regulation of BMP-Smad pathway is unimpaired in cellular models of PD. Additionally, the results emanating from this body of work also identify NME1 as a new growth factor with potential neuroprotective and restorative properties. Finally, we demonstrated that GDF5 regulates mitochondrial respiration by promoting differentiation in neuronal cells and that GDF5 rescues neurite growth impairments in cells stably expressing G2019S-LRRK2. Therefore, we conclude that GDF5 and NME1 are, and remain, candidate neurotrophic factors for neuroprotection in PD.
Axonal degeneration , Axon , Dopaminergic neurons , GDF5 , NME1 , Neurotrophic growth factor therapy , Parkinson's disease
Anantha, J. 2021. Characterisation of the molecular mechanisms of dopaminergic axonal growth and their impairment in Parkinson's disease. PhD Thesis, University College Cork.
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