Investigating the mechanisms by which PINK1 protects against Parkinson's disease

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dc.check.date2025-01-14T11:41:41Z
dc.check.embargoformatApply the embargo to both hard bound copy and e-thesis (If you have submitted an e-thesis and a hard bound thesis and want to embargo both)en
dc.check.entireThesisEntire Thesis Restricted
dc.check.infoRestricted to everyone for five yearsen
dc.check.opt-outNot applicableen
dc.check.reasonThis thesis is due for publication or the author is actively seeking to publish this materialen
dc.contributor.advisorO'Neill, Coraen
dc.contributor.advisorSullivan, Aideen M.en
dc.contributor.authorFurlong, Rachel
dc.contributor.funderIrish Research Councilen
dc.date.accessioned2020-01-16T11:41:41Z
dc.date.issued2019
dc.date.submitted2019
dc.description.abstractPINK1 (PTEN-induced putative kinase-1) functions as a ubiquitous serine-threonine kinase, with pro-survival, neuroprotective and anti-stress signalling functions. Autosomal recessive mutations in PINK1, resulting in a loss of PINK1 function, cause early onset Parkinson’s disease (PD). This thesis aimed to investigate the mechanisms by which PINK1 protects against PD, with a focus on PI3-kinase/Akt signalling. Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in PD. Akt activation is reduced in the PD brain, and by many PD-causing genes, including PINK1. However, it is not yet known how PINK1 regulates PI3-kinase/Akt signalling. Chapter Two investigated the mechanisms by which PINK1 regulates Akt signalling in PINK1 modified mouse embryonic fibroblasts (MEFs). Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors and enhances Akt activation in normal growth medium. In PINK1 modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to significantly impaired PINK1 kinase-dependent increases in PI(3,4,5)P3 at both the plasma membrane and Golgi. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This demonstrates a new role for PINK1 as an upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD. Despite its discovery nearly two decades ago, there is a lack of cohesive information on PINK1 binding partners. Determining how PINK1 interacts with proteins involved in certain signalling pathways or cellular processes is crucial for understanding how loss of PINK1 function leads to PD. Chapter 3 aimed to assemble a comprehensive list of known PINK1- interacting proteins and to identify new candidates that are bound to PINK1, using bioinformatics analysis of existing data sets coupled with mass spectrometry approaches. This study identified PINK1-interacting proteins that are heavily involved in receptor tyrosine kinase (RTK) signalling pathways, such as PI3-kinase/Akt, VEGF, PDGF, ERRB2/4 and EGF, indicating that PINK1 plays a key functional role through these pathways. Further analysis highlighted two PINK1-interacting proteins (PTEN and IRS4) that are involved in PI3-kinase/Akt signalling and which may mediate PINK1’s regulation of this pathway through lipid binding with PIP3. Using mass spectrometry approaches, several new binding proteins for PINK1 were identified, including RIPK3, calmodulin, EGFR, and interferon regulatory genes, all of which have been implicated in PD and as components of the Akt signalling system. Accumulation of α-synuclein is central to the development of PD, and mutations in SNCA, which encodes α-synuclein, cause PD. There is a need to identify the mechanisms by which α-synuclein overexpression induces neurodegeneration in PD and to determine whether αsynuclein and PINK1 share common mechanisms. This information will be critical for future identification of therapeutic targets. In Chapter 4, we identified significant Golgi fragmentation, mitochondrial fission and reductions in neurite length in cultured ventral midbrain neurons, in response to overexpression of wildtype or mutant α-synuclein, and to deletion of PINK1. Increased Golgi fragmentation and mitochondrial fission induced by the PD risk genes were significantly correlated with reductions in neurite length. Levels of PI(3,4,5)P3 were significantly decreased by overexpression of wildtype or mutant αsynuclein. α-synuclein overexpression in combination with knockdown of PINK1 induced a “dual-hit”, to further reduce neurite length and increase Golgi fragmentation. This provides novel data showing that α-synuclein overexpression and PINK1 deletion converge to induce significant increases in Golgi fragmentation and mitochondrial fission, with concomitant decreases in neurite length and defects in PI3-kinase/Akt signalling. This thesis therefore demonstrates that studies on the function of PD-causing genes such as PINK1 and α-synuclein enable the discovery of molecular pathways that may be involved in the pathogenesis of PD and could be targets for future therapies.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Version
dc.format.mimetypeapplication/pdfen
dc.identifier.citationFurlong, R. 2019. Investigating the mechanisms by which PINK1 protects against Parkinson's disease. PhD Thesis, University College Cork.en
dc.identifier.endpage218en
dc.identifier.urihttps://hdl.handle.net/10468/9523
dc.language.isoenen
dc.publisherUniversity College Corken
dc.relation.projectIrish Research Council (Government of Ireland Postgraduate Scholarship)en
dc.rights© 2019, Rachel Furlong.en
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/en
dc.subjectPINK1en
dc.subjectAkten
dc.subjectParkinson's diseaseen
dc.subjectPIP3en
dc.subjectAlpha-synucleinen
dc.thesis.opt-outfalse
dc.titleInvestigating the mechanisms by which PINK1 protects against Parkinson's diseaseen
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhDen
ucc.workflow.supervisorc.oneill@ucc.ie
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