Gregori, Martina;
(2023)
Dysregulated Ca2+ Signalling in LRRK2 Parkinson's.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Disrupted cellular calcium (Ca2+) signalling is believed to play an important role in a number of neurodegenerative diseases, including Parkinson’s disease (PD). Mutations in the leucine rich repeat kinase (LRRK2) gene are the most common genetic cause of familial PD and are also found in a subset of sporadic cases; however, the precise biological functions of LRRK2 involved in PD pathogenesis remain unclear. Here I first investigate the impact of mutant LRRK2 on Ca2+ signalling in neuronal-like cells. Single-cell Ca2+ imaging was performed on SH-SY5Y cells stably transfected with various LRRK2 constructs. I observed increased depolarisation-induced Ca2+ signals in mutant LRRK2-expressing cells [G2019S (GS) and R1441G/C (RG/C)] compared to cells expressing wild-type LRRK2. These changes are likely due to LRRK2s’ kinase activity, as they were absent when a kinase-dead LRRK2 mutant was expressed. Importantly, I found that compromising lysosomal integrity using different pharmacological interventions corrected the Ca2+ signalling defect in GS LRRK2 cells, whereas ER Ca2+ store depletion had no effect. I also showed that pharmacological and molecular inhibition of the lysosomal two pore channel 2 (TPC2) reverses the Ca2+ defects in GS LRRK2 cells, implicating the cation channel in pathology. Intriguingly, five distinct methods known to boost lysosomal exocytosis also improved the GS LRRK2 Ca2+ signalling phenotype. To determine the in vivo consequences of TPC2 gain-of-function, I leverage the natural absence of TPC2 in Drosophila melanogaster. By transiently expressing human TPC2 in the Drosophila S2R+ cell line, I found that the heterologous cation channel is correctly trafficked and functional in the fruit fly system. Furthermore, I recorded spontaneous Ca2+ signals in dopaminergic neurons using two-photon microscopy of ex vivo Drosophila larval brains. Collectively, these results uncover a novel interplay between LRRK2, voltage-gated Ca2+ channels and TPC2, and introduce a new in vivo model for studying TPC2 in the context of LRRK2-PD.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Dysregulated Ca2+ Signalling in LRRK2 Parkinson's |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10168758 |
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