Yates, Elizabeth Lucy; (2017) Lysosomal Ca2+ Signalling and Neurodegeneration: A Global View. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Dysfunction of the lysosomal Ca2+ channels TRPML1 and TPC2 has been implicated in neurodegenerative disease. However, there is little information about the involvement of these channels in cell-wide global Ca2+ signalling and it is unknown whether their dysfunction contributes to neurodegeneration by disturbing it. First, by using synthetic compounds, I demonstrate that TRPML1 activation causes global Ca2+ signals. In contrast with the predominant lysosomal localisation of the channel these Ca2+ signals comprised a small lysosomal contribution and a large Ca2+ entry component. Examination of TRPML1-mediated Fe2+ entry posed the possibility that divalent cation entry can occur directly through TRPML1 on the plasma membrane. Second, I identified enlarged and clustered lysosomes in fibroblasts derived from people with sporadic Parkinson’s disease (PD). This was appropriately quantified from microscopy images by creating an automated sequence of image processing functions. By inhibiting TPC expression in fibroblasts I demonstrated their involvement in the propagation of physiological global Ca2+ signals evoked by bradykinin. In sporadic and familial PD patient fibroblasts these TPCdependent Ca2+ signals were subtly modulated. Finally, in a neuronal cell line, reduced TPC expression inhibited the propagation of physiological global Ca2+ signals evoked by carbachol. These Ca2+ signals were also blocked by a recently identified TPC blocker and by putative TPC blockers that were screened by collaborators. In cells expressing the PD-associated mutant, LRRK2 G2019S, these TPC-dependent Ca2+ signals were potentiated. In contrast, bradykinin-evoked Ca2+ signals in this neuronal cell line were not inhibited by TPC blockers, nor were they potentiated in the LRRK2 G2019S cells. Therefore, physiological global Ca2+ signalling in PD may be perturbed by TPC dysfunction, and be a compounding factor in neurodegeneration. Collectively this research suggests that lysosomal Ca2+ signalling through TRPML1 and TPCs plays a role in global Ca2+ signalling and that this may be disturbed in neurodegenerative disease.

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