The cellular quality-control process of mitophagy, entailing selective degradation of defective mitochondria (following damage to their DNA) by the process of autophagy, has attracted considerable research interest. In particular, mitochondrial deficits and specifically reduced mitophagy are evident in both sporadic and familial Parkinson’s disease (PD), rendering aberrant mitophagy an important therapeutic target for preventing neuronal cell death and formation of ‘Lewy Bodies’, abnormal aggregations of protein e.g. α-synuclein that form inside neurons affected by PD. Studies showed mitophagy proteins interact with α-synuclein, to further strengthen links between abnormal mitophagy and PD‘s underlying pathology. However, limited availability of suitable chemical probes has restricted our understanding of the molecular mechanisms involved in mitophagy in healthy brains and when affected by PD. Although mitophagy can be initiated through acute dissipation of the mitochondrial membrane potential (ΔΨm) using mitochondrial uncouplers and drugs such as oligomycin to impair respiration, both approaches impair mitochondrial homeostasis, limiting the scope for dissecting out subtle, bioenergy-related regulatory phenomena. Small molecule pharmacological tools offer considerable value for dissecting complex biological processes and identifying potential therapeutic interventions. Recently, novel mitophagy activators acting independently of the respiration collapse have been reported, offering new opportunities to understand the process and potential for therapeutic exploitation. This project will use several cellular and biochemical methods to probe the effects and molecular mechanisms of novel mitophagy modulators in cell models of PD. Our lab recently optimised neuronal cell lines that are either pheno-chemotypically ‘dopaminergic’ (producing and releasing the neurotransmitter, dopamine) or ‘cholinergic’ (producing and releasing the neurotransmitter, acetylcholine), with both neuronal types that degenerate and eventually die during PD. Intriguingly, our lab’s published work showed that the mitochondrial DNA profile of these neuronal types differ when affected by PD (Bury et al. 2017; 82:1016-21), suggesting that dopaminergic versus cholinergic neurons’ mitophagy-related homeostatic mechanisms (which are regulated by specific nuclear-encoded genes) differ in response to PD. This also suggests that mitophagy-regulating drugs might exert different effects when applied to PD-affected dopaminergic versus cholinergic neurons, whilst aspects such as adjusting the dose to the neuronal type, should also be considered. A therapeutic screen of the modulators will be performed in dopaminergic versus cholinergic cell lines, against various PD-relevant mitochondrial toxins. Output variables will include cell survival and -viability and ΔΨm, indicating mitochondrial activity, since it reflects electron transport and oxidative phosphorylation (OXPHOS), which generates ATP, the cell‘s major energy currency. Also, we will conduct high-resolution respirometry measures of the cells to decipher if and to what degree the mitophagy regulars may improve OXPHOS and/or reduce oxidative stress. Mitophagy flux will be quantified using a flow cytometry-based approach, using the mitochondria-selective probe, MitoTracker Deep Red (Mauro-Lizcano et al. 2015; Autophagy, 11:833-43). In addition, the ability of the modulators to reduce aggregated α-synuclein will be assessed; all measures will be performed comparatively, to determine for a neuronal-type specific profile. The current work holds immense potential for understanding the underlying defective mitophagy seen in PD, and will allow for a major leap in our understanding as to how such critical cell-regulatory processes might differ between vulnerable neuronal types in PD brains. The work will also pave the way for identifying and characterising small molecules capable of improving defective mitophagy in PD-affected brains, which will allow for next step testing in animal models of PD as well as clinical studies.