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  • br Conclusion The development discovery


    Conclusion The development/discovery of compounds targeting small GTPases is challenging [43,44]. Our data point to RBC8 being efficient and potent as a Ral inhibitor in human and mouse platelets, but that it exhibits some activity beyond just Rals, particularly in mouse platelets. It is however possible that species differences in Ral function and structure could partly explain our observations in human platelets, in which wider functions for Rals may be present than in mouse platelets. For functional assessment of Rals in tissues it is advisable therefore to use a combination of genetic and pharmacological approaches and to be aware of possible species differences.
    Conflict of interest
    Acknowledgements We would like to thank Elizabeth Aitken and David Phillips for technical assistance and maintaining the RalAB mouse colony. We would also like to thank Prof. Theodorescu for generously providing the RBC8 compound. Finally, we want to thank the British Heart Foundation for funding this research in grants awarded to A.W·P (FS/14/23/30756, FS/16/66/32520 and RG/15/16/31758).
    Introduction Membrane trafficking plays an essential role in maintaining cellular homeostasis. As such, trafficking is thought to be particularly critical in human diseases such as neurodegenerative disorders. Currently, vesicle-mediated transport has emerged as a key pathway altered in different types of neurodegenerative diseases [1]. In particular, Parkinson’s disease (PD) has been suggested to arise from defects in membrane trafficking using human genetic data [2,3], suggesting that there is a causal relationship between altered membrane trafficking and PD pathogenesis. Specifically, mutations in PINK1, VPS13C, VPS35, DNAJC6, DNAJC13, PRKN, FBXO7, ATP13A2, or PLA2G6 have been discovered in familial cases of PD (for a review [4]) and linked to various aspects of trafficking. In addition, genome wide association studies (GWAS) in sporadic PD have identified risk variants present in loci that include several membrane trafficking salubrinal such as TMEM175, RAB7L1, GAK, CHMP2B, CTSB, GALC, VPS13C, SH3GL2, SYT4, ATP6V0A1 or VAMP4 [5,6,]. Additionally previous work proposed a functional connection between Rab GTPases and PD [8,9].
    Rab35 as a link between LRRK2 and α-synuclein Several independent sources of data suggest that Rab35 may be important in PD pathogenesis. For example, Rab35 protein levels are increased in the serum of PD patients compared to matched controls and patients with other parkinsonism disorders and Rab35 serum levels significantly correlate with age-at-onset of the disease [45]. Rab35 is also one of the potential LRRK2 Rab substrates. Mutations in the LRRK2 phosphorylation site for multiple Rabs cause neurotoxicity in primary neurons that is especially severe in the Rab35 phospho-mutants (both phosphomimetic and phospho-null) [46]. Additionally, overexpression of Rab35 phospho-mutants in the murine substantia nigra by viral delivery causes neurodegeneration. One proposed mechanism for PD progression in the brain is the propagation of α-synuclein aggregates [47], and LRRK2 pathogenic mutations have been proposed to increase α-synuclein fibrils propagation in primary neurons [48]. Recently, it has been suggested that LRRK2-mediated α-synuclein propagation occurs through Rab35 phosphorylation at its T72 site []. A proximity labelling screening for potential α-synuclein interactors in rat cortical neurons showed that native α-synuclein is likely to interact with Rab35, among other Rab GTPases (Rab3A, Rab3B, Rab3C, Rab4B, Rab6A, Rab8A, and Rab15) [50]. Even though the molecular mechanism of how phosphorylated Rab35 is able to propagate α-synuclein aggregates remains unclear, it is known that endocytosed α-synuclein is degraded in the lysosome after transfer through the endosomal pathway and that ESCRT-III depletion blocks α-synuclein degradation and increases its release from the cell [51]. ESCRT-III promotes the biogenesis of multivesicular bodies (MVB), and Rab35 stimulates the release of MVB material to the extracellular space [52,53]. It is, therefore, possible that phosphorylated Rab35 impairs α-synuclein clearance by promoting its release through exosomes thus enhancing α-synuclein propagation (Figure 3, Table 1).