mct2 inhibitor The contribution of LRRK to

The contribution of LRRK2 to PD pathology has yet to be fully elucidated. LRRK2 has been proposed to act upstream of SNCA and tau to promote aggregation, but it may also act independently to promote neuron loss in the absence of aggregate pathology (Taymans and Cookson, 2010). Additionally, in some experimental cell culture systems, LRRK2 G2019S mutation has been shown to generate numerous protein mct2 inhibitor (Greggio et al., 2006), whereas in others it does not (Kett et al., 2012). Together, these data indicate a need for better understanding of cell-type-specific LRRK2 functions. Our data also suggest that the aggregates are distinct from traditional Lewy bodies and form independent of SNCA expression levels. The lack of aggregate formation in SNCA (3×) iPSC sensory neurons may be further indication of divergent LRRK2 and SNCA pathways leading to cellular dysfunction in PD. The low level of SNCA-positive aggregations in the LRRK2 iPSCs is somewhat surprising as many LRRK2 cases show SNCA Lewy body pathology (Giasson and Van Deerlin, 2008; Hasegawa et al., 2009). However, some LRRK2 PD patients do not develop Lewy body pathology (Cookson et al., 2008), and a number of experimental models of LRRK2-associated PD exhibit protein aggregations and/or inclusions that do not associate with SNCA (Waxman et al., 2009; Tsika et al., 2015). Differences in experimental models, such as species, neuron subtype, LRRK2 expression levels, and antibody epitopes and/or specificity may contribute to variations in results (Zhu et al., 2006; Melrose et al., 2007). Nevertheless, these data all indicate mutant LRRK2 can disrupt neurite integrity in multiple neuronal systems and may promote neuron dysfunction and death. Specifically, how LRRK2 is affecting microtubule stability in sensory neurons needs further investigation, but the robust aggregate phenotype found in LRRK2 G2019S iPSC neurons will be a valuable system to address this. Using live-cell calcium imaging analysis, we found that LRRK2 G2019S iPSC-derived sensory neurons display diminished calcium responses to KCl depolarization. As calcium is critical for proper neuronal signaling and function, any perturbation could be detrimental. Dysregulated calcium is evident in mutant LRRK2 neurons. For example, others showed that mutant LRRK2-expressing mouse cortical neurons had reduced calcium recovery and efflux (Cherra et al., 2013). Altered calcium levels can lead to aberrations in lysosomal clearance, followed by an increase in proteostatic stress. In this regard, Gómez-Suaga et al. (2012) showed that overexpression of wild-type or G2019S LRRK2 caused an increase in autophagosomes through calcium-dependent activation of the CaMKK/AMPK pathway, which could be inhibited by calcium chelation. Autophagosome accumulation in the LRRK2 G2019S iPSC-derived sensory neurons further indicates impairment of the autophagy-lysosome system in LRRK2-mediated PD. However, the neuron-subtype-specific consequences of calcium dysregulation and subsequent altered autophagy signaling remain to be determined in relation to pathological mechanisms in PD. Finally, our findings further support the idea that kinase activity is playing a role in LRRK2-G2019S-induced neuronal dysfunction. Inhibition of LRRK2 kinase activity using LRRK2-IN-1, GSK2578215A, or CZC25146 resulted in partial but significant functional and morphological rescue in homozygous and heterozygous LRRK2 G2019S iPSC-derived sensory neurons. Individual kinase inhibitors show differing efficacy in the parameters tested, but treatment with GSK2578215A most consistently resulted in significant aggregate reduction and calcium signaling improvement in both the homozygous and heterozygous contexts. However, neither outcome measure was rescued to control levels, nor were levels of autophagosome markers improved. This could be due to suboptimal inhibitor dosages or treatment paradigms, but it is also possible that other functional domains of LRRK2 contribute to sensory neuron dysfunction. For example, mutations in the GTPase domain result in neurite aggregations in transgenic mice (Li et al., 2009b). GTPase activity has been shown to modulate kinase activity, and it has been proposed that the GTPase and kinase domains may reciprocally regulate each other to direct the function of LRRK2 (Biosa et al., 2013), thereby necessitating further consideration of the potential pathogenic interplay between LRRK2 functional domains.