sirtuin We show that the GCase defect was

We show that the GCase defect was associated with impaired autophagosome formation in the N370S GBA1 mutation dopaminergic haNCSCs, in agreement with Schondorf et al. (2014). We have shown that ambroxol rescues the biochemical abnormalities resulting from the N370S mutation. Ambroxol increased GCase activity in GD and PD fibroblasts and reduced α-synuclein levels in SHSY-5Y sirtuin (McNeill et al., 2014). Ambroxol has also been shown to activate transcription factor EB and therefore to induce transcriptional upregulation of the CLEAR (coordinated lysosomal expression and regulation) network and saposin C levels (Ambrosi et al., 2015; McNeill et al., 2014). In the dopaminergic neuronal model derived from haNCSCs, ambroxol significantly increased GCase protein in the idiopathic PD and N370S GBA1 mutant lines. There were parallel increases in activity in the N370S and iPD lines, with a trend in the controls that did not reach significance. We also found that ambroxol can significantly increase mature cathepsin D protein in N370S GBA1 mutant lines. This may be a consequence of the transcriptional effects of ambroxol and may contribute to the mechanisms by which this drug reduces α-synuclein levels. An alternative or additional mechanism for the reduction in α-synuclein may be an increase in cathepsin D due to elevated GCase and ceramide levels induced by ambroxol. Autophagy is considered to be a dynamic process comprising three sequential steps: formation of autophagosomes, the fusion of autophagosomes with lysosomes, and degradation. During this process, cytosolic form of LC3 is conjugated with phosphatidylethanolamine to form LC3-II, which is recruited to autophagosome membranes. p62 is a ubiquitin-binding scaffold protein acting as a cargo receptor, which is recruited in the formation of autophagosomes (Itakura and Mizushima, 2011). Ambroxol treatment significantly increased the steady-state levels of both LC3-II and p62 proteins in our model, suggesting that ambroxol increases autophagy by promoting autophagosome formation and turnover. This interpretation is further supported by a substantial increase in p62 mRNA. The unaltered LC3B gene expression suggests that the influence of ambroxol on LC3 synthesis is minimal. The autophagic pathway has been increasingly implicated in a number of neurodegenerative diseases including PD (Mizushima et al., 2008), and the deregulation of autophagy is evident in the brains of PD patients (Levy et al., 2009). Autophagy has three different pathways, namely macroautophagy, microautophagy, and CMA. Previous studies have found that both macroautophagy and CMA pathways contribute to α-synuclein degradation in primary postnatal ventral midbrain neurons (Xilouri et al., 2008). Directly applying bafilomycin A1, an inhibitor of autophagy, to the cortex of α-synuclein transgenic mice increased α-synuclein levels in brain (Ebrahimi-Fakhari et al., 2012), suggesting that the autophagic pathway plays an important role in degradation of α-synuclein in neurons. Ambroxol treatment restores autophagosome formation in GBA1 mutant lines, which in turn would enhance the degradation of α-synuclein and contribute to the decreased α-synuclein levels in the GBA1 mutant lines. These results are important in confirming in patient-derived dopaminergic neuronal cells that ambroxol can rescue defects due to GCase deficiency, and appears to do so by several mechanisms. The reduction in neuronal α-synuclein levels offers the potential for ambroxol or similar drugs to be tried as a disease-modifying therapy in PD patients with GBA1 mutations (Schapira and Gegg, 2013). The strategy to reduce α-synuclein in PD is common to a number of approaches to slow progression in PD (Schapira et al., 2014). Ambroxol is currently under investigation to confirm blood-brain barrier penetration before use in a larger trial. However, the haNCSC dopaminergic model used here provides the opportunity to test similar drugs to manipulate the GCase pathway for neuroprotection in PD.