Homozygous GBA mutations are the

Homozygous GBA1 mutations are the cause of the autosomal recessive lysosomal storage disorder Gaucher disease (GD). Both homozygous and heterozygous GBA1 mutation carriers have a similar risk for the development of PD in later life, although onset in GD patients may be earlier (Alcalay et al., 2014). The PD expressed in GBA1-positive patients is clinically indistinguishable from sporadic PD, except for slightly earlier onset and more cognitive dysfunction. Pharmacological responses are identical, imaging and pathology being the same as for sporadic PD (Schapira, 2015). Interest has focused on the molecular mechanisms by which GBA1 mutations and reduced activity of the glucocerebrosidase enzyme (GCase) increase the risk for PD. A reciprocal relationship between GCase activity and α-synuclein levels has emerged as an important candidate that may influence the development and progression of PD pathology (Sardi et al., 2015). There are several potential processes by which reduced GCase activity may result in increased α-synuclein levels and vice versa, including GCase trafficking defects, lysosomal dysfunction, substrate accumulation, and disordered lipid membrane function (Siebert et al., 2014). To investigate further biochemical effects of GBA1 mutations, we have developed dopaminergic neuronal lines from neural crest stem cells (NCSCs) obtained from PD and GBA1 mutation subjects. This model has also been used to examine the potential to manipulate the GCase/α-synuclein interaction to provide candidate molecules for further investigation as disease-modifying therapies in PD. We demonstrate that these patient-derived dopaminergic cells recapitulate the main biochemical abnormalities seen in PD postmortem GSK503 and that through the use of a small-molecule chaperone, GCase activity can be increased and α-synuclein levels reduced.
Results
Discussion Our study describes the preparation and analysis of dopaminergic neurons from NCSCs isolated from human adipose tissue, their use in the investigation of the biochemical consequences of the GBA1 N370S mutation, and the potential for the small-molecule GCase chaperone, ambroxol, to reverse these effects. The use of haNCSCs provides direct access to pluripotent stem cells and provides an alternative source to those derived from fibroblast lines for the creation of neuronal lines. The haNCSC approach has advantages in terms of ease of access to neural crest cells, no requirement to reprogram, retention of the intact host genome, simple protocols for neuronal differentiation, production of homogeneous colonies, and less cost. We have used this technique to develop both haNCSCs and iPSCs from controls and patients with the N370S GBA1 mutation that causes GD and increases the risk for PD. Cell lineages were differentiated into dopaminergic neurons and compared with data from iPSCs derived from fibroblasts from the same donors. Compared with controls, the N370S mutant lines from both sources showed reduced GCase activity and protein levels and increased α-synuclein levels. These results are in agreement with a previous study using PD-derived iPSCs (Schondorf et al., 2014). We also show that the small-molecule GCase chaperone ambroxol can rescue GCase activity and reduce α-synuclein levels in human neurons. A recent study (Mazzulli et al., 2016) used a non-inhibitory small modulator to activate GCase in synucleinopathy culture model and found that activation of GCase enhanced the clearance of pathological α-synuclein. These findings support the hypothesis that increasing GCase activity reduces α-synuclein levels in dopaminergic neurons, and has significant implications for the use of GCase chaperones as treatments to reduce α-synuclein in PD patients. Our results also have several important implications for the future study of GBA1 mutations in PD. The ability easily to generate dopaminergic neurons from stem cells derived directly from the host provides an important source for the study of the biochemical consequences of GCase deficiency. We show that the N370S mutation reduces GCase protein levels and activity, and is associated with elevated α-synuclein levels. These are the core biochemical features in the study of GBA1 mutations in PD as expressed in PD brain (Gegg et al., 2012) and provide a model with which to test therapeutic interventions to manipulate the GCase-α-synuclein pathway.