Frontiers in Cellular Neuroscience (Oct 2023)
Live cell in situ lysosomal GCase activity correlates to alpha-synuclein levels in human differentiated neurons with LRRK2 and GBA1 mutations
Abstract
IntroductionHeterozygous mutations in GBA1, which encodes the lysosomal hydrolase glucocerebrosidase (GCase), are a common risk factor for the neurodegenerative movement disorder Parkinson's disease (PD). Consequently, therapeutic options targeting the GCase enzyme are in development. An important aspect of this development is determining the effect of potential modifying compounds on GCase activity, which can be complicated by the different methods and substrate probes that are commonly employed for this purpose.MethodsIn this study, we employed the GCase substrate probe 5-(pentafluorobenzoylamino)fluorescein di-D-glucopyranoside (PFB-FDGlu) in combination with live cell imaging to measure GCase activity in situ in the lysosome.ResultsThe live cell assay was validated using the GCase inhibitor conduritol-B-epoxide and with GBA1 knockout neural cells and was then used to assess GCase activity in iPSC differentiated into neural stem cells and neurons that were obtained from idiopathic PD patients and PD patients with the LRRK2 G2019S and GBA N370S mutations, as well as controls (n = 4 per group). Heterogeneity in GCase activity was observed across all groups. However, a significant inverse correlation between GCase activity and levels of alpha-synuclein protein was observed.DiscussionThe live cell imaging assay for GCase activity could be useful for further understanding the role of GCase in PD and screening potential modifying compounds in differentiated human cell models.
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