Fig. 5: GFPT2 restores ER-Golgi trafficking of lysosomal hydrolases, GCase activity and reduces α-syn accumulation in PD iPSn.

A Western blot analysis of GFPT2 and GFP in day 90 A53T iPSn infected with lentivirus expressing GFP as a control, and GFPT2 at MOI-3. iPSn were analyzed 15 days after infection. GAPDH is a loading control. Quantification is shown on the right (GFP, n = 3; GFPT2, n = 4). B A53T iPSn were infected with lentivirus expressing GFP and GFPT2. Glucose levels were measured from lysates by enzymatic production of glucose-derived NADH in vitro followed by conversion of water-soluble tetrazolium (WST) to formazan, and normalized to total protein (GFP, n = 3; GFPT2, n = 4). C Western blot of N-glycosylated proteins using biotinylated Con-A, in A53T iPSn infected with lentivirus expressing GFP and GFPT2. GAPDH is a loading control. Quantification is shown on the right (n = 3). D Calnexin (CANX) binding activity was measured in PD iPSn (A53T) infected with GFP or GFPT2 as in (A). Quantification of N-glycan binding was done by Con-A pull-down/CANX western blot (n = 3 culture wells). Western blot for GCase was done on the same precipitates and quantified on the right (n = 6 culture wells). E GCase levels and maturation were measured by western blot analysis using endoglycosidase H (endo H) digestion of lysates from GFP and GFPT2 infected A53T iPSn. GAPDH is a loading control. n = 3, ±SEM, *p < 0.05. Total GCase levels were quantified from the non-endo H digested lanes. F Western blot analysis of Hex B in A53T iPSn infected with GFP and GFPT2, using GAPDH as a loading control. Quantifications of mature Hex-B is shown on the right (GFP, n = 3; GFPT2, n = 4). Note that the Immature Hex B bands are shown at a higher intensity compared to the mature Hex B bands, to avoid saturation of the mature forms. G Quantitative RT-PCR analysis of GBA1, HEXB, and GFPT2 in A53T iPSn at day 90, infected with control GFPT2 lentivirus at MOI-3, 15 days after infection (n = 3). H Analysis of lysosomal GCase activity in living A53T iPSn infected with empty vector or GFPT2 lentivirus at MOI-3. Fluorescent substrate degradation was evaluated in a microplate reader for 3 h. Activity within acidic cellular compartments was determined by quantifying the response to bafilomycin A1 (Baf A1) (n = 4). I Western blot analysis of α-syn from Triton X-100 soluble (T-sol) (GFP, n = 3; GFPT2 n = 4) and insoluble (T-insol) (n = 4) fractions using the C20 antibody, in A53T iPSn infected with vector and GFPT2 lentivirus. Coomassie brilliant blue (CBB) is a loading control. Quantification is shown on the right. The line indicates that the T-sol and T-insol samples were run on different gels/blots. (J) Neuron viability was assessed in A53T iPSn infected with vector and GFPT2 lentivirus, by quantification of neurofilament (NFM) content and normalized to a general cell volume stain (cell-tag). Quantification is shown on the right (n = 20). Scatter plots represent individual culture well replicates. For all quantifications, values are the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s two-sided t-test was used for all comparisons. Source data are provided as a Source Data file.