Figure 6.

Brief inhibition of lysosomal activity with CHQ interferes with lysosomal processing of pffs and enhances the recruitment of endogenous α-syn to intracellular inclusions. A, representative images of intracellular mSyn–GFP puncta in live neurons 24 h pTd without (left) and with (right) a brief pulse of CHQ to inhibit lysosomal proteases (30 min, 100 μm). Images were acquired immediately after treatment with 500 μm TB to quench fluorescence of extracellular mSyn–GFP signal. B, quantification of detectable intracellular mSyn–GFP puncta after pff transduction (55.9 ± 24.7, mean ± S.D.) compared with pff transduction followed by CHQ pulse (202.5 ± 60.1, mean ± S.D.). Error bars, S.D. of three independent experiments, each consisting of transductions performed in duplicate, at least five images per transduction. Statistical significance was determined by Student's t test (*, p = 0.0174). C, immunofluorescence staining of fixed cells 3 days post-transduction with WT mSyn pffs (0.5 μg/coverslip) reveals a substantial increase in the amount of p-syn–positive inclusions (81A; Ser(P)-129) resulting from a 3-h transduction period followed by a 30-min CHQ pulse (+CHQ) relative to neurons transduced with pffs followed by a pulse with PBS vehicle (−CHQ). No difference in the appearance of neuronal morphology was indicated by staining for MAP2. D, >70% of total coverslip surface area was quantified using HALO analysis of scanned coverslips, revealing a 2.63 ± 1.19-fold (mean ± S.D., n = 4) increase in signal relative to PBS control (81A background) as a function of pff transduction. A 30-min CHQ pulse applied 3 h pTd increased pathology on day 3 pTd to 20.17 ± 11.12-fold over PBS control (mean ± S.D., n = 4, p < 0.01 (analysis of variance)). E, coverslips quantified using a similar protocol to determine somatodendritic morphology showed no significant changes in MAP2 coverage as a function of any treatment (n = 4, analysis of variance).