LC3 II levels slightly increase following GCase inhibition in differentiated SH-SY5Y cells.

<p>(A–B) Differentiated SH-SY5Y cells were exposed to increasing doses of CBE (50, 100, 200 µΜ) for 72 h and 7 days respectively. Untreated cells served as control (ctl). Cells were lysed, separated with SDS-PAGE and immunoblotted with the LC3 polyclonal antibody. ERK = loading control. (C–D) Graphs show quantification of LC3 II immunoreactivity (vs. ERK) via densitometric analysis at 72 h and 7 day time point respectively. Statistical analysis made via One Way Anova showed a significant increase in LC3 II levels for the 100 µΜ dose at the 7 day time point only, compared to ctl cultures (P<0,05; n = 8).</p

LC3 II shows no change following GCase inhibition in rat embryonic cortical cultures.

<p>(A) Cultured cortical neurons (E18) were exposed to increasing doses of CBE (100, 200 µΜ) for 7 and 10 days respectively. Untreated cells served as control (ctl). Cells were lysed, separated with SDS-PAGE and immunoblotted with the LC3 polyclonal antibody. ERK = loading control. (B) Graphs show quantification of LC3 II immunoreactivity (vs. ERK) via densitometric analysis at the 7 and 10 day time points respectively. Statistical analysis made via One Way Anova showed no difference in the LC3 II levels for the corresponding doses and time points of CBE in comparison to the control samples (n = 6 samples/2 different culture preparations with 3 independent biological samples per condition).</p

Lysosomal activity is not compromised following GCase inhibition in differentiated SH-SY5Y cells.

<p>Differentiated SH-SY5Y cells were treated or not (control cells) with CBE (200 µm) for 5 days. Following this, cells were radiolabelled with [³H] leucine supplemented with either 3MA (10 mM) or Baf (500 nM) and then assayed for long-lived protein degradation as described analytically in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060674#s2" target="_blank">Materials and Methods</a>”. Untreated cells were also used as control (ctl). Values are the mean ±SE of two independent experiments that were represented in triplicate per condition. Statistical analysis made via One Way Anova revealed no difference in the activity of the different lysosomal pathways between CBE-treated and control cells.</p

ASYN levels remain unchanged following GCase inhibition in rat embryonic cortical cultures.

<p>(A) Rat cortical neuron cultures were prepared from embryonic day 18 (E18) rat foetuses and were exposed at the 6^th day of culture to increasing doses of CBE (100, 200 µΜ) for 72 h and 7 days respectively. Untreated cells served as control (ctl). Cell pellets were assessed for GCase enzymatic activity at the aforementioned doses and time points. (B) Cell lysates were separated with SDS-PAGE and immunoblotted with the C-20 polyclonal antibody to ASYN. ERK = loading control. (C) Graph shows quantification of ASYN immunoreactivity (vs ERK) via densitometric analysis at the 7 and 10 day time points respectively. Statistical analysis made via One Way Anova showed no significant difference in ASYN levels at the given doses and time points (n = 6 samples/2 different culture preparations with 3 independent biological samples per condition).</p

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats

<p>Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.</p