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. 2013 Apr 22;110(19):E1817–E1826. doi: 10.1073/pnas.1305623110

Fig. 1.

Fig. 1.

α-Synuclein impairs autophagy by sequestrating TFEB. (A) qPCR analysis showing mRNA expression of ALP markers (Beclin-1, Cathepsin B, Cathepsin D, Lamp-1, and TFEB) and dopaminergic markers (TH and Aldh1) in the ventral midbrain 10 d, 3 wk, and 8 wk after intranigral injection of AAV-GFP (white bar) or AAV–α-syn vectors at high (2.1 × 1012 gc/mL) (black bar) and low concentration (3.8 × 1011 gc/mL) (gray bar). *P < 0.05 compared with AAV-GFP group (Student t test) (n = 5 per group). (B) Protein expression of Beclin-1, Lamp-1, Cathepsin D, LC3I/II, and TH in the midbrain 10 d (presymptomatic stage), and 3 wk (initiation of neurodegeneration) after overexpression of GFP or α-syn. (C–E) Fluorescent immunohistochemistry showing the colocalization of human α-syn with Beclin-1 (C), Cathepsin D (D), and p62 (E) 3 wk after intranigral injection of AAV–α-syn. (Scale bars, 15 μm.) (F) Fluorescent immunohistochemistry illustrating the accumulation of dense ubiquitin-positive structures inside DA neurons overexpressing α-syn 8 wk after AAV–α-syn injection. (Scale bar, 15 μm.) (G) Western blot showed an important accumulation of the autophagy substrate p62 8 wk after AAV–α-syn injection in the SN compared with the GFP and control group. (H) Western blot analysis illustrating the progressive accumulation of high molecular weight (HMW) α-syn oligomers in the striatum and p62 in midbrain extracts at 10 d, 3 wk, and 8 wk after AAV–α-syn injection (high titer). (I) Western blot analysis showed an increased nuclear expression of TFEB at 10 d, and a reduced nuclear localization of TFEB at 3 wk after AAV–α-syn injection at high titer. Shift in the molecular weight corresponds to the phosphorylated and dephosphorylated forms of TFEB in the cytoplasm and nucleus, respectively. Control of nuclear extraction was performed by analysis of the nuclear marker HDAC1. (J) Coimmunoprecipitation (IP) of TFEB with α-syn and 14-3-3 in α-syn–overexpressing midbrain tissue (3 wk postinjection). Lysates were subjected to IP for TFEB or control IgG, and immunoblotted for α-syn and 14-3-3 (Inp: input, no I.P).