Figure 3

Tubastatin A treatment up-regulates chaperone-mediated autophagy members and reduces phosphorylation of alpha-synuclein at serine 129. (A) Schematic of the chaperone-mediated autophagy. Proteins degraded by the chaperone-mediated autophagy are identified in the cytosol by Hsc70, the constitutive member of the cytosolic family of chaperones of 70 kDa. Hsc70 and co-chaperones bind to the targeting KFERQ-like motif in the substrate protein and bring it to the surface of lysosomes. Binding of the substrate to the cytosolic tail of the receptor protein Lamp2A promotes Lamp2A multimerization to form a translocation complex. Upon unfolding, substrate proteins cross the lysosomal membrane and reach the lysosomal matrix where they undergo complete degradation. (B) Representative immunoblots for Hsc70, Lamp2A and Gapdh in the ipsilateral (Ipsi) side of alpha-synuclein-injected rats, and (C,D) quantification of Hsc70 and Lamp2A normalized with Gapdh, expressed as relative protein level, in the contralateral (Cont) and ipsilateral sides of alpha-synuclein-injected rats treated with vehicle (Veh) or Tubastatin A (TubA) (*P < 0.05; **P < 0.01; ***P < 0.001; n = 6–7/group). (E) Representative immunoblots for human alpha-synuclein (h-aSyn) and phosphorylated alpha-synuclein at serine position 129 (pS129-aSyn), and (F) quantification of pS129-aSyn normalized with h-aSyn, expressed as relative protein level, in the ipsilateral side of alpha-synuclein-injected rats treated with Veh or TubA (***P < 0.001; n = 6–7/group). (G,H) Representative immunofluorescence confocal microscopy images showing the double staining of Hsc70 (green) and pS129-aSyn (red) (G), or the double-staining of Lamp2A (red) and human alpha-synuclein (green) (H), in the substantia nigra of alpha-synuclein-injected animals (purple square on brain representation) treated with vehicle (top) or Tubastatin A (bottom) on the ipsilateral side. Scale bar = low magnification, 100 µm; high magnification, 20 μm.