title: Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron.
abstract: BACKGROUND
  Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level.
  
  
  RESULTS
  We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant γPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs.
  
  
  CONCLUSIONS
  In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neuronal functions and neural disease pathogenesis.
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. tseki@hiroshima-u.ac.jp
authors.forename: Takahiro
authors.initials: T
authors.lastname: Seki
authors.forename: Ken-ich
authors.initials: KI
authors.lastname: Yoshino
authors.forename: Shigeru
authors.initials: S
authors.lastname: Tanaka
authors.forename: Eisuke
authors.initials: E
authors.lastname: Dohi
authors.forename: Tomoya
authors.initials: T
authors.lastname: Onji
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Izumi
authors.initials: I
authors.lastname: Hide
authors.forename: Henry L
authors.initials: HL
authors.lastname: Paulson
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
authors.forename: Norio
authors.initials: N
authors.lastname: Sakai
mesh_terms: D000818:Animals; D001343:Autophagy; D003600:Cytosol / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D018832:Molecular Chaperones / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000378:metabolism* / Q000473:pathology*; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics; D021381:Protein Transport; D011689:Purkinje Cells / Q000201:enzymology / Q000473:pathology; D059010:Single-Cell Analysis / Q000379:methods*; D020754:Spinocerebellar Ataxias / Q000201:enzymology / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D050883:HSC70 Heat-Shock Proteins; D018832:Molecular Chaperones; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); C087857:protein kinase C gamma; D011493:Protein Kinase C