The Autophagy-Lysosomal Pathway Plays a Critical Role in the Cytotoxicity of a Tau-derived Peptide J. R. Veloria1, L. Li1, X. Tan1, E. Meermans1, W. J. Goux2, Gail A. M. Breen1* Departments of Molecular and Cell Biology1 and Chemistry2, The University of Texas at Dallas, Richardson, TX 75080 ABSTRACT T-peptide Induces Autophagy and Localizes to Autophagosomes Autophagy-Lysosomal Inhibitors Abrogate the Cytotoxicity of T-peptide Tau pathology (such as aggregation) is involved in many neurodegenerative diseases, including Alzheimer’s disease and other tauopathies. The mechanisms by which Tau induces neurodegeneration are not fully understood. The ability of Tau to aggregate has been attributed to short sequences in the microtubule-binding repeats (MTBR). We have found that a hexapeptide derived from the third MTBR of Tau (VQIVYK; PHF6) can rapidly self-nucleate into fibrils in vitro (Zhao et al., 2010). Furthermore, we have determined that this peptide was highly neurotoxic if a polyarginine tag was added to increase cell permeability (VQIVYK-R9; T-peptide). In contrast, a peptide with a single amino acid substitution (VQIVKK-R9; K-peptide) did not aggregate and was not neurotoxic (Zhao et al., 2010). We have determined that T-peptide was internalized into cells where it partially localized to the nucleolus and caused the displacement of nucleolar Tau. T-peptide also partially localized to lysosomes and induced lysosomal membrane permeabilization. T-peptide induced the synthesis of autophagosomes and T-peptide was found in autophagosomes. ROS levels were significantly increased in T-peptide-treated cells. We also found that the levels of endogenous Tau protein were decreased in cells treated with T-peptide. Furthermore, T-peptide induced the formation of cytoplasmic amyloid aggregates composed of hyperphosphorylated and caspase-cleaved Tau. Interestingly, inhibitors of the autophagy-lysosomal pathway (including leupeptin, NH4Cl, Chloroquine and Bafilomycin A1) abrogated the cytotoxicity of T-peptide. Autophagy-lysosomal inhibitors also protected cells against the down-regulation of Tau. Fig 8. Inhibitors of the Autophagy-Lysosomal Pathway Protect Cells against T-peptide-induced Cytotoxicity. Cells were pretreated with either vector alone or leupeptin, ammonium chloride or Bafilomycin A1 before treatment with T-peptide. Cell viability was determined using MTT assays. Leupeptin, ammonium chloride and Bafilomycin A1 rescued cells from T-peptide-induced cytotoxicity. Fig. 3. T-peptide Induces Autophagic Flux. A. The number of GFP-LC3-positive fluorescent punctate dots/cell was increased in HeLa/GFP-LC3 cells treated with T-peptide. B. The number of GFP-LC3 fluorescent punctate dots/cell was further increased in cells treated with T-peptide together with Bafilomycin A1. Autophagy-Lysosomal Inhibitors Protect Against the Down-regulation of Tau Fig 9. Inhibitors of the Autophagy-Lysosomal Pathway Protect Cells against T-peptide-mediated Down-regulation of Tau. Cells were pretreated with either leupeptin (A) or Bafilomycin A1 (B) before treatment with T-peptide. Tau was detected using either the Tau-1 or PHF-1 antibodies. The levels of Tau protein were restored in cells treated with leupeptin (A) or Bafilomycin A1 (B). Fig. 4. T-peptide is Present in Autophagosomes. Rh-T-peptide partially colocalizes with GPF-LC3 in autophagosomes (yellow color in the merged image). T-peptide Partially Localizes to Lysosomes and Induces LMP Fig. 5A. T-peptide Localizes to Late Endosomes/Lysosomes. Fl-T-peptide partially colocalizes with Rh-Dextran in late endosomes/lysosomes (yellow color in the merged image). CONCLUSIONS RESULTS An amyloidogenic peptide derived from Tau protein (T- peptide) is highly cytotoxic T-peptide was internalized into cells and partially localized to autophagosomes and lysosomes T-peptide induced autophagic flux and LMP The levels of endogenous Tau protein were down- regulated in T-peptide-treated cells T-peptide induced the aggregation of endogenous Tau protein Inhibitors of the autophagy-lysosomal pathway protected cells against both T-peptide-induced cytotoxicity and the down-regulation of Tau protein Fig. 5B. T-peptide induces LMP. Cells were stained with acridine orange to detect lysosomes. Note the loss of the red punctate staining in the T-peptide-treated cells, indicating permeabilization of the lysosomal membrane. T-peptide Localizes to the Nucleolus and Induces the Translocation of Nucleolar Tau ROS Levels Are Increased in T-peptide-treated Cells Fig. 6. Reactive Oxygen Species (ROS) Levels are Increased in T-peptide-treated Cells. HT22 cells were treated with various concentrations of T-peptide for 3 h. ROS levels were measured using CM-H2DCFDA. Fig. 1. T-peptide Partially Localizes to the Nucleolus and Induces the Translocation of Nucleolar Tau. A. HeLa cells were labeled with Rhodamine-labeled T-peptide. Tau was detected using the Tau-1 antibody. Rh-T-peptide colocalizes with nucleolar Tau (yellow color in merged). B. T-peptide induces the translocation of dephosphorylated Tau from the nucleolus into the nucleoplasm and the cytoplasm. T-peptide Induces the Aggregation of Endogenous Tau Tau Protein Levels Are Down-regulated in T- peptide-treated Cells Tau-1 Tau H-150 * ** Fig. 10. Model for the Cytotoxicity of a Tau-derived Peptide. REFERENCES K. Zhao, G. Ippolito, L. Wang, V. Price, M. Kim, G. Cornwell, S. Fulenchek, G. A. Breen, W. J. Goux and S. R. D’Mello (2010) J. Neurosci. Res. 88: 3399-3413 Fig. 2. The Levels of Tau Protein are Decreased in T-peptide-treated Cells. The levels of dephosphorylated Tau (detected with the Tau-1 antibody) were decreased in cells treated with T-peptide, but not in cells treated with K-peptide or AcPHF6. Total Tau levels (detected with the Tau H-150 antibody) were also decreased in T-peptide-treated cells, but not in K-peptide or PHF6-treated cells. SUPPORT/ ACKNOWLEDGEMENTS Fig. 7. Endogenous Tau is Found in the Intracellular Amyloid Aggregates Formed in T-peptide-treated Cells. HeLa cells were treated with either vehicle alone (Control) or T-peptide. Amyloid aggregates were detected using Thioflavin–S (ThS) staining. Tau was detected using either the Tau H-150, PHF-1, TauC3, or MC-1 antibodies. Tau that is present in the aggregates is hyperphosphorylated and truncated at aspartic acid residue 421. This work was supported by grants from The University of Texas at Dallas to GAMB and WJG. JRV, XT and EM received undergraduate research scholar awards from The University of Texas at Dallas.