Fig 2: Acinar cell apoptosis is delayed in post-ligated SMGs of ATG5-deficient mice. (a) ATG5 status impinges upon duct ligation-triggered acinar apoptosis. Apoptosis by ApopTag Peroxidase (brown nuclear staining) was visualized using an In situ Apoptosis Detection Kit (left panel). Peak apoptosis was detected in L1 and L3 SMGs of Atg5WT mice, whereas the strongest ApopTag signals were detected in L3 and L7 SMGs of Atg5KO mice. Magnification: × 100, and enlarged view (inset): × 400. Bar: 100 μm. Percent ApopTag-positive cells quantified by dividing the total ApopTag-positive cells by total number of cells examined from 10 randomly chosen fields are shown (right panel). Student's t-test was employed to determine statistically significant differences in percentage of ApopTag-positive cells between Atg5WT and Atg5KO groups. Results are shown as mean±S.D.; *P<0.05. (b) ATG5 deficiency delays caspase-3 activation. Equal amounts of whole SMG lysates from Ctrl, L1, L3 and L7 SMGs of Atg5WT and Atg5KOmice were analyzed on western blots using indicated antibodies. The cleavage of caspase-3 represents caspase-3 activation. (c) Gene expression analyses of Prol1/Muc-10 and Klk1, markers for acinar and duct cells, respectively. SMG duct ligation was performed as described in Figure 2. Expression of the indicated messages in ligated and control SMGs was analyzed by quantitative RT-PCR analyses. Relative gene expression was calculated where respective mean value for control SMG set to 1 for each amplification (N≥4). Non-parametric Mann–Whitney test was performed to compare expression levels between respective ligated and control SMGs (* below bars), and between same-day ligated SMGs and control SMGs from Atg5WT and Atg5KO (* above bars). Results are shown as mean±S.D.; *P<0.05; **P<0.01. (d) Autophagy inhibition correlates with resistance to H2O2-induced cell death. Cell viability was measured in m5-7, Atg7-KO MEF, WT MEF and salivary Pa-4 cells treated with indicated concentrations of H2O2 in combination with chloroquine (CQ; 20 μM) or baflomycin A1 (BafA1; 10 nM) for 48 h and compared with that of the control cells. Data were analyzed with ANOVA followed by Bonferroni t-test to determine the statistical differences between treatment and control group (left panels). Representative western blots (right panels), in which total lysates from cells treated for 6 h with vehicle, CQ (20 μM) or BafA1 (10 nM) were stained with indicated antibodies, are shown. Results are shown as mean±S.E.M.; N=3; *P<0.05; **P<0.01; ***P<0.001

Fig 3: Summary of the findings. Cardiac I/R induces mitochondrial damage and ROS production. Directly activated autophagic flux by a novel and powerful autophagy-inducing peptide, Tat-Beclin 1, reduces I/R-induced ROS level and increases mitochondrial homeostasis during cardiac I/R injury. Tat-Beclin 1’s cardioprotective effects are dependent on the crucial autophagy-related gene, ATG7. Induction of autophagic flux also seems to induce mitochondrial biogenesis mediated by PGC1α and possibly increase mitochondrial dynamics (fission and fusion). The mechanisms of autophagic flux-induced mitochondrial biogenesis and mitochondrial dynamics need further investigation.

Fig 4: The beneficial effects of Tat-Beclin 1 on mitochondria in mouse hearts depend on autophagy. ATG7 cKO (KO) (αMHC-merCremer+; ATG7F/F injected with tamoxifen) were generated. ATG7F/F treated with tamoxifen injection (WT) were used as control. Mice were treated with Tat-Scrambled or Tat-Beclin 1 for 24 h, and the left ventricle was isolated for analysis. (A), ATG7 gene expression in mouse heart. n = 9–10. WT vs. KO, p = 0.0002. (B), PGC1α gene expression in mouse heart. n = 4–6. TS vs. TB, WT, p = 0.023. KO, p = NS (not significant). (C,D), mitochondrial dynamics fission-related gene expression in mouse heart. n = 4–6. TS vs. TB, Drp1, WT, p = 0.0421. KO, p = NS. Fis1, WT, p = 0.0216. KO, p = NS. (E–G), mitochondrial dynamics fusion-related gene expression in mouse heart. n = 4–6. TS vs. TB, Opa1, WT, p = 0.0205. KO, p = NS. Mfn1, WT, p = 0.0121. KO, p = NS. Mfn2, WT, p = 0.0459. KO, p = NS. (H), mtDNA copy numbers in mouse hearts were analyzed by ATP6 qPCR. n = 4–6. TS vs. TB, WT, p = 0.0095. KO, p = NS. TS, Tat-Scrambled; TB, Tat-Beclin 1; NS (not significant).

Fig 5: Htt secretion is autophagy dependent.A, Htt secretion is inhibited by ATG7 KO. WT and ATG7 KO MEF cells were transfected with GFP-Htt-Q74 and analyzed for Htt secretion by Western blot. B, quantitation of Htt secretion in A. C, Htt secretion is reduced by autophagy inhibition. WT and 55KO HeLa cells expressing GFP-Htt-Q74 were treated with 0, 400, or 500 nM BafA1 for 4 h and analyzed for Htt secretion by Western blot. Note that BafA1 reduced Htt secretion in WT but not 55KO cells. D, quantitation of Htt secretion in C. E, Htt colocalizes with autophagosomes. Htt-expressing cells were prepermeabilized with 0.1% saponin for 2 min, fixed, and immunostained with a myc antibody. F, quantitation of Htt and LC3 colocalization in E. G, Htt is likely released by lysosome exocytosis. WT HeLa cells expressing GFP or GFP-Htt-Q74 were stained for LAMP1 without permeabilization. H, quantitation of cell surface LAMP1 level in G, with LAMP1 GFP–expressing WT cells normalized to 1. Results are presented as mean ± SEM; statistical analysis was performed using Student’s t test. ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001. I, Htt secretion is independent of exosomes. Conditioned medium from GFP-Htt-Q74–expressing cells was harvested and subjected to differential centrifugation. Equal proportions of each pellet and soluble fraction were analyzed by Western blot for actin, TSG101, and GFP. L, whole cell lysate; 10 K, 10,000g 30 min pellet; 100 K, 100,000g 60 min pellet; SN, 100,000g 60 min supernatant. J, secreted Htt is not enclosed in membrane structures. Conditioned medium from GFP-Htt-Q74–expressing cells was treated with 2.5 μg/ml proteinase K (PK) for 10 min in the presence or the absence of Triton X-100 followed by Western blot of GFP. BafA1, bafilomycin A1; Htt, huntingtin; 55KO, GRASP55 KO; LAMP, lysosomal membrane–associated protein; MEF, mouse embryonic fibroblast.