Journal: Molecular Neurodegeneration

Article Title: Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy

doi: 10.1186/s13024-019-0354-0

Figure Lengend Snippet: Hyperactive p300/CBP Is Associated with Impairment in the Autophagy-Lysosomal Pathway in Tauopathy Brains a Representative immunoblots of p300, CBP, acH3K18, and H3 in lysates of p300 F/F /CBP F/F primary neurons infected with lenti-control (ctrl) or lenti-Cre. b Levels of acH3K18, measured by ELISA, in CSF samples from normal controls (normal, n = 14) and patients with AD ( n = 13). *** p < 0.001 by unpaired t test. Values are mean ± SEM. c Pearson correlation analysis of acH3K18 and CSF p-tau/Aβ42 levels in CSF samples from normal controls (normal, n = 3) and AD patients ( n = 6). d Representative immunoblots of acH3K18, total H3, LC3-I, LC3-II, SQSMT/p62, and GAPDH in hippocampal lysates of 10-month-old wildtype (WT) and PS19 mice. e – g Levels of acH3K18 relative to total H3 ( e ), LC3-II ( f ) and p62 ( g ) relative to GAPDH, normalized to WT. h – j Pearson correlation analysis of LC3-II and p62 levels ( h ), acH3K18 and LC3-II levels ( i ), and acH3K18 and p62 levels ( j ). ( d – j ) n = 7 mice per group. ** p < 0.01, *** p < 0.001 by unpaired t test. Values are mean ± SEM

Article Snippet: Antibodies for p300 (N-15, SCBT), CBP (Cell Signaling), acetylated H3/K18 (Abcam), H3 (Cell Signaling), LC3B (Acris), LC3A (Novus Biologicals), p62/SQSTM1 (Novus Biologicals), GAPDH (Sigma), actin (DSHB), Tau-5 (Bio-Source), HT7 (Thermo Fisher), BT2 (Thermo Fisher), AT8 (Thermo Fisher), caspase-3 (Cell Signaling), α-tubulin (Abcam), MAP-2 (Millipore), GST-D2 (Cisbio), and europium cryptate-labeled anti-rabbit IgG-EuK (Cisbio) were purchased.

Techniques: Western Blot, Infection, Control, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy

doi: 10.1186/s13024-019-0354-0

Figure Lengend Snippet: p300/CBP Inhibits Autophagic Flux in Neurons a Schematic diagram showing autophagic flux with changes in autophagic markers. b – e p300/CBP double knockout in primary mouse neurons reduces LC3 and p62 accumulation. b Representative immunoblots of LC3-I, −II, p62, and GAPDH in lysates of p300 F/F /CBP F/F primary neurons infected lenti-ctrl or lenti-Cre. c – e Quantification of LC3-II ( c ), p62 ( e ) relative to GAPDH, and LC3-II/I ( d ), normalized to control. n = 6 wells from three independent experiments. * p < 0.05, *** p < 0.001 by unpaired t test. f Measuring autophagic flux by LC3 turnover assay. g , h p300/CBP double knockout in primary mouse neurons increases autophagic flux. g Representative immunoblots of LC3-I, −II and actin in lysates of p300/CBP double knockout neurons that were treated with BafA1 (10 nM) or DMSO for 24 h. h Quantification of autophagic flux based on the difference (increase) of LC3-II in response to BafA1 treatment, normalized to control (Cre-, BafA1-). n = 4 wells from two independent experiments. ** p < 0.01 by unpaired t test. i – n CTB treatment in primary neurons reduces autophagic flux. i Representative immunoblot of LC3-I, −II, p62 and actin in lysates of primary mouse neurons treated with DMSO (control) or CTB (50 uM) for 24 h. j – l Quantification of acH3K18 relative to H3 ( j ), LC3-II ( k ) and p62 ( l ) relative to actin, normalized to control. n = 6 wells from four independent experiments. * p < 0.05, ** p < 0.01 by unpaired t test. m Representative immunoblot of LC3-I, −II and actin in lysates of primary mouse neurons treated with DMSO (control), CTB (50 uM), BafA1 (10 nM), or CTB + BafA1 for 24 h. n Quantification of autophagic flux by the difference (increase) of LC3-II in response to BafA1, normalized to control (CTB-, BafA1-). n = 3 wells from three independent experiments. ** p < 0.01 by unpaired t test. o Using mCherry-GFP-LC3 color change to measure autophagic flux. p Representative images of primary mouse neurons infected with lentivirus expressing mCherry-GFP-LC3 treated with CTB (50 uM) or DMSO (control) for 24 h. Scale bar: 10 μm. q Quantification of autophagic vesicles (AV). Autophagosomes (APG) are identified as yellow vesicles retaining both mCherry and GFP fluorescence. Autolysosomes (AL) are identified as red vesicles in which GFP fluorescence is quenched by the low pH in lysosomes. * p < 0.05, *** p < 0.001, two-way ANOVA, Tukey-Kramer post hoc analysis. r Ratio of the number of red vesicles to yellow vesicles per cell. ** p < 0.01, unpaired t test. From two independent experiments, n = 12 cells (control), n = 17 cells (CTB). Values are mean ± SEM

Article Snippet: Antibodies for p300 (N-15, SCBT), CBP (Cell Signaling), acetylated H3/K18 (Abcam), H3 (Cell Signaling), LC3B (Acris), LC3A (Novus Biologicals), p62/SQSTM1 (Novus Biologicals), GAPDH (Sigma), actin (DSHB), Tau-5 (Bio-Source), HT7 (Thermo Fisher), BT2 (Thermo Fisher), AT8 (Thermo Fisher), caspase-3 (Cell Signaling), α-tubulin (Abcam), MAP-2 (Millipore), GST-D2 (Cisbio), and europium cryptate-labeled anti-rabbit IgG-EuK (Cisbio) were purchased.

Techniques: Double Knockout, Western Blot, Infection, Control, LC3 Turnover Assay, Expressing, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy

doi: 10.1186/s13024-019-0354-0

Figure Lengend Snippet: New p300 Inhibitor 37892 Reduces Tau Secretion ( a ) Structure of SMDC37892. b 37892 inhibited tau acetylation by p300 with an IC 50 of 35 μM. c Concentration-response curve (CRC) for 37892, quantified from p300 activity in HEK293T cells. d – g 37892 treatment in human iPSC-derived neurons. d Representative immunoblot of ac-tau (K174), t-tau, and GAPDH in lysates of human iPSC-induced neurons treated with DMSO (control) or 37892 (50 μM) for 3 days. Quantification of levels of ac-tau ( e ) and intracellular t-tau ( f ) after 3 days of treatment, normalized to control. g Quantification of tau secretion over 3 h, normalized to intracellular tau levels, and normalized to control. n = 6 wells from three independent experiments. * p < 0.05, *** p < 0.001 by unpaired t test. h – j 37892 treatment increases autophagic flux in hTau-expressing mouse primary neurons. h Representative immunoblots of p62, LC3-II and actin in primary neurons treated with 37892 (50 μM), BafA1 (5 nM), or both, for 24 h. i Autophagic flux in control and 37892-treated neurons quantified from the increase of LC3-II levels in response to BafA1 treatment, normalized to control. n = 6 wells from three independent experiments. ** p < 0.01 by unpaired t test. j Quantification of tau secretion over 3 h in p300/CBP double knockout neurons treated with 37892. p300 F/F /CBP F/F primary neurons were infected with lenti-ctrl or lenti-Cre, and treated with DMSO (−) or 37892 (50 μM) for 24 h. n = 4 wells from 2 independent experiments. * p < 0.05, ns, non-significant, by one-way ANOVA and Sidak’s multiple comparisons test. Values are mean ± SEM

Article Snippet: Antibodies for p300 (N-15, SCBT), CBP (Cell Signaling), acetylated H3/K18 (Abcam), H3 (Cell Signaling), LC3B (Acris), LC3A (Novus Biologicals), p62/SQSTM1 (Novus Biologicals), GAPDH (Sigma), actin (DSHB), Tau-5 (Bio-Source), HT7 (Thermo Fisher), BT2 (Thermo Fisher), AT8 (Thermo Fisher), caspase-3 (Cell Signaling), α-tubulin (Abcam), MAP-2 (Millipore), GST-D2 (Cisbio), and europium cryptate-labeled anti-rabbit IgG-EuK (Cisbio) were purchased.

Techniques: Concentration Assay, Activity Assay, Derivative Assay, Western Blot, Control, Expressing, Double Knockout, Infection

Journal: Molecular Neurodegeneration

Article Title: Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy

doi: 10.1186/s13024-019-0354-0

Figure Lengend Snippet: Blocking Autophagic Flux Increases Tau Secretion and Occludes the Effects of p300 a Schematic diagram showing autophagic flux and predicted changes in autophagic markers with baflomycin A1 (BafA1) treatment. b – f BafA1 treatment in primary neurons. b Representative immunoblots of LC3-I, LC3-II, p62 and actin in lysates of rat primary neurons treated with BafA1 (10 nM) or control (ctrl, DMSO) for 24 h. Quantification of levels of LC3-II ( c ) and p62 ( d ) relative to actin, normalized to control. e Quantification of intracellular tau levels by ELISA, normalized to control. f Quantification of tau secretion over 3 h by ELISA, normalized to intracellular tau levels and normalized to control. ( b – f ) n = 6 wells from three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05 by unpaired t test. g Quantification of tau secretion over 3 h in primary neurons treated with BafA1 (10 nM), CTB (50 μM) or combined, normalized to intracellular tau levels and normalized to control (CTB- BafA1-). n = 6 wells from three independent experiments. ** p < 0.01, * p < 0.05, ns, non-significant by one-way ANOVA and Sidak’s multiple comparisons test. h Quantification of tau secretion over 3 h in neurons treated with BafA1 (10 nM), 37892 (50 μM) or combined, normalized to intracellular tau levels and normalized to control (37892- BafA1-). n = 6 wells from three independent experiments. *, # p < 0.05, *** p < 0.001 by one-way ANOVA and Sidak’s multiple comparisons test. Values are mean ± SEM

Article Snippet: Antibodies for p300 (N-15, SCBT), CBP (Cell Signaling), acetylated H3/K18 (Abcam), H3 (Cell Signaling), LC3B (Acris), LC3A (Novus Biologicals), p62/SQSTM1 (Novus Biologicals), GAPDH (Sigma), actin (DSHB), Tau-5 (Bio-Source), HT7 (Thermo Fisher), BT2 (Thermo Fisher), AT8 (Thermo Fisher), caspase-3 (Cell Signaling), α-tubulin (Abcam), MAP-2 (Millipore), GST-D2 (Cisbio), and europium cryptate-labeled anti-rabbit IgG-EuK (Cisbio) were purchased.

Techniques: Blocking Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy

doi: 10.1186/s13024-019-0354-0

Figure Lengend Snippet: Enhancing Autophagy Flux Mitigates p300-mediated Tau Secretion a Schematic diagram showing autophagic flux and predicted changes in autophagic markers with rapamycin (rapa) treatment. b – f Rapa treatment in primary neurons. b Representative immunoblots of LC3-I, LC3-II, p62 and actin in lysates of rat primary neurons treated with rapa (0.25 μM) or DMSO (ctrl) for 24 h. Quantification of levels of LC3-II/I ( c ) and p62 ( d ) relative to actin, normalized to control. e Quantification of intracellular tau levels by ELISA, normalized to control. f Quantification of tau secretion over 3 h by ELISA, normalized to intracellular tau levels and normalized to control. ( b – f ) n = 6 wells from three independent experiments. *** p < 0.001, ** p < 0.01 by unpaired t test. g , h Rapamycin treatment in HEK293T cells with and without p300 overexpression. g Representative immunoblots of LC3-I, LC3-II, p62, total tau (t-tau) and GAPDH in lysate and conditioned medium of HEK293T cells transfected with tau alone or tau+p300. Serum-starved HEK293T cells were treated with DMSO (ctrl) or rapamycin (1 μM) for 24 h. h Quantification of t-tau secretion in ( g ) normalized to intracellular levels and normalized to control. n = 3 wells from three independent experiments. *, # p < 0.05 by one-way ANOVA and Sidak’s multiple comparisons test. i , j Primary neurons treated with CTB and rapa. i Representative immunoblots of LC3-I, LC3-II, p62, and actin in lysates of mouse primary neurons treated with DMSO (ctrl), CTB (50 μM), rapa (0.25 μM) and CTB + rapa. j Quantification of tau secretion over 3 h by ELISA of neurons as in ( i ), normalized to intracellular tau levels and normalized to control (CTB- rapa-). n = 6 wells from three independent experiments. *, # p < 0.05, ** p < 0.01 by one-way ANOVA and Sidak’s multiple comparisons test. Values are mean ± SEM

Article Snippet: Antibodies for p300 (N-15, SCBT), CBP (Cell Signaling), acetylated H3/K18 (Abcam), H3 (Cell Signaling), LC3B (Acris), LC3A (Novus Biologicals), p62/SQSTM1 (Novus Biologicals), GAPDH (Sigma), actin (DSHB), Tau-5 (Bio-Source), HT7 (Thermo Fisher), BT2 (Thermo Fisher), AT8 (Thermo Fisher), caspase-3 (Cell Signaling), α-tubulin (Abcam), MAP-2 (Millipore), GST-D2 (Cisbio), and europium cryptate-labeled anti-rabbit IgG-EuK (Cisbio) were purchased.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Transfection

Journal: Kidney & blood pressure research

Article Title: Urotensin II Induces Mice Skeletal Muscle Atrophy Associated with Enhanced Autophagy and Inhibited Irisin Precursor (Fibronectin Type III Domain Containing 5) Expression in Chronic Renal Failure.

doi: 10.1159/000499880

Figure Lengend Snippet: Fig. 3. Expression of UII, FNDC5, LC3 and p62 in 3 groups detected by immunochemistry. NC: sham opera- tion as NC. WT CRF: wild-type mice with 5/6 nephrectomy. UTKO CRF: UTKO with 5/6 nephrectomy mice (* p < 0.05 vs. NC, # p < 0.05 vs. NC, & p < 0.05 vs. WT CRF). Scale bar = 100 μm. FNDC5, fibronectin type III domain containing 5; IOD, integral optical density; UII, urotensin II; NC, normal control; WT CRF, wild-type chronic renal failure; UTKO CRF, UII receptor gene knockout chronic renal failure group.

Article Snippet: Following the immersion of membranes with 5% BSA (AMRESCO Inc., OH, USA), membranes were incubated with primary rabbit polyclonal anti-UII antibody (ab194676, Abcam), primary rabbit polyclonal anti-LC3 Pan et al.: UII Induces Autophagy and Inhibits FNDC5 Expression in Skeletal Muscle antibody (ab48394, Abcam), primary mouse monoclonal anti-p62 antibody (ab56416, Abcam), and primary rabbit polyclonal anti-FNDC5 antibody (ab131390, Abcam) overnight at 4 ° C. Membranes were then incubated with fluorescence-conjugated antirabbit and antimouse antibodies (LI-COR, NE, USA).

Techniques: Expressing, Control, Gene Knockout

Journal: Kidney & blood pressure research

Article Title: Urotensin II Induces Mice Skeletal Muscle Atrophy Associated with Enhanced Autophagy and Inhibited Irisin Precursor (Fibronectin Type III Domain Containing 5) Expression in Chronic Renal Failure.

doi: 10.1159/000499880

Figure Lengend Snippet: Fig. 4. Comparison of UII, FNDC5, LC3II and p62 expression among 3 groups by western blot. NC: sham op- eration as NC. WT CRF: wild-type mice with 5/6 nephrectomy. UTKO CRF: UTKO with 5/6 nephrectomy mice. * p < 0.05 vs. NC, # p < 0.05 vs. NC, & p < 0.05 vs. WT CRF. FNDC5, fibronectin type III domain containing 5; UII, urotensin II; NC, normal control; WT CRF, wild-type chronic renal failure; UTKO CRF, UII receptor gene knockout chronic renal failure group.

Article Snippet: Following the immersion of membranes with 5% BSA (AMRESCO Inc., OH, USA), membranes were incubated with primary rabbit polyclonal anti-UII antibody (ab194676, Abcam), primary rabbit polyclonal anti-LC3 Pan et al.: UII Induces Autophagy and Inhibits FNDC5 Expression in Skeletal Muscle antibody (ab48394, Abcam), primary mouse monoclonal anti-p62 antibody (ab56416, Abcam), and primary rabbit polyclonal anti-FNDC5 antibody (ab131390, Abcam) overnight at 4 ° C. Membranes were then incubated with fluorescence-conjugated antirabbit and antimouse antibodies (LI-COR, NE, USA).

Techniques: Comparison, Expressing, Western Blot, Control, Gene Knockout

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: The hypothalamic–pituitary–gonadal axis controls muscle stem cell senescence through autophagosome clearance

doi: 10.1002/jcsm.12653

Figure Lengend Snippet: Regulation of autophagy–lysosome system in muscle stem cells (MuSCs) by sex steroid receptor signalling. (A–I) Three‐month‐old WT and dKO mice were orally administered with tamoxifen for 5 days, and then, MuSCs were isolated from hindlimb muscles 6 months after tamoxifen treatment. Freshly isolated MuSCs were analysed by RNA sequencing 2868 genes satisfying fc 2. Z‐score were used for normalization. Gene ontology (GO) analysis (A). Heat map for relative expression of autophagy‐related and lysosomal genes in WT and dKO MuSCs (B). Autophagy genes were obtained from the Human Autophagy Database ( http://autophagy.lu ) and the mouse Autophagy Database ( http://www.tanpaku.org/autophagy ). Lysosomal genes were obtained from Lysosomal gene database ( http://lysosome.unipg.it ). The confirmation of autophagy and lysosomal genes in MuSCs by qPCR analysis (C). Western‐blot analysis of Atg5‐12 complex, p62, polyubiquitinated proteins in isolated MuSCs. Relative band densities were indicated below each blot (D). Immunostaining (E and G) and quantified mean fluorescence intensity (MFI) for LC3 and p62/poly‐ubiquitin (F and H) in MuSCs with (Veh) or without Bafilomycin (Baf), respectively. IPA analysis for bioinformatic prediction of putative upstream regulators of down‐regulating autophagy and lysosomal genes in dKO MuSCs compared with WT MuSCs (I). (J) Representative images and (K) quantifications of GFP and mCherry fluorescent. MuSCs expressing mCherry‐GFP‐LC3 were treated with indicated inhibitors for 24 h. Flutamide, an Ar inhibitor; MPP, an Esr1 inhibitor; PHTPP, an Esr2 inhibitor; AL, autolysosome, AP; autophagosome. Comparisons by one‐way ANOVA with Tukey's post hoc test (F, H, and K) and unpaired t ‐test (C). Scales, 10 μm, bars, mean ±SEM. n = 4 animals per group; * P < 0.05, ** P < 0.01. The detailed procedure and sample size of MFI quantification applied throughout this study are described in . dKO, double knockout; WT, wild‐type.

Article Snippet: The fixed MuSCs were washed with PBST (0.1% Tween20) several times and blocked at room temperature with blocking buffer (5% horse and goat serum in PBS) for 1 h and incubated with primary LC3 (1:200, MBL), p62 (1:200, sc‐25575, Santa Cruz Biotech.) and polyubiquitinated conjugates (Ub (n) , 1:100, FK1, Enzo) antibodies overnight at 4°C.

Techniques: Isolation, Muscles, RNA Sequencing, Expressing, Western Blot, Immunostaining, Fluorescence, Ubiquitin Proteomics, Double Knockout

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: The hypothalamic–pituitary–gonadal axis controls muscle stem cell senescence through autophagosome clearance

doi: 10.1002/jcsm.12653

Figure Lengend Snippet: A proposed model. In youthful environment, sex steroid hormones controlled by the HPG axis transcriptionally induce the expression of Tfeb in muscle stem cells (MuSCs). Tfeb drives the expression of the entire network of genes that contain the coordinated lysosomal expression and regulation (CLEAR) motif in their promoters. Accordingly, the autophagy–lysosome pathway maintains stemness and quiescence of MuSCs. However, at early stage, decreased sex hormone signalling by functional decline of the HPG axis at old age or inhibition of GnRH results in decrease of Tfeb and autophagy and lysosomal genes, which leads to impaired autophagosome clearance and accumulation of bulk autophagy substrates (LC3, p62/Ub) and ROS. At late stage, the gradual increase of ROS by prolonged decrease of the HPG axis reaches the level similar to that of geriatric MuSCs and then eventually provokes entry into senescence, which is relevant to an organism's normal aging process: impaired autophagy clearance at an early stage and then cellular senescence at the late stage. Importantly, Tfeb overexpression in geriatric or sex hormone signalling‐disrupted MuSCs rescues impaired autophagy–lysosome system and reverts MuSC function. Therefore, the HPG‐Tfeb axis would be a key contributor to the aging of MuSCs. HPG, hypothalamic–pituitary–gonadal.

Article Snippet: The fixed MuSCs were washed with PBST (0.1% Tween20) several times and blocked at room temperature with blocking buffer (5% horse and goat serum in PBS) for 1 h and incubated with primary LC3 (1:200, MBL), p62 (1:200, sc‐25575, Santa Cruz Biotech.) and polyubiquitinated conjugates (Ub (n) , 1:100, FK1, Enzo) antibodies overnight at 4°C.

Techniques: Expressing, Functional Assay, Inhibition, Over Expression

Journal: Autophagy

Article Title: BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

doi: 10.1080/15548627.2017.1357792

Figure Lengend Snippet: BNIP3L is required in ischemia-reperfusion-induced mitophagy. (A) Bnip3l+/+, Bnip3l+/−, and bnip3l−/− mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 1 h, and expression of SQSTM1, TOMM20, COX4I1, and LC3B in ischemic penumbra was assessed by western blot after 6 h of reperfusion. (B–E) Semi-quantitative analysis of SQSTM1, TOMM20, COX4I1, and LC3B bands are shown, respectively. (F and G) Primary cultured mice cortical neurons were subjected to 2 h of oxygen and glucose deprivation followed by 6 h of reperfusion (OGD-Rep). (F) The SQSTM1, TOMM20, and COX4I1 protein levels in both Bnip3l+/+ and bnip3l−/− neurons treated with OGD-Rep were determined. (G) The relative mitochondrial DNA (mtDNA) levels, which as indicated by the mt-Atp6 (mitochondria-encoded DNA):Rpl13 (nucleus-encoded DNA) ratio, were assessed by real-time PCR in both Bnip3l+/+ and bnip3l−/− neurons. (H) Adeno-associated viruses expressing GFP-BNIP3L or GFP were intracerebrally injected into Bnip3l+/+ and bnip3l−/− mice 2 wk before tMCAO. After 6 h of reperfusion, the TOMM20, COX4I1, and cleaved-CASP3 expression in ischemic penumbra were determined by western blot. The data are expressed as means ± SD. Statistical comparisons were performed with one-way ANOVA followed by Dunnett's t test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.

Article Snippet: The following primary antibodies were used: phos-BNIP3L (S81, 1:2000; Abcam, ab208190), LC3 (1:1,000; Sigma, L7543), SQSTM1 (1:1,000; Cell Signaling Technology, 5114), cleaved CASP3 (1:1,000; Cell Signaling Technology, 9661), COX4I1 (1:1,000; Cell Signaling Technology, 4850), TOMM20 (1:1,000; Anbo Biotechnology, c16678), PARK2 (1:1,000; Sigma, P5748), or GAPDH (1:3,000; KangChen, KC-5G4).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Injection

Journal: Autophagy

Article Title: BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

doi: 10.1080/15548627.2017.1357792

Figure Lengend Snippet: PARK2 is dispensable in BNIP3L-mediated mitophagy evoked by ischemia-reperfusion. (A) Park2+/+ and park2−/− neurons were treated with 2 h of OGD. LC3B (green) and BNIP3L (red) were stained by immunocytochemistry at the indicated time after reperfusion, and the images were taken by confocal microscopy. (B) Columns represent the Manders overlap coefficient of BNIP3L and LC3B in Park2+/+and park2−/− neurons. At least 30 cells from 3 independent experiments for each group were included. (C and D) park2−/− mice and primary cultured neurons were infected with AAVs expressing GFP-BNIP3L or GFP. The mice were subjected to one h of tMCAO with 6 h of reperfusion. The cultured neurons were subjected to 2 h of OGD and 6 h of reperfusion. The SQSTM1, TOMM20 and COX4I1 levels in (C) brain tissue and (D) cultured neurons were determined by western blot. (E) Another set of mice were killed 24 h after MCAO and infarct volumes were determined by TTC staining. (F) Primary cultured wild-type and park2−/− neurons were infected with GFP-BNIP3L or GFP-expressing AAVs 48 h in advance. After 2 h of OGD and 24 h of reperfusion, the cell viability was measured by MTT assay. The data are expressed as means ± SD. Statistical comparisons were performed with unpaired Student t tests. *p < 0.05 vs. the indicated group.

Article Snippet: The following primary antibodies were used: phos-BNIP3L (S81, 1:2000; Abcam, ab208190), LC3 (1:1,000; Sigma, L7543), SQSTM1 (1:1,000; Cell Signaling Technology, 5114), cleaved CASP3 (1:1,000; Cell Signaling Technology, 9661), COX4I1 (1:1,000; Cell Signaling Technology, 4850), TOMM20 (1:1,000; Anbo Biotechnology, c16678), PARK2 (1:1,000; Sigma, P5748), or GAPDH (1:3,000; KangChen, KC-5G4).

Techniques: Staining, Immunocytochemistry, Confocal Microscopy, Cell Culture, Infection, Expressing, Western Blot, MTT Assay

Journal: Autophagy

Article Title: BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

doi: 10.1080/15548627.2017.1357792

Figure Lengend Snippet: Mitophagy is further impaired in bnip3l and park2 double-knockout ischemic brain. park2 and bnip3l double-knockout mice (DKO) were generated by hybridizing park2−/− with bnip3l−/− mice. (A) Primary cultured cortical neurons from the indicated germline mice were previously transfected with GFP-LC3B and Mito-DsRed by viral vector infection. After 3 h of reperfusion, fluorescent images were captured by confocal microscopy. Images show representative examples from 3 independent experiments. (B) Columns represent the number of GFP-LC3B-positive puncta per cell (left panel) and the proportion of LC3B puncta on mitochondria of the total LC3B puncta (right panel). At least 5 random fields from one section and 3 to 6 sections were averaged in each independent experiment. The bar chart shows mean ± SD values of puncta number from at least 3 independent experiments; at least 50 cells were analyzed in each group. (C and D) Brain corticostriatal slices from the indicated mice were subjected to 15 min of OGD plus 1 h reperfusion. (C) The protein levels of SQSTM1, TOMM20, and COX4I1 in the indicated mice were assessed by western blot. (D) After one h of reperfusion, tissue viability was quantified by TTC conversion assay. (E) DKO mice were injected with AAVs expressing GFP, GFP-BNIP3L or GFP-PARK2. The mice were then subjected to one h of tMCAO with 24 h of reperfusion. The representative images of ischemic brains are shown and the infarct volumes were determined by TTC staining. The data are expressed as means ± SD. Statistical comparisons were performed with one-way ANOVA followed by Dunnett's t test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.

Article Snippet: The following primary antibodies were used: phos-BNIP3L (S81, 1:2000; Abcam, ab208190), LC3 (1:1,000; Sigma, L7543), SQSTM1 (1:1,000; Cell Signaling Technology, 5114), cleaved CASP3 (1:1,000; Cell Signaling Technology, 9661), COX4I1 (1:1,000; Cell Signaling Technology, 4850), TOMM20 (1:1,000; Anbo Biotechnology, c16678), PARK2 (1:1,000; Sigma, P5748), or GAPDH (1:3,000; KangChen, KC-5G4).

Techniques: Double Knockout, Generated, Cell Culture, Transfection, Plasmid Preparation, Infection, Confocal Microscopy, Western Blot, Injection, Expressing, Staining