Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: ISG15 is upregulated in the brains of patients with AD and in AD models in vivo and in vitro . (A and B) immunohistochemistry staining revealed increased ISG15 levels in the brains of patients with AD compared to controls ( n = 3 patients per group). (C) qRT-pcr analysis showed elevated Isg15 mRNA levels in the hippocampus of 6-month-old 5×FAD mice ( n = 6 mice per group). (D and E) Western blotting confirmed increased ISG15 protein levels in the hippocampus of 6-month-old 5×FAD mice ( n = 6 mice per group). (F) ISG15 level was increased and colocalized with microglia around amyloid plaques in AD transgenic mice, as indicated by white arrows. Representative images of immunostaining in the hippocampal DG regions of 6-month-old wild-type (WT) and 5×FAD mice. Amyloid deposits (magenta false color) were visualized using ThioS staining. Microglia (green false color) were stained with anti-AIF1/IBA1 antibody. ISG15 (red false color) stained with anti-ISG15 antibody. Inset box corresponds to zoom image. (G and H) increased ISG15 was detected in the hippocampal subsets of 10-month-old 3×Tg-ad mice compared with WT mice ( n = 3 mice per group). (I) elevated neuronal ISG15 levels were associated with increased phospho-MAPT/tau expression in AD transgenic mice. Representative images show immunostainings for ISG15 (red) and p-T205-MAPT/tau (green) in the prefrontal cortex of 10-month-old WT and 3×Tg-ad mice. (J) overexpression of wild-type full-length human MAPT/tau (HsMAPT/tau, also termed MAPT/tau441, MAPT/tau40, or MAPT/tau2N4R) in primary cultured hippocampal neurons increased Isg15 mRNA levels, as examined by qRT-pcr ( n = 5 wells per group). (K and L) overexpressing HsMAPT (probed by HT7) in primary cultured hippocampal neurons increased ISG15 protein level examined by western blotting. The purity of neurons was determined by the expression of specific neuronal marker, RBFOX3 ( n = 3 wells per group of neurons culture). (M and N) overexpression of HsMAPT (probed by HT7) in HEK293WT cells elevated ISG15 protein levels, as examined by western blotting ( n = 3 wells per group). All data are presented as mean ± SEM. Unpaired t-test. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: In Vivo, In Vitro, Immunohistochemistry, Staining, Quantitative RT-PCR, Western Blot, Transgenic Assay, Immunostaining, Expressing, Over Expression, Cell Culture, Marker

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: ISG15 overexpression leads to cognitive impairment, along with neuronal loss, and dendrite damage in mice. (A) virus constructs, including AAV-CMV- ISG15 -2A-GFP ( ISG15 ) or the empty vector AAV-CMV-MCS-2A-GFP (vec), were stereotaxically injected into the hippocampus of 2-month-old C57 mice. Behavioral tests were conducted one month later. Experimental procedures of virus injection and behavioral tests: NOR for spatial memory, MWM for spatial learning and memory, and FC for contextual fear memory. (B) the expression of virus was confirmed by GFP fluorescence. (C) ISG15 overexpression did not significantly affect novel object recognition preference ( n = 12–15 mice per group). (D) during the 5-day learning test in the MWM, ISG15-overexpressing mice showed increased latency to find the hidden platform on the 5 th day ( n = 12–15 mice per group). (E) Representative swimming paths of mice in each group during the MWM probe test. (F-H) ISG15 overexpression significantly impaired spatial memory during probe trial carried out at day 7 by removing the platform, indicated by increased latency to reach the platform location (F), decreased platform crossing times (G) and reduced duration in the target quadrant (H) ( n = 12–15 mice per group). (I) ISG15 overexpression did not affect swimming speed ( n = 12–15 mice per group). (J and K) in the FC test, ISG15 overexpression impaired contextual fear memory, evidenced by decreased freezing time on the second day ( n = 12–15 mice per group). (L-P) ISG15 overexpression in the hippocampus led to decreased spine density and dendrite impairment. Representative images of golgi staining in the hippocampal DG subset (L) and quantitative analysis of spine density (M). 30 neurons from three mice per group were analyzed. The representative morphology of dendritic arbors was shown (N), the numbers of dendrite crossings (O) and the average dendrite length (P) were measured by Sholl analyses. 20 neurons from three mice per group were analyzed. (Q and R) ISG15 overexpression decreased levels of synapse-associated proteins, as measured by western blotting ( n = 4 mice per group). All data are presented as mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test for D and O, *, p < 0.05, **, p < 0.01, vs vec. Unpaired t-test for others, *, p < 0.05, **, p < 0.01.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Over Expression, Virus, Construct, Plasmid Preparation, Injection, Expressing, Fluorescence, Staining, Western Blot

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: Downregulating ISG15 ameliorates HsMAPT-induced cognitive deficits, neuronal loss, and dendrite damage. (A) AAV-U6-shRNA (vector)-CMV-GFP (vec), AAV-U6-shRNA ( Isg15 )-CMV-GFP (sh- Isg15 ), AAV-CMV- human mapt -mCherry ( HsMAPT ), or a combination of AAV-CMV- human mapt -mCherry and AAV-U6-shRNA ( Isg15 )-CMV-GFP ( HsMAPT +sh- Isg15 ) were stereotaxically injected into the hippocampus of 2-month-old C57 mice. Virus expression was confirmed by immunofluorescence imaging after one month. (B) downregulating ISG15 or overexpressing HsMAPT did not significantly affect novel object recognition preference ( n = 13–15 mice per group). (C) downregulating ISG15 mitigated HsMAPT-induced spatial learning deficits, as shown by decreased escape latency during water maze training ( n = 13–15 mice per group). (D) Representative swimming paths of mice in each group during the MWM probe test. (E-G) downregulating ISG15 mitigated HsMAPT-induced spatial memory impairment during the probe trial on day 7, indicated by decreased latency to reach the platform location (E), increased platform crossing times (F), and increased duration in the target quadrant (G) ( n = 13–15 mice per group). (H) swimming speed was unaffected by ISG15 knockdown or HsMAPT overexpression ( n = 13–15 mice per group). (I and J) in the FC test, downregulating ISG15 improved contextual fear memory in HsMAPT-overexpressing mice, as evidenced by increased freezing time on the second day ( n = 13–15 mice per group). (K-O) downregulating ISG15 in the hippocampus attenuated HsMAPT-induced reduction of spine density and dendrite impairment. Representative images of golgi staining in the hippocampal DG subset (K) and quantitative analysis of spine density (L). 30 neurons from three mice per group were analyzed. The representative morphology of dendritic arbors was shown (M), the numbers of dendrite crossings (N) and the average dendrite length (O) were measured by Sholl analyses. 20 neurons from three mice per group were analyzed. (P and Q) downregulating ISG15 mitigated HsMAPT-induced reduction of synapse-associated proteins, as measured by western blotting ( n = 3 mice per group). All data are presented as mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test for C and N, *, p < 0.05, **, p < 0.01, vs Vec; &, p < 0.05, &&, p < 0.01, vs HsMAPT . One-way ANOVA followed by Bonferroni’s post hoc test for others, *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: shRNA, Plasmid Preparation, Injection, Virus, Expressing, Immunofluorescence, Imaging, Knockdown, Over Expression, Staining, Western Blot

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: ISG15 regulates MAPT level via the autophagy pathway. (A and B) HEK293/MAPT cells were transfected with either the plasmid pcDNA3.1- ISG15- ha ( ISG15 ) or the empty vector pcDNA3.1-ha (vec) for 48 h. Total HsMAPT levels (detected by HT7) were assessed by western blotting (A) and quantitatively analyzed (B) ( n = 4 wells per group). (C) overexpressing ISG15 in the hippocampus of mice did not affect mapt mRNA levels, as examined by qRT-pcr ( n = 6 mice per group). (D and E) HEK293/MAPT cells transfected with pcDNA3.1- ISG15 -ha ( ISG15 ) or pcDNA3.1-ha (vec) were treated with cycloheximide (CHX, 100 μg/ml) for the indicated times. Total HsMAPT levels were assessed by western blotting (D) and quantitatively analyzed (E) ( n = 3 wells per group). (F and G) HEK293WT cells were transfected with either pcDNA3.1- ISG15 -ha ( ISG15 ) or pcDNA3.1-ha (vec) for 48 h. MAP1LC3-II and SQSTM1 levels were assessed by western blotting (F) and quantitatively analyzed (G) ( n = 4 wells per group). (H-J) HEK293WT cells were co-transfected with ISG15 siRNA (si- ISG15 ) and p3×flag- mapt -3flag ( HsMAPT ) for 24 h, then treated with CQ (40 μM) or baf A1 (100 μM) for an additional 24 h. Total HsMAPT (detected by HT7), MAP1LC3-II, and SQSTM1 levels were analyzed by western blotting (H) and quantitatively analyzed (I and J) ( n = 6 wells per group). (K and L) HEK293WT cells were co-transfected with ISG15 siRNA (si- ISG15 ) and p3×flag- mapt -3flag ( HsMAPT ) for 42 h, followed by treatment with MG132 (20 μM) for 6 h. Total HsMAPT (detected by HT7), MAP1LC3-II, and SQSTM1 levels were assessed by western blotting (K) and quantitatively analyzed (L) ( n = 3 wells per group). All data are presented as mean ± SEM. Unpaired t-test for B, C and G, *, p < 0.05, **, p < 0.01. Two-way ANOVA test followed by Bonferroni’s post hoc test for E, *, p < 0.05, **, p < 0.01, vs Vec+CHX. One-way ANOVA followed by Bonferroni’s post hoc test for I, J and L, *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: Downregulating ISG15 promotes the clearance of HsMAPT by enhancing autophagic flux. (A and B) Downregulation of ISG15 reduced levels of exogenous MAPT (human MAPT), including total MAPT (detected by Tau5) and phospho-mapt (p-S262, p-S396, p-S404), in the virus-infected hippocampal region of HsMAPT mice ( n = 3 mice per group). (C and D) Representative images (C) and quantitatively analyzed (D) of immunohistochemical staining demonstrated that ISG15 knockdown reduced total MAPT levels in the hippocampal DG subset of mice ( n = 3 mice per group). (E and F) Downregulation of ISG15 reversed HsMAPT-induced elevation in protein levels of MAP1LC3-II and SQSTM1 in the infected hippocampal area, as measured by western blotting ( n = 6 mice per group). (G and H) the empty vecror siRNA (vec), ISG15 siRNA (si- ISG15 ), the plasmid p3×flag- mapt ( human wild-type 2N4R MAPT )-3flag ( HsMAPT , non-gfp tag), or ISG15 siRNA plus the plasmid p3×flag- mapt ( human wild-type 2N4R MAPT )-3flag ( HsMAPT +si- ISG15 ) were co-transfected with mCherry- MAP1LC3 into HEK293WT cells for 48 h, and the mCherry-MAP1LC3 puncta were measured by direct fluorescence imaging. At least 40 cells were analyzed for each group. (I and J) HEK293WT cells were transfected with either empty vector siRNA (vec), ISG15 siRNA (si- ISG15 ), the plasmid p3×flag- mapt ( HsMAPT , non-gfp tag), or co-transfected with ISG15 siRNA and the plasmid p3×flag- mapt ( HsMAPT +si- ISG15 ) for 48 h. Double immunofluorescence was performed with an anti-p-S396-mapt antibody (green) and anti-MAP1LC3B antibody (red). 25 cells were analyzed for each group. (K and L) the empty vecror siRNA (vec), ISG15 siRNA (si- ISG15 ), the plasmid p3×flag- mapt -3flag ( HsMAPT , non-gfp tag), or ISG15 siRNA plus the plasmid p3×flag- mapt -3flag ( HsMAPT +si- ISG15 ) were co-transfected with gfp-mCherry- MAP1LC3 into HEK293WT cells for 48 h, the GFP + mCherry + puncta and the GFP − mCherry + puncta were measured by direct fluorescence imaging. At least 30 cells were analyzed for each group. All data are presented as mean ± SEM. One-way ANOVA followed by Bonferroni’s post hoc test. ***, p < 0.001, vs Vec; &&, p < 0.01, vs HsMAPT . *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Virus, Infection, Immunohistochemical staining, Staining, Knockdown, Western Blot, Plasmid Preparation, Transfection, Fluorescence, Imaging, Immunofluorescence

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: ISG15 induces autophagic defect by arresting AP-LY fusion. (A and B) overexpression of ISG15 increased levels of endogenous MAPT (approximately 55kDa, mouse MAPT), including total MAPT (detected by Tau5) and phospho-mapt (p-S262, p-S396, p-S404), in the infected hippocampal region of C57 mice ( n = 6 mice per group). (C and D) Representative images (C) and quantitative analysis (D) of immunohistochemical staining showed that ISG15 overexpression increased total MAPT levels in the hippocampus of mice ( n = 3 mice per group). (E and F) Western blotting revealed increased levels of MAP1LC3-II and SQSTM1 in the virus-infected hippocampal region of ISG15-overexpressing mice compared to vec mice ( n = 6 mice per group). (G and H) the plasmid pcDNA3.1- ISG15 -ha ( ISG15 ), or its empty vector pcDNA3.1-ha (vec) was transfected into HEK293WT cells for 24 h followed by a 24 h treatment with CQ (40 μM) or solvent control, MAP1LC3-II, and SQSTM1 levels were analyzed by western blotting (G) and quantitatively analyzed (H) ( n = 3 wells per group). (I and J) the plasmid pEGFP- ISG15 ( ISG15 ), or its empty vector pEGFP-C1 (vec), was co-transfected with mCherry- MAP1LC3 into HEK293WT cells for 24 h followed by a 24 h treatment with CQ (40 μM) or solvent control, the mCherry-MAP1LC3 puncta were measured by direct fluorescence imaging. At least 15 cells were analyzed for each group. (K and L) the plasmid pcDNA3.1- ISG15 -ha ( ISG15 , non-gfp tag), or its empty vector pcDNA3.1-ha (vec), was co-transfected with gfp-mCherry- MAP1LC3 into HEK293WT cells for 48 h, the GFP + mCherry + puncta and the GFP − mCherry + puncta were measured by direct fluorescence imaging. At least 40 cells were analyzed for each group. (M and N) the plasmid pcDNA3.1- ISG15 -ha ( ISG15 , non-gfp tag), or its empty vector pcDNA3.1-ha (vec), was transfected into HEK293WT cells for 48 h. Double immunofluorescence was performed with an anti-MAP1LC3B antibody (red) and anti-ctsb antibody (green) (M). Quantitative analysis of overlap coefficient of MAP1LC3 and CTSB (N). Inset box corresponds to zoom image. At least 20 cells were analyzed for each group. All data are presented as mean ± SEM. Unpaired t-test. ****, p < 0.0001, vs vec. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Over Expression, Infection, Immunohistochemical staining, Staining, Western Blot, Virus, Plasmid Preparation, Transfection, Solvent, Control, Fluorescence, Imaging, Immunofluorescence

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: ISG15 reduces the recruitment of CTTN and F-actin to lysosomes by inhibiting HDAC6 activity. (A and B) ISG15 bound to HDAC6 in HEK293WT cells. HEK293WT cells were transfected with pcDNA3.1- ISG15 -ha (A) or pcDNA3.1- HDAC6 -3flag (B), and then, ISG15 was immunoprecipitated with anti-ha and blotted with anti-ISG15 and anti-HDAC6 (A), or HDAC6 was immunoprecipitated with anti-flag and blotted with anti-HDAC6 and anti-ISG15 (B). The target protein band was indicated by an arrow. (C) ISG15 bound to HDAC6 in vivo . AAV-CMV- ISG15 -2A-GFP ( ISG15 ) or AAV-CMV-MCS-2A-GFP (vec) was stereotaxically injected into the hippocampi of 2-month-old C57 mice. Homogenates from the infected hippocampal area were immunoprecipitated with anti-ISG15 and blotted with anti-ISG15 and anti-HDAC6 one month later. (D) schematic of ha-tagged ISG15 with wild type or mutants. The two ubiquitin-like domains were shown as closed blue boxes. *: amino acids were deleted from the LRLRGG peptide sequences at the C-terminal end (c-end). D: aspartic acid insertion. (E and F) HDAC6 bound ISG15 at the C-terminal LRLRGG motif. HEK293WT cells were co-transfected flag-tagged HDAC6 with ha-tagged ISG15-WT , I SG15-LRLR or ISG15-LRLRGGD . ISG15 was immunoprecipitated with anti-ha and blotted with anti-ISG15 and anti-HDAC6 (E), or HDAC6 was immunoprecipitated with anti-flag and blotted with anti-HDAC6 and anti-ISG15 (F). The target protein band was indicated by an arrow. (G) overexpressing ISG15 inhibited HDAC6 activity in HEK293WT cells. HDAC6 activity was assayed in HEK293WT cells transfected with pcDNA3.1- ISG15 -ha ( ISG15 ) or pcDNA3.1-ha (vec) for 48 h. Trichostatin a served as a positive control ( n = 3 biological replicates per group). (H) overexpressing ISG15 inhibited HDAC6 activity in vivo . HDAC6 activity was assayed in the virus-infected hippocampal area of mice infected with AAV-CMV- ISG15 -2A-GFP ( ISG15 ) or AAV-CMV-MCS-2A-GFP (vec) for one month. Trichostatin a served as a positive control ( n = 3 mice per group). (I and J) overexpressing ISG15 increased CTTN acetylation in vitro . HEK293WT cells were transfected with pcDNA3.1- ISG15 -ha ( ISG15 ) or pcDNA3.1-ha (vec) for 48 h, cell lysates were immunoprecipitated with anti-acetylated lysine (ace-lys) and blotted with anti-ace-lys and anti-cttn. The target protein band was indicated by an arrow ( n = 3 independent replicate experiments). (K and L) overexpressing ISG15 increased CTTN acetylation in vivo . Homogenates from the virus-infected hippocampal region of vec and ISG15-overexpressed mice were immunoprecipitated with anti-ace-lys and blotted with anti-ace-lys and anti-cttn. The target protein band was indicated by an arrow ( n = 4 mice per group). (M and N) overexpressing ISG15 reduced the lysosomal localization of CTTN and F-actin in vitro . HEK293WT cells transfected with pcDNA3.1- ISG15 -ha ( ISG15 ) or pcDNA3.1-ha (vec) were fractionated to isolate cytoplasm (cyto) and lysosome (lys). Fraction lysates were immunodetected for HDAC6, CTTN, F-actin, ISG15, TOMM70, CANX, GAPDH, and LAMP1 (M). TOMM70, CANX, and GAPDH were used as organelle/cell compartment-specific markers for mitochondria, endoplasmic reticulum, and cytoplasm, respectively. The relative levels of HDAC6, CTTN, and F-actin in the lysosomal fraction were calculated (N) ( n = 5 independent replicate experiments). (O and P) overexpressing ISG15 reduced the lysosomal localization of CTTN and F-actin in vivo . The virus-infected hippocampal area from vec and ISG15-overexpressed mice was fractionated to isolate cyto and lys. Fraction lysates were immunodetected for HDAC6, CTTN, F-actin, ISG15, TOMM70, CANX, GAPDH, and LAMP1 (O). The relative levels of HDAC6, CTTN, and F-actin in the lysosomal fraction were calculated (P) ( n = 5 mice per group). (Q and R) inhibition of HDAC6 reduced the lysosomal localization of CTTN and F-actin in vitro . HEK293WT cells treated with tubastatin a (a specific HDAC6 inhibitor, 5 μM) for 24 h were fractionated to isolate cyto and lys. Fraction lysates were immunodetected for HDAC6, CTTN, F-actin, ISG15, TOMM70, CANX, GAPDH, and LAMP1 (Q). The relative levels of HDAC6, CTTN, and F-actin in the lysosomal fraction were calculated (R) ( n = 4 independent replicate experiments). All data are presented as mean ± SEM. Two-way ANOVA test followed by Bonferroni’s post hoc test for G and H, **, p < 0.01, vs vec. Unpaired t-test for for others. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Activity Assay, Transfection, Immunoprecipitation, In Vivo, Injection, Infection, Ubiquitin Proteomics, Positive Control, Virus, In Vitro, Inhibition

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: Downregulating ISG15 restores the recruitment of CTTN and F-actin to lysosomes by rescuing the HsMAPT-induced inhibition of HDAC6. (A) Downregulating ISG15 rescued the inhibition of HDAC6 activity by HsMAPT in vivo . The virus-infected hippocampal regions from vec, sh- Isg15 , HsMAPT and HsMAPT +sh- Isg15 mice were used to assay HDAC6 activity. Trichostatin a served as a positive control ( n = 3 mice per group). (B and C) Downregulating ISG15 reduced the acetylation of CTTN induced by HsMAPT in HEK293WT cells. HEK293WT cells were co-transfected with ISG15 siRNA (si- ISG15 ) and p3×flag- mapt -3flag ( HsMAPT ) for 24 h, followed by treatment with tubastatin a (5 μM) for 24 h. Cell lysates were immunoprecipitated with anti-acetylated lysine (ace-lys) and blotted with anti-ace-lys and anti-cttn. The target protein band was indicated by an arrow ( n = 4 independent replicate experiments). (D and E) Downregulating ISG15 rescued the inhibition of lysosomal recruitment of CTTN and F-actin induced by HsMAPT in HEK293WT cells. HEK293WT cells transfected with vec, si- ISG15 , HsMAPT , or HsMAPT +si- ISG15 were fractionated to isolate cytoplasm (cyto) and lysosome (lys). Fraction lysates were immunodetected for HDAC6, CTTN, F-actin, ISG15, TOMM70, CANX, GAPDH, and LAMP1 (D). The relative levels of HDAC6, CTTN, and F-actin in the lysosomal fraction were calculated (E) ( n = 5 independent replicate experiments). (F and G) Downregulating ISG15 rescued the inhibition of lysosomal recruitment of CTTN and F-actin induced by HsMAPT in vivo . The infected hippocampal regions from vec, sh- Isg15 , HsMAPT and HsMAP T+sh- Isg15 mice were fractionated to isolate cyto and lys. Fraction lysates were immunodetected for HDAC6, CTTN, F-actin, ISG15, TOMM70, CANX, GAPDH, and LAMP1 (F). The relative levels of HDAC6, CTTN, and F-actin in the lysosomal fraction were calculated (G) ( n = 5 mice per group). All data are presented as mean ± SEM. Two-way ANOVA test followed by Bonferroni’s post hoc test for A, **, p < 0.01, vs Vec; &&, p < 0.01, vs HsMAPT . One-way ANOVA test followed by Bonferroni’s post hoc test for others. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Inhibition, Activity Assay, In Vivo, Virus, Infection, Positive Control, Transfection, Immunoprecipitation

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: The proposal working model of the vicious circle between MAPT accumulation and autophagy. MAPT accumulation increases ISG15 expression. ISG15 binds to HDAC6 via its C-terminal LRLRGG domain, hindering HDAC6’s deacetylase activity and leading to elevated acetylation levels of CTTN. This heightened acetylation disrupts CTTN’s recruitment with F-actin to lysosomes, inhibiting the fusion of autophagosomes and lysosomes, and impeding MAPT protein degradation. Consequently, a vicious cycle is established between increased ISG15 expression, autophagy impairment, and MAPT accumulation, ultimately resulting in cognitive deficits in mice.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques: Expressing, Histone Deacetylase Assay, Activity Assay

Journal: Autophagy

Article Title: Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in Alzheimer disease

doi: 10.1080/15548627.2024.2431472

Figure Lengend Snippet: Antibodies used in the present study.

Article Snippet: The virus constructs including AAV-CMV- ISG15 -2A-GFP or its empty vector AAV-CMV-MCS-2A-GFP, AAV-CMV- human MAPT -mCherry or its empty vector AAV-CMV-CMS-mCherry, AAV-U6-shRNA ( Isg1 5)-CMV-GFP (Knockdown sequence 5′- GGGACCTAAAGGTGAAGATGC-3′) or its empty vector AAV-U6-shRNA were synthesized by OBIO Technology.

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

doi: 10.1016/j.apsb.2023.05.012

Figure Lengend Snippet: The role of BEST1 in PT-induced acute ischemic damage. (A) Scatter plot (top, n = 5) and representative blots (bottom) showing the time course of BEST1 expression in the peri-infarct cortex after PT. (B) The scheme of experimental design. (C) Representative fluorescence image showing sh Best1 -GFP expression in the peri-infarct cortex of mice infected with AAV-CMV-sh Best1 -GFP 3 days after PT. Similar results were observed with 3 mice. (D, E) Representative fluorescence images (D) and Western blots (E, n = 4) showing BEST1 expression in the mice infected with AAV-CMV-GFP or AAV-CMV-sh Best1 -GFP 3 days after PT. (F–H) Scatter plots showing forelimb asymmetry (F) in cylinder task, foot faults of left (G) and right (H) forelimb in grid-walking task for mice in 4 groups: sham, PT, PT + GFP (injected with AAV-CMV-GFP) and PT + sh Best1 -GFP (injected with AAV-CMV-sh Best1 -GFP). n = 13–15. Two mice in PT + GFP group and two mice in PT + sh Best1 -GFP group were excluded because the brain region was not correctly targeted in microinjection. (I, J) Representative immunofluorescence images (I) and scatter plot (J) showing infarct volume on Day 3 after PT surgery. n = 13–15. Data are mean ± SEM; ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns = no significance, compared with sham in (A). Scale bars, 100 μm in (C), 50 μm in (D), and 400 μm in (I).

Article Snippet: Recombinant AAV-CMV-sh Best1 -GFP was generated by GeneChem Co., Ltd. (Shanghai, China) to knockdown BEST1 expression in vivo .

Techniques: Expressing, Fluorescence, Infection, Western Blot, Injection, Microinjection, Immunofluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

doi: 10.1016/j.apsb.2023.05.012

Figure Lengend Snippet: The effects of neuron-specific knockdown of BEST1 against PT ischemia. (A) The scheme of experimental design. (B) Representative fluorescence image showing sh Best1 -GFP expression in the peri-infarct cortex of mice infected with mixed AAV-Syn-Cre and AAV-Flex-sh Best1 -GFP 4 days after PT. Similar results were observed with 4 mice. (C) Scatter plot showing the ratio of BEST1 + neurons and BEST1 + astrocytes in mice infected with AAV-Syn-Cre + AAV-Flex-sh Best1 -GFP or AAV-Syn-Cre + AAV-Flex-GFP 4 days after PT. n = 6. (D) Representative fluorescence images showing specific knockdown of BEST1 expression in neurons by the combination of AAV-Syn-Cre and AAV-Flex-sh Best1 -GFP. Similar results were observed with 3 mice. (E–G) Scatter plots showing forelimb asymmetry (E) in cylinder task, foot faults of left (F) and right (G) forelimb in grid-walking task for mice in 4 groups: sham, PT, PT + Syn-Cre + Flex-GFP (infused with mixed AAV-Syn-Cre and AAV-Flex-GFP) and PT + Syn-Cre + Flex-sh Best1 -GFP (infused with mixed AAV-Syn-Cre and AAV-Flex-sh Best1 -GFP). n = 13–15. One mouse in PT + Syn-Cre + Flex-GFP group and one mouse in PT + Syn-Cre + Flex-sh Best1 -GFP group were excluded because brain region was not correctly targeted in microinjection. Two mice in PT group and one mouse in PT + Syn-Cre + Flex-GFP group were excluded because of no movement in tests. (H) Scatter plot showing infarct volume assessed by NeuN-labeling on Day 3 after PT surgery. n = 13–15. Data are mean ± SEM; ∗∗∗ P < 0.001, ns = no significance. Scale bars, 200 μm in (B), 50 μm in (D) (top) and 20 μm in (D) (left bottom and right bottom).

Article Snippet: Recombinant AAV-CMV-sh Best1 -GFP was generated by GeneChem Co., Ltd. (Shanghai, China) to knockdown BEST1 expression in vivo .

Techniques: Knockdown, Fluorescence, Expressing, Infection, Microinjection, Labeling

Journal: Acta Pharmaceutica Sinica. B

Article Title: Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

doi: 10.1016/j.apsb.2023.05.012

Figure Lengend Snippet: The neuroprotection of BEST1 knockdown in neurons. (A, B) Representative immunofluorescence images (A) and scatter plot (B, n = 6) showing the number of NeuN + cells in the peri-infarct cortex. (C) Representative traces of mEPSCs recorded on pyramidal neurons in the peri-infarct cortex (or the corresponding location) on Day 3 after PT (or sham) surgery. (D, E) Cumulative fraction plots of mEPSC amplitude (D, mean amplitude in inset) and interevent intervals (E, mean frequency in inset). n = 10–12 neurons (from 5 to 6 mice). The mixed AAV-Syn-Cre and AAV-Flex-sh Best1 -GFP (or AAV-Flex-GFP) was microinjected 14 days before PT surgery. (F–J) Representative immunofluorescence images (F) and scatter plot for Western blot (G, n = 3) showing BEST1 expression of cultured cortex primary neurons infected with LV-CMV-sh Best1 -GFP or it control LV-CMV-GFP at day 14 in vitro . (H) Scatter plot showing LDH release ratio of cultured neurons during 8–24 h after OGD exposure. n = 4 (from 2 independent experiments). (I, J) Scatter plot (I) and representative immunofluorescence images (J) showing the spine density of cultured neurons 24 h after OGD exposure. n = 28–29 neurons from 3 independent experiments. Neurons in Control + GFP, OGD + GFP and OGD + sh Best1 -GFP group were infected with LV-CMV-GFP, LV-CMV-GFP, and LV-CMV-sh Best1 -GFP, respectively. Data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Scale bars, 50 μm in (A, F, and J) (left), 10 μm in (J) (right).

Article Snippet: Recombinant AAV-CMV-sh Best1 -GFP was generated by GeneChem Co., Ltd. (Shanghai, China) to knockdown BEST1 expression in vivo .

Techniques: Knockdown, Immunofluorescence, Western Blot, Expressing, Cell Culture, Infection, Control, In Vitro

Journal: Acta Pharmaceutica Sinica. B

Article Title: Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

doi: 10.1016/j.apsb.2023.05.012

Figure Lengend Snippet: The contribution of BEST1 channel permeability to acute ischemia-impaired motor function. (A) Representative current traces were recorded with whole-cell patch-clamp on pyramidal neurons of the peri-infarct cortex 12–24 h after PT or sham surgery. Internal solution for all groups contained ∼4.5 μmol/L Ca 2+ , except 0 Ca 2+ group. For NPPB group, 100 μmol/L NPPB was contained in external solution. (B) Current-voltage curves (left) and current amplitudes at −80 mV (right) for the recordings shown in (A). n = 10 neurons (from 4 to 5 mice). (C) Representative current traces recorded on pyramidal neurons in peri-infarct cortex of AAV-CMV-sh Best1 -GFP- or AAV-CMV-GFP-infected PT mice. AAV-CMV-sh Best1 -GFP or AAV-CMV-GFP was microinjected 14 days before ischemic surgery, and the whole-cell patch-clamp was recorded 12–24 h after surgery. (D) Current-voltage curves (left) and current amplitudes at −80 mV (right) for the recordings shown in C. n = 10 neurons (from 4 to 5 mice). (E) Representative immunofluorescence images showing effective BEST1 W93C -3Flag expression in the neurons in peri-infarct cortex of mice infected with AAV-CMV-BEST1 W93C -3Flag-T2A-GFP 3 days after PT. Similar results were observed with 4 mice. (F–H) Scatter plots showing forelimb asymmetry (F) in cylinder task, foot faults of left (G) and right (H) forelimb in grid-walking task for mice in 4 groups: sham, PT, PT+3Flag-T2A-GFP (infused with AAV-CMV-3Flag-T2A-GFP) and PT + BEST1 W93C -3Flag-T2A-GFP (infused with AAV-CMV-BEST1 W93C -3Flag-T2A-GFP). n = 14–15. One mouse in each group except sham group was excluded because of no movement in tests. Data are mean ± SEM; ∗∗∗ P < 0.001. Scale bars, 200 μm in (E) (top) and 50 μm in (E) (bottom).

Article Snippet: Recombinant AAV-CMV-sh Best1 -GFP was generated by GeneChem Co., Ltd. (Shanghai, China) to knockdown BEST1 expression in vivo .

Techniques: Permeability, Patch Clamp, Infection, Immunofluorescence, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

doi: 10.1016/j.apsb.2023.05.012

Figure Lengend Snippet: BEST1-mediated extrasynaptic glutamate release. (A–D) Characteristic peaks of glutamate (A) and GABA (C) from ion flow chromatograph by UPLC–MS/MS and scatter plots showing extracellular glutamate (B) and GABA (D) concentration by microdialysis. AAV-CMV-sh Best1 -GFP or AAV-CMV-GFP was microinjected 14 days before PT surgery, and dialysate was collected 12–24 h after surgery. n = 5–6. (E–H) Representative traces of sEPSCs with baseline shift (E and G) and scatter plot (F and H) showing the change of glutamate tonic excitation after DCPIB (50 μmol/L) or TBOA (30 μmol/L) application. Whole-cell currents were recorded on the pyramidal neurons of peri-infarct cortex (or the corresponding location) during 12–24 h after PT (or sham) surgery. n = 10 neurons (from 4 mice). (I, J) Representative traces of sEPSCs with baseline shift (I) and scatter plot (J) showing the change of glutamate tonic excitation after NPPB (100 μmol/L) application in PT (or sham) mice. The mice were microinjected with the mixed AAV-Syn-Cre and AAV-Flex-sh Best1 -GFP (or AAV-Flex-GFP) 14 days before PT surgery, and whole-cell currents were recorded on the pyramidal neurons of peri-infarct cortex during 12–24 h after PT (or sham) surgery. n = 12–14 neurons (from 6 to 7 mice). Data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Recombinant AAV-CMV-sh Best1 -GFP was generated by GeneChem Co., Ltd. (Shanghai, China) to knockdown BEST1 expression in vivo .

Techniques: Tandem Mass Spectroscopy, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

doi: 10.1016/j.apsb.2023.05.012

Figure Lengend Snippet: The role of chloride flux in BEST1-mediated glutamate release. (A) Representative current traces were recorded with whole-cell patch-clamp in HEK293 cells expressing mouse BEST1 WT or BEST1 W93C . To show glutamate permeability, the internal solution contained 146 mmol/L CsGlutamate instead of 146 mmol/L CsCl. Internal solution for all groups contained ∼4.5 μmol/L Ca 2+ , except 0 Ca 2+ group. For NPPB group, 100 μmol/L NPPB was contained in external solution. (B) Current-voltage curves (left) and current amplitudes at −80 mV (right) quantified for the recordings are shown in (A). n = 10 cells (from 3 independent experiments). (C, D) Representative immunofluorescence image (C) and Western blots (D) showing effective BEST1 WT -3Flag expression in the cortex neurons of mice infected with AAV-CMV-BEST1 WT -3Flag-T2A-GFP 21 days after microinjection. Similar results were observed with 3 mice in each group. (E–H) Representative traces (E, G) and the scatter plot (F, H) showing OGD-induced AD current were recorded on cortex pyramidal neurons 21 days after the infection of AAV-CMV-BEST1 WT -3Flag-T2A-GFP or AAV-CMV-3Flag-T2A-GFP. n = 7 or 14 neurons (from 5 to 6 mice) for (F) and n = 10 or 13 neurons (from 5 to 6 mice) for (H). The concentration of AP-5, CNQX and NPPB was 50, 10 and 100 μmol/L, respectively. (I, J) Representative traces of sEPSCs with baseline shift (I) and scatter plot (J) showing the glutamate tonic excitation after AP-5 (50 μmol/L) and CNQX (10 μmol/L) application recorded on the pyramidal neurons of peri-infarct cortex 21 days after the infection of AAV-CMV-BEST1 WT -3Flag-T2A-GFP or AAV-CMV-3Flag-T2A-GFP. NaCl (125 mmol/L) in normal solution was replaced with NaGluconate (125 mmol/L) to obtain a low external Cl – concentration. n = 6–10 neurons (from 3 to 5 mice). Data are mean ± SEM; ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns = not significant. Scale bar, 20 μm.

Article Snippet: Recombinant AAV-CMV-sh Best1 -GFP was generated by GeneChem Co., Ltd. (Shanghai, China) to knockdown BEST1 expression in vivo .

Techniques: Patch Clamp, Expressing, Permeability, Immunofluorescence, Western Blot, Infection, Microinjection, Concentration Assay