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• Research Paper Volume 7, Issue 10 pp 839-853

  Blocking the association of HDAC4 with MAP1S accelerates autophagy clearance of mutant Huntingtin

  Relevance score: 4.963355

  Fei Yue, Wenjiao Li, Jing Zou, Qi Chen, Guibin Xu, Hai Huang, Zhen Xu, Sheng Zhang, Paola Gallinari, Fen Wang, Wallace L. McKeehan, Leyuan Liu

  Keywords: acetylation, AGERA, aggregate, apicidin, autophagy, C19ORF5, CRISP/Cas9 system, deacetylase, HDAC4, huntingtin, huntington's disease, LC3, MAP1S, N2a, stability

  Published in Aging on October 24, 2015

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  Autophagy controls and executes the turnover of abnormally aggregated proteins. MAP1S interacts with the autophagy marker LC3 and positively regulates autophagy flux. HDAC4 associates with the aggregation-prone mutant huntingtin protein (mHTT) that causes Huntington's disease, and colocalizes with it in cytosolic inclusions. It was suggested HDAC4 interacts with MAP1S in a yeast two-hybrid screening. Here, we found that MAP1S interacts with HDAC4 via a HDAC4-binding domain (HBD). HDAC4 destabilizes MAP1S, suppresses autophagy flux and promotes the accumulation of mHTT aggregates. This occurs by an increase in the deacetylation of the acetylated MAP1S. Either suppression of HDAC4 with siRNA or overexpression of the MAP1S HBD leads to stabilization of MAP1S, activation of autophagy flux and clearance of mHTT aggregates. Therefore, specific interruption of the HDAC4-MAP1S interaction with short peptides or small molecules to enhance autophagy flux may relieve the toxicity of mHTT associated with Huntington's disease and improve symptoms of HD patients.

  

  (A-D) Overexpression of HDAC4 increases levels of GFP-HTT72Q (HTT72Q) in HeLa cells stably expressing HTT72Q. Representative fluorescent images (A) and immunoblot results (C) and their respective quantification (B,D) are shown. Bars in (A) were 50 or 2 μm in the normal view on the left half and enlarged view on the right. HTT72Q aggregates retained in stacking gel (stk) and soluble HTT72Q (sol) were labeled. Data here or throughout are the average ± standard deviation of at least three repeats. Statistical significance was determined by Student's t-test. *, p ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001. The 45 KD HTT72Q formed aggregates that failed to penetrate stacking gel. (E, F) Suppression of HDAC4 decreases levels of HTT72Q aggregates in HeLa cells stably expressing HTT72Q. Representative results of immunoblots (E) and quantification (F) when HDAC4 is suppressed with siRNA are shown. (G-I) Overexpression of HDAC4 increases levels of GFP-HTT74Q (HTT74Q) in N2a cells transiently expressing HTT74Q. Representative results from normal immunoblot analyses of aggregates in stacking gel (G) or AGERA (H) and their respective quantification (I) were shown. (J) Lysosomal inhibitor Bafilomycin A1 (BAF) causes accumulation of both HTT72Q and LC3-II in cells expressing HTT72Q. None, without BAF. (K, L) HTT72Q aggregates colocalize with LAMP2-labelled lysosomes (red) in cells stably expressing HTT72Q and transiently expressing Flag-HDAC4 in the presence of BAF. Representative fluorescent images are shown and white arrows indicate HTT72Q aggregates that colocalize with LAMP2 (K). Statistical significance of difference in the percentages of HTT72Q aggregates associated with LAMP2-labelled lysosomes to total aggregates was assessed between control and BAF-treated cells (I).

  
  
  

  

  (A-D) HDAC4 affects levels of MAP1S and LC3-II in HeLa cells in the absence (None) or presence of BAF. Representative immunoblot results (A, C) and their respective quantification (B, D) of the impact of HDAC4 overexpression (A, B) and HDAC4 suppression with siRNA (C, D) are shown. (E-H) HDAC4 affects levels of MAP1S and LC3-II in N2a cells in the absence (None) or presence of BAF. Representative immunoblot results (E, G) and their respective quantification (F, H) of the impact of HDAC4 overexpression (E, F) or HDAC4 suppression with siRNA (G, H) are shown. (I, J) HDAC4 overexpression reduces punctate foci of RFP-LC3 in HeLa cells stably expressing RFP-LC3. Fluorescence microscopy images (I) and quantification (J) of punctate foci of RFP-LC3 in the absence or presence of BAF are shown. (K-N) HDAC4 overexpression (K, L) or siRNA suppression (M, N) alters the vacuolar areas in HeLa cells. Transmission electron microscopic images (K, M) and quantification (L, N) are shown. Symbol “*” indicates vacuoles.

  
  
  

  

  (A-D) MAP1S is required for HDAC4 to affect LC3-II. Representative immunoblot results (A, C) and quantification (B, D) of the impact of MAP1S deletion on the effect of HDAC4 overexpression in wild-type and MAP1S−/− MEF cells (A, B) or suppression with siRNA in wild-type and MAP1S−/− HeLa cells (C, D) on LC3-II in the absence or presence of BAF. (E, F) MAP1S suppression with siRNA reduces levels of LC3-II in cells stably expressing HTT72Q. Representative immunoblots (E) and their quantification (F) are shown. (G, H) MAP1S suppression with siRNA increases levels of HTT72Q aggregates in cells stably expressing HTT72Q. Representative immunoblots (G) and their quantification (H) are shown. (I-L) MAP1S depletion increases levels of HTT74Q aggregates in MEF cells transiently expressing GFP-HTT74Q (HTT74Q). Representative immunoblot (I, K) and quantification (J, L) of the levels of HTT74Q aggregates in wild-type and MAP1S−/− MEF cells analyzed by stack gel (I, J) or AGERA (K, L). (M, N) MAP1S is required for the HDAC4-dependent increases in levels of HTT74Q aggregates. Representative immunoblots (M) and quantification (N) of the differences between wild-type and MAP1S−/− MEF cells are shown.

  
  
  

  

  (A) A diagram showing the sequence domains of MAP1S proteins. FL, full length; HC, heavy chain; SC, short chain; LC, light chain; 4G1, region recognized by MAP1S monoclonal antibody 4G1; HBD, HDAC4-binding domain within the R653-Q855 fragment. All sequence numbers were deduced based only on our experimental results. (B) Endogenous HDAC4 interacts with MAP1S. HeLa cell lysates were precipitated with a MAP1S-specific antibody 4G1 or IgG control antibody. (C, D) Endogenous HDAC4 interacts with MAP1S. Lysates from brain tissues of wild-type (+/+) and MAP1S−/− mice (−/−) (C) or N2a cells (D) were precipitated with a specific antibody against HDAC4 or MAP1S, or IgG control antibody. (E, F) Interaction of HDAC4 and MAP1S in cells overexpressing both HDAC4 and MAP1S FL. In (E) lysates from 293T cells overexpressing HA-MAP1S were precipitated with HA or MAP1S-specific antibody or IgG control. In (F) lysates from normal 293T cells and 293T cells overexpressing both HDAC4 and HA-MAP1S were precipitated with MAP1S-specific antibody or IgG control. (G) HDAC4 interacts with MAP1S isoforms SC, HC and FL. Lysates from 293T cells overexpressing HDAC4 and HA-fused MAP1S isoforms were precipitated with MAP1S-specific antibody or IgG control. (H) HDAC4 interacts with MAP1S SC but not LC. Lysates from 293T cells overexpressing HDAC4 and HA-fused MAP1S SC and LC were precipitated with a HA-specific antibody or IgG control. (I, J) HDAC4 in cell lysates (I) is pulled down by GSH beads bound with purified GST-fused MAP1S SC, but not LC (J). HDAC4 was revealed on blots with specific antibody and GST fusion proteins were visualized with Coomassie Blue staining. (K) HDAC4 interacts with an overlapping domain between MAP1S HC and SC. Lysates from 293T cells overexpressing HDAC4 and HA-fused MAP1S sequence R653-Q855 (HA-HBD) were precipitated with a HA-specific antibody or IgG control and blotted with HA or MAP1S-specific antibody. (L) The Interaction of MAP1S with HDAC4 is abolished when the HBD is deleted. Lysates from 293T cells overexpressing HDAC4 and HA-fused full length MAP1S (HA-FL) or mutant with sequence R653-Q855 deleted (HA-HBDΔ) were precipitated with a HA or HDAC4-specific antibody or IgG control and blotted with HA and HDAC4 antibodies.

  
  
  

  

  (A, B) MAP1S depletion has no effect on HDAC4 levels in HeLa cells treated with MAP1S-specific siRNA (A) or MEF cells derived from wild-type or MAP1S−/− mice (B). (C) Overexpression of MAP1S isoforms has no impact on levels of HDAC4 in HeLa cells. (D, E) Increasing expression of HDAC4 causes dose-dependent reduction in levels of MAP1S in 293T cells. Representative immunoblots (D) and quantification (E) are shown. (F) HDAC4 depletion with siRNA increases levels of MAP1S in HeLa or COS7 cells. (G) HDAC4 suppression or overexpression does not alter levels of MAP1S mRNA. The relative levels of MAP1S mRNA in HeLa cells transfected with siRNA to suppress the expression of HDAC4 or with plasmid for HDAC4 overexpression. (H, I) HDAC4 depletion increases the stability of MAP1S proteins. HeLa cells were treated with random (Mock) or HDAC4-specific siRNA (HDAC4), and cellular protein translation was then terminated with cycloheximide (CHX). Samples were collected at different times after cycloheximide treatment. Representative immunoblots (H) and quantification (I) are shown.

  
  
  

  

  (A-D) HDAC4 reduces levels of acetylated MAP1S. HDAC4 was purified from 293T cells overexpressing HDAC4 by immunoprecipitation with antibody against HDAC4; purified HDAC4 (Active) was inactivated by boiling (Inactive). MAP1S purified from MAP1S overexpressing cells with MAP1S antibody was incubated with increasing amounts of active HDAC4 for 2 hrs (A, B) or same amount of HDAC4 for increasing amounts of time (C,D). Representative immunoblots (A, C) and quantification (B, D) are shown. (E, F) HDAC4 mutants with increased or reduced activity exhibit reduced or enhanced levels of acetylated MAP1S, respectively. HDAC4 mutant proteins purified in same way as the wildtype were incubated with purified MAP1S for 1 hr. Representative immunoblot results (E) and quantification (F) are shown.

  
  
  

  

  (A, B) Overexpression of HBD but not HA-HBDΔ reduces HDAC4-bound endogenous full length MAP1S. Equal amounts of lysates collected from HeLa cells expressing control HA, HBD and HA-HBDΔ were subjected to immunoprecipitation with HDAC4-specific antibody. Representative immunoblots (A) and quantification of the precipitated MAP1S (B) were shown. (C, D) Overexpression of HBD but not HA-HBDΔ enhances stability of endogenous MAP1S protein in HeLa cells. Lysates from HeLa cells transiently transfected with HA vector and HA-HBD and HA-HBDΔ were collected at different times after cycloheximide treatment. Representative immunoblots (C) and quantification (D) are shown. (E-G) Overexpression of HBD but not HA-HBDΔ enhances autophagy flux in the presence of MAP1S. Lysates were collected from control (MAP1S^+/+) or MAP1S knockout HeLa cells (MAP1S^−/−) transiently transfected with HA vector and HA-HBD and HA-HBDΔ in the absence (Ctrl) or presence of BAF. Representative immunoblots (E) and quantification of the relative levels of MAP1S in control HeLa cells in the absence of BAF (F) or relative levels of LC3-II in the presence of BAF (G) are shown. (H-J) Overexpression of HBD but not HA-HBDΔ prevents the HDAC4-induced MAP1S destabilization and autophagy flux suppression. Lysates were collected from HeLa cells transiently co-transfected with HDAC4 and HA vector, HA-HBD or HA-HBDΔ in the absence (Ctrl) or presence of BAF. Representative immunoblots (H) and quantification of the relative levels of MAP1S in the absence of BAF (I) or LC3-II in the presence of BAF (J) are shown. (K-M) Overexpression of HBD but not HA-HBDΔ reduces levels of mHTT aggregates. N2a cells transiently expressing GFP-HTT74Q were simultaneously transfected with two additional plasmids with one to express control, HBD or HA-HBDΔ, and another to express control or HDAC4, respectively. Equal amounts of cell lysates are subjected to immunoblot. Representative blots of GFP-74Q resolved by stack gel or AGERA (K) and their quantification in stack gel (L) or AGERA (M) are shown. (N) A diagram showing the potential mechanism by which HDAC4 regulates MAP1S-mediated autophagy turnover of mHTT aggregates. Under normal condition, isolation membrane-associated acetylated-MAP1S is deacetylated and destabilized by microtubule or aggregate-associated HDAC4. In the presence of excessive HBD, HBD competes with acetylated MAP1S for interaction with HDAC4, which leads to the exposure of mHTT aggregates to be packaged by the MAP1S-associated isolation membrane. The resulted mHTT-containing autophagosomes are connected to microtubules by acetylated MAP1S and fuse with lysosome to become autolysosomes in which mHTT aggregates are degraded by lysosomal enzymes.