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. 2019 Aug 9;18(8 suppl 1):S92-S113.
doi: 10.1074/mcp.RA118.001253. Epub 2019 Apr 30.

Hdac4 Interactions in Huntington's Disease Viewed Through the Prism of Multiomics

Joel D Federspiel  1 , Todd M Greco  1 , Krystal K Lum  1 , Ileana M Cristea  2
Affiliations

Hdac4 Interactions in Huntington's Disease Viewed Through the Prism of Multiomics

Joel D Federspiel et al. Mol Cell Proteomics. .

Abstract

Huntington's disease (HD) is a monogenic disorder, driven by the expansion of a trinucleotide (CAG) repeat within the huntingtin (Htt) gene and culminating in neuronal degeneration in the brain, predominantly in the striatum and cortex. Histone deacetylase 4 (Hdac4) was previously found to contribute to the disease progression, providing a potential therapeutic target. Hdac4 knockdown reduced accumulation of misfolded Htt protein and improved HD phenotypes. However, the underlying mechanism remains unclear, given its independence on deacetylase activity and the predominant cytoplasmic Hdac4 localization in the brain. Here, we undertook a multiomics approach to uncover the function of Hdac4 in the context of HD pathogenesis. We characterized the interactome of endogenous Hdac4 in brains of HD mouse models. Alterations in interactions were investigated in response to Htt polyQ length, comparing mice with normal (Q20) and disease (Q140) Htt, at both pre- and post-symptomatic ages (2 and 10 months, respectively). Parallel analyses for Hdac5, a related class IIa Hdac, highlighted the unique interaction network established by Hdac4. To validate and distinguish interactions specifically enhanced in an HD-vulnerable brain region, we next characterized endogenous Hdac4 interactions in dissected striata from this HD mouse series. Hdac4 associations were polyQ-dependent in the striatum, but not in the whole brain, particularly in symptomatic mice. Hdac5 interactions did not exhibit polyQ dependence. To identify which Hdac4 interactions and functions could participate in HD pathogenesis, we integrated our interactome with proteome and transcriptome data sets generated from the striata. We discovered an overlap in enriched functional classes with the Hdac4 interactome, particularly in vesicular trafficking and synaptic functions, and we further validated the Hdac4 interaction with the Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex. This study expands the knowledge of Hdac4 regulation and functions in HD, adding to the understanding of the molecular underpinning of HD phenotypes.

Keywords: Hdac4; Hdac5; Huntington's Disease; Label-free quantification; Multiomics; Neurodegenerative diseases*; Omics; Protein-Protein Interactions*; Systems biology*; WASH complex.

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Figures

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Graphical abstract
Fig. 1.
Fig. 1.
Optimization workflow for the analyses of endogenous Hdac4 and Hdac5 interaction in the brain of HD mouse models. A, Isolations of endogenous Hdacs were optimized in whole mouse brain lysates. Multiple antibodies and buffers were tested to arrive at an optimized set of conditions demonstrated in the Western blotting. B, Using the optimized isolation conditions, IPs of Hdac4 and Hdac5, along with IgG controls, were performed from whole brain, analyzed by LC-MS/MS, filtered for interaction specificity, and bioinformatically assessed for known interactions and functional enrichments.
Fig. 2.
Fig. 2.
Investigation of Hdac4 protein interaction in whole brain. A, Comparison of endogenous Hdac4 interactions in Q20 and Q140 mice at 2 and 10 months of age. Each interacting protein is shown as a ring plot with the relative median MS1 abundance levels in each isolation condition depicted as indicated on the Hdac4 ring at the center of the network. Gene names shown in red are Hdac4 specific interactions that are also reported Htt interactions. Edges represent known protein-protein interactions and other associations present in the STRING database. Protein interactions have been functionally grouped and labeled in blue text. B, Relative Hdac4 pSer629 levels in mouse brain are over 400-fold higher than in cultured human T cells. C, PCA of Hdac4 interactions in whole brain shows no overt Q- or age-dependent trend.
Fig. 3.
Fig. 3.
Alterations in Hdac4 whole-brain protein interactions with Htt polyQ length and age. A, Line plots and heatmaps of selected interaction categories are shown. Each category is broken down into three or more groups of proteins with similar abundance profiles and these are shown on the line graphs depicting the relative association with Hdac4 (scaled 0–1) in each condition with a different color for each group. The number of proteins examined in each functional grouping is listed below the title of each. Below these graphs are heatmaps depicting all members of one or more groups shown in the line graph above. The heatmaps show the relative changes in each protein across conditions with the minimal amount associated with Hdac4 getting a blue color and the maximal association with Hdac4 a red color. B, A slight trend of decreased relative interaction abundances is observed across age and Q length. *** p ≤ 0.001, Kruskal-Wallis test.
Fig. 4.
Fig. 4.
The Hdac4 protein interaction network in the striatum of HD mouse models. A, PCA of specificity-filtered Hdac4 interactions revealed Q length-dependent trends in the interactome. B, A Q-length dependent effect on overall Hdac4-interaction associations was observed in striatum. *** p ≤ 0.001, Kruskal-Wallis test. C, Comparison of endogenous Hdac4 interactions in Q20 and Q140 mice at 2 and 10 months age. Each interacting protein is shown as a ring plot with the relative median MS1 abundance levels in each isolation condition depicted as indicated on the Hdac4 ring at the center of the network. Gene names shown in red are Hdac4 specific interactions that are also reported Htt interactions. Edges represent known protein-protein interactions and other associations present in the STRING database. Protein interactions have been functionally grouped and labeled in blue text. Interactions were truncated so that a specificity score of 0.97 was required for 10-month IPs for illustrative purposes. The additional interactions are provided in supplemental Fig. S5.
Fig. 5.
Fig. 5.
Comparison and characterization of Hdac4 interactomes in the whole brain and striatum. A, Biological process gene ontology analysis of shared and unique Hdac4 interactions in whole brain and striatum. Blue represents interactions in striatum and yellow interactions in whole brain. Pie charts show relative proportion of interactions in each functional class that were present in whole brain or striatum. B, Examination of the regulation of synapse organization GO term shows an enrichment in striatal IPs of proteins involved in dendrite generation. C, Examination of receptor-mediated endocytosis and organelle localization GO terms shows an enrichment in striatal IPs of vesicle trafficking and synaptic proteins.
Fig. 6.
Fig. 6.
Hdac4 displays a polyQ-dependent enriched association with the Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex. A, Line graphs of neuronal function associated proteins show differential patterns of Hdac4 association in striatum. The heatmap below depicts the relative changes in each protein across conditions for group3 of the neuronal function associated interactions with the minimal amount associated with Hdac4 getting a blue color and the maximal association with Hdac4 a red color. B, Components of the WASH complex present in striatum data set show concerted relative abundance changes in association with Hdac4 and an overall increased association in Q140 mice. C, Reciprocal IPs of FLAG-tagged WASH complex members in HEK-293T cells confirm association of the WASH complex with HDAC4.
Fig. 7.
Fig. 7.
Multiomic analysis of striata from HD model mice. A, Integration of proteome and transcriptome from HD model mice resulted in 7939 quantifiable protein-mRNA pairs for analysis. B, Venn diagrams of significantly differential (adjusted p ≤ 0.1) proteins and transcripts show both concerted and non-concerted changes in gene expression. C, Functional analysis of genes with concerted protein and RNA changes revealed co-regulated processes. D, STRING functional interaction network of individual co-regulated transcripts/proteins at 10 months of age that were annotated to the GO Biological Processes from (C). Nodes were color-coded and grouped by k-means clustering, labeled with respective genes and heatmaps (squares) of the relative abundances (Q140/Q20) for the Proteome (Pr) and Transcriptome (Tr). *, represents proteins that were uniquely detected in either Q140 or Q20 10 month proteome.
Fig. 8.
Fig. 8.
Integration of the Hdac4 interactome with proteome and transcriptome alterations in HD. A, Workflow of data sets utilized to generate multiomics-informed data set. B, Number of significantly increased and decreased proteins and transcripts in the omics data sets. C, Heatmap of Q140/Q20 fold changes at 2 and 10 months of age in interactome, proteome, and trancriptome. D, Heatmap of most changed Hdac4 interactions following background proteome abundance correction. Original and adjusted values are shown.
Fig. 9.
Fig. 9.
Potential model of role for Hdac4 in Huntington's Disease. Our observations in this study identified Hdac4 associations with RNA binding proteins, proteins involved in vesicle trafficking, as well as various proteins involved in synapse function. Integrating these observations with our multiomic analysis and known hallmarks of HD pathogenesis suggests a model whereby mHtt aggregation leads to an average striatum-specific increase in Hdac4 interactions, potentially representing gain-of-function effect that together results in defects in vesicle transport/recycling and decreased synaptic transmission.
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