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* This work was supported by a grant from the CHDI Foundation to IMC. The CHDI Foundation is a not-for-profit biomedical research organization dedicated to Huntington's disease research and the development of therapeutics for treatment of this disease. CHDI participated in study design and data collection. This article contains supplemental material.
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.
). This expansion results in a mutant protein (mHtt) with an expanded tract of glutamine (polyQ) residues. The number of polyQ repeats is inversely correlated with age of onset of the disease symptoms (
). For example, in individuals that have mutant Htt containing 40–50Q, age of onset is usually in mid-life, whereas individuals with a juvenile age of onset may have upwards of 250 Q residues. The expanded polyQ tract of mHtt increases its self-aggregation and propensity to form oligomers and eventually insoluble aggregates (
) and the development of symptoms can be patient-specific. Moreover, despite the observed selectivity of HD neurodegeneration, the root-cause pathogenic protein, mHtt, is widely expressed in most tissues. Taken together, this suggests that although HD etiology is monogenic, HD pathophysiology is multifactorial. Indeed, mHtt is known to have pleotropic effects on “omics” cellular networks, exerting significant polyQ-dependent effects on the transcriptome (
). Broad spectrum inhibition of lysine deacetylases has been found to ameliorate HD-related phenotypes in R6/2 transgenic mice, which was also associated with degradation of specific histone deacetylases, Hdac2 and Hdac4 (
). Yet, the underlying molecular mechanisms that convey improved HD phenotypes with reduced Hdac4 levels are not known. One aspect of Hdac4 biology that has not been sufficiently studied in the brains of HD or non-affected animal models, particularly with unbiased experimental approaches, is the regulation of its protein interactions. Although studies of Hdac4 interactions isolated from cell culture models have been performed, usually via epitope-tagged Hdac4 (
), endogenous Hdac4-containing protein complexes have not been studied in tissues.
To address this limited understanding of Hdac protein interactions and their relevance to HD pathophysiology, we undertook a systems level study of Hdac4 in the whole brain and striatum of HD mouse models with normal (Q20) and mutant (Q140) Htt at pre-symptomatic and symptomatic ages. To explore the modulation of endogenous Hdac4 interactions, we pursued an immunoaffinity purification-mass spectrometry (IP-MS) approach. Because there are no prior reports of IP-MS studies of endogenous Hdac4 in any tissues, this study required an initial extensive optimization of an experimental workflow for the efficient solubilization and enrichment of endogenous Hdac4 from brain tissues. To discover interactions underlying specific Hdac4 functions, we performed parallel interaction studies of endogenous Hdac5, a related class IIa Hdac that was found not to have an impact on HD phenotypes. Overall, our results identified specific molecular targets that have known roles in synaptic plasticity and vesicle trafficking. These enriched functional associations with Hdac4 coincided with an overall dysregulation of synaptic processes in the Q140 HD mouse models, as we determined by integration of proteome and transcriptome data sets from striata of the same HD mouse series, further strengthening the relevance of Hdac4 in HD. More broadly, these results expand the knowledge of Hdac4 regulation and functions in the context of HD and may provide a framework for understanding the molecular underpinning of HD phenotypes and for designing therapeutic interventions.
Our multiomic study of Hdac4 highlights the unique role that this protein performs in neuronal tissues, as compared with its previously known functions in human cell culture (
). In HD mouse brain, we see enrichments for proteins involved in RNA binding, vesicle trafficking, synaptic function, and other related neuronal functions. By comparing interactomes generated from both whole brain and a single brain region, we were able to identify Hdac4 interactions that are enriched in the striatum, giving us clues into region-specific Hdac4 roles. Performing our interactome analysis in four different conditions, non-disease and disease polyQ lengths at pre- and post-symptomatic ages, further allowed us to identify sets of functionally related proteins that have altered associations with Hdac4 in response to age or disease progression. Finally, the integration of whole proteome and transcriptome data sets from these HD mouse models offered a perspective of the impact of mutant Htt and age on protein abundance and gene expression patterns, and how these synergize with functional enrichments observed in our Hdac4 interactomes. Below we discuss some of the main findings from this multiomic study.
We observed a strong enrichment in cytoplasmic Hdac4 protein interactions, in conjunction with increased phosphorylation levels (in brain when compared with cultured T-cells) on a residue important for Hdac4 cytoplasmic shuttling. These findings support previous reports of predominant cytoplasmic Hdac4 localization in the mouse brain and proposed cytoplasmic function in HD mouse models (
). Our identification of proteins involved in vesicle trafficking, synaptic function, and cytoskeletal regulation (Fig 2A) offers hypotheses for the mechanisms underlying the previously observed rescue of synaptic function in HD mice on Hdac4 knockdown. A possible interpretation is that Hdac4 may regulate synaptic vesicle trafficking or neurotransmitter recycling and that this function becomes disrupted in HD. In support of this hypothesis, another study observed neurons with synaptic localization of Hdac4 (
), indicating that this may be a normal function for Hdac4. However, it is also possible that these interactions represent the active trafficking of Hdac4 to different subcellular regions, and that this proper trafficking is disrupted by aggregated Htt, resulting in Hdac4 mis-localization or possibly accumulation at the synapse, with subsequent disruption of synaptic function. It should be taken into account that, as only one antibody was found to be suitable for IP-MS experiments for each Hdac, we cannot rule out the possibility that a subset of the proteins that passed the specificity filtering may be derived from possible antibody cross-reactivity rather than by association with Hdac4 or Hdac5. We hope that the protein interactions identified in our study, particularly those that functionally correlate with the perturbations identified in the multiomic data, provide a set of potential targets that will aid future studies aimed at elucidating the roles of Hdac4 at the synapse.
Additional support for an Hdac4 role in vesicle trafficking and synaptic function was provided by our interactome study in dissected striata (Fig 4C), which demonstrated strong enrichment in proteins involved in these functional categories (Fig 5A). The enrichment in synapse organization included proteins like Snx27, a known binding partner of the WASH complex that we also found in the Hdac4 interaction network. The WASH complex is an actin-regulating multiprotein complex involved in endosomal sorting and trafficking. Of note, the WASH complex and its associated binding proteins have been previously implicated in two other neurological disorders, Parkinson's Disease (
). In our study, we observed a clear concerted pattern of association with Hdac4 for all members of the WASH complex, in which the Q140 mice had a significantly higher level of complex components associated with Hdac4. We further confirmed this association of Hdac4 and the WASH complex by reciprocal isolations of HDAC4 by several members of the WASH complex (WASHc2 and Washc3, and the accessory factor Snx27). Of note, a recent study found that association of Snx27, the WASH complex, and retromer facilitated the recycling of endosomal cargos to the plasma membrane (
). It is tempting to speculate that this increased Hdac4-WASH association could play a role in the synaptic dysregulation in HD, and this would require further investigation.
Along with the increased Hdac4-WASH complex association, we noted a general trend of elevated interactions in the Q140 mice (Fig 4B). Among these interactions were those with cation channel complex proteins, particularly the N-type calcium channel proteins. Some of these, Cacna1a and Cacna1b, were also present in the synaptic vesicle transport GO term that was enriched in the striatal interactome (Fig 5C). This family of proteins is of interest in this context, having been previously implicated in HD, though only Cacna2d1 was reported as an Htt-interacting protein. Previous work has shown that mutant Htt can cause alterations to the cell surface expression of Cacna1b and disrupt its interactions with regulatory proteins (
). Although we do not have cell surface measurements of Cacna1b abundance, we did note a slight increase in protein abundance in our proteome data set. The association of Hdac4 with these calcium channel proteins provides additional evidence that Hdac4 has a role in synaptic function in HD.
In addition to Cacna2d1, we observed several previously reported Htt-interacting proteins in our Hdac4 interactomes. This suggests that, although we did not detect Htt in our Hdac4 interactomes, a possible indirect interaction (either physical or functional) between Htt and Hdac4 may exist. Interestingly, the Htt-interacting proteins that also associated with Hdac4 was increased in the Q140 10-month-old condition (supplemental Fig. S8E–S8F). This was not observed for Hdac5, adding additional weight to the possibility of a functional Hdac4 association with Htt via these common interacting proteins.
The Hdac5 interaction networks were substantially distinct from Hdac4, with only the 14–3-3 proteins and five other proteins present in both interactomes and no major Q-length dependence in the interaction levels observed. Hdac5 also appears to be primarily cytoplasmic in the brain and associated prominently with tubulin and the centrosome. The almost completely unique set of interactions for Hdac4 and Hdac5 in the brain does not mirror observations from human cell culture experiments with T cells and HEK293 cells, where overlapping interactions were detected, though these were primarily nuclear associations (
). Other than the 14–3-3 proteins, only Catalase (Cat) and Protein furry homolog (Fry) were consistent among all of the interactomes generated. Fry may act as a scaffold protein to bridge Polo-like kinase 1 and Aurora kinase A, and aurora kinases have been shown to phosphorylate class IIa Hdacs in cells (
), thus it is possible that a similar regulation occurs in this biological system.
A primary goal of HD research is to identify those proteins that are mostly directly involved in conveying the cellular pathology of mutant Htt. Omics studies have the potential to significantly contribute to this goal. Yet the inherent nature of these approaches to provide large, unbiased data sets can present challenges for data summarization and interpretation. Although targeted omics investigations, such as subcellular proteomes or interactomes are powerful, as illustrated by our protein network analyses of Hdac4 interactions in the striatum (see Fig 4C), the functional assessment of putative interactions still covers a reasonable proteome space across diverse biological processes. Therefore, additional reductionist strategies are required to maximize biological insights. The integration of multi-omics data sets has the potential to provide these insights and expedite the design of tractable, hypothesis-testing studies. In the current study, we viewed our Hdac4 interactions from the perspective of proteomes and transcriptomes perturbed by HD to identify common feature sets at the pathway and gene level. Though not yet fully published, dual omic data sets for the same HD mouse models at 2 and 10 months of age were available in public repositories. This also highlights the value of making omic data sets available to the scientific community for re-analysis from different biological perspectives. We performed re-processing of the raw quantitative proteomics data to allow for equivalent comparison to our interactome data set. Pathway and biological process enrichment analyses on differential genes identified with concerted changes at the proteome and transcriptome level identified key enriched processes that were also over-represented in the striatal interactome (Fig 4C versus 7C), most notably processes associated with synaptic functions such as vesicle trafficking and recycling and synaptic transmission. However, it is also interesting to consider why there are nearly as many proteome changes in the younger mice as the old despite almost no difference in the transcriptome at 2 months of age. HD has been shown to have both proteostatic and transcriptional dysfunctions, and it is possible that the proteostatic/translational differences are more apparent in the younger mice and the transcriptional effects are detected at more advanced age. Indeed, the transcriptome analysis by Langfelder et al., which examined striatum, cortex, and liver at three different ages, showed almost no HD-dependent transcriptional differences at the 2-month time point (
). However, the authors observed that by 6 months of age, all tissues had detectable levels of transcriptional dysregulation. The Langfelder study also showed that the 6-month age striatum had more differentially regulated transcripts than the 10-month-old mice, further highlighting the temporal nature of this dysregulation. Altogether, these previous reports and our current study show the need for integrating multiomic data sets for understanding the contribution of complex biological processes to disease phenotypes.
Another interesting result from our Hdac4 striatal interactome that we further refined using multi-omics comparisons was the observation of polyQ-dependent increases in the protein interaction levels. As discussed above, this was not observed for proteins associating with Hdac5, which suggested that many of the Hdac4 interactions are uniquely regulated in HD. Yet, the possibility existed that differential regulation at the transcriptome and proteome levels could influence these Hdac4 interaction changes. Our integration of the transcriptome, proteome, and Hdac4 interactome abundances strongly suggest that the polyQ-dependent modulation of interactions occurs largely at the post-translational level. For most interacting proteins, their proteome abundance levels were relatively unchanged. An exception to this was the cohesin subunit SA-1 (Stag1), which showed opposing omic abundance changes. Stag1 exhibited a 2-fold increase in association with Hdac4, whereas its overall abundance within the proteome was decreased by 2-fold, suggesting that the potentiation of this interaction is precisely modulated. The interaction could be potentiated within specific cellular compartments, or given our model system, in specific cell types. Functionally, Stag1 is a member of the cohesion complex, which is well-known for its role in chromosome structure maintenance during the cell cycle (
), the latter of which would be relevant for post-mitotic cells. Indeed, CNS and limb developmental defects are observed when cohesin functions are disrupted, such as in individuals with Cornelia de Lange syndrome (
). Given there is currently no known functional link between Stag1, or the cohesin complex, and HD pathogenesis, understanding the functional significance of the Hdac4-Stag1 will require further investigation.
Overall, our observation that polyQ-dependent effects on Hdac4 interaction abundances are largely independent of their respective proteome changes suggests that the primary influence of mutant Htt on Hdac4 is to alter the composition and/or stability of its associated proteins. This could be achieved through several mechanisms, including changes in post-translational modifications (PTMs) or conformation, as well as protein sequestration. For example, as exemplified by class IIa Hdacs, phosphorylation status largely determines nuclear versus cytoplasmic localization (
), though whether re-localization occurs within cytoplasmic compartments, such as at pre- or post-synaptic densities, remains a possibility. It is also possible that the PTM status of Hdac interacting partners are altered in the Q140 mice, potentiating their interaction with Hdac4. Although the broad impact of mutant Htt on the phosphoproteome is not known, Htt phosphorylation does play a critical role in modulating its aggregation and neuronal toxicity (
). For Hdac4 interactions that are decreased under conditions of Q140 Htt, mutant Htt may directly or indirectly sequester the interaction, preventing their normal cellular function(s). However, given the majority of Hdac4 interactions are potentiated and that Hdac4 reduction ameliorates HD phenotypes, a more likely hypothesis involves Hdac4 interactions acting as gain-of-function Htt toxicity modifiers (Fig. 9).
In all, the data presented here represents the first unbiased assessment of Hdac4 and Hdac5 interactions in mouse brain and highlights the potential for a specialized role of Hdac4 in the striatum. Our integrated multiomic assessment of Hdac4 interactions, protein abundance changes, and transcriptome changes in HD mouse models provides a comprehensive and contextualized view of the role Hdac4 is performing in HD and provides a resource for future investigation into the molecular mechanisms underlying these functions.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (