The Two Faces of HDAC3: Neuroinflammation in Disease and Neuroprotection in Recovery

Abstract

Histone deacetylase 3 (HDAC3) is a critical regulator of gene expression, influencing a variety of cellular processes in the central nervous system. As such, dysfunction of this enzyme may serve as a key driver in the pathophysiology of various neuropsychiatric disorders and neurodegenerative diseases. HDAC3 plays a crucial role in regulating neuroinflammation, and is now widely recognized as a major contributor to neurological conditions, as well as in promoting neuroprotective recovery following brain injury, hemorrhage and stroke. Emerging evidence suggests that pharmacological inhibition of HDAC3 can mitigate behavioral and neuroimmune deficits in various brain diseases and disorders, offering a promising therapeutic strategy. Understanding HDAC3 in the healthy brain lays the necessary foundation to define and resolve its dysfunction in a disease state. This review explores the mechanisms of HDAC3 in various cell types and its involvement in disease pathology, emphasizing the potential of HDAC3 inhibition to address neuroimmune, gene expression and behavioral deficits in a range of neurodegenerative and neuropsychiatric conditions.

Plain Language Summary

Histone deacetylase 3 (HDAC3) is an essential enzyme that helps regulate gene expression in the brain, influencing a variety of processes critical for brain health. Dysfunction of this enzyme may contribute to various brain disorders and diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal cord injury, stroke and major depressive disorder. HDAC3 is involved in the brain's response to injury and disease through dual roles in neuroinflammation and neuroprotection. Recent evidence suggests that inhibiting HDAC3 can mitigate detrimental immune processes in the brain, making it a promising target for new therapies. By understanding HDAC3 in the healthy brain, we can better identify how its dysfunction contributes to disease and how its inhibition may aid in recovery. This review explores the mechanisms of HDAC3 in various brain cell types and explores its potential as a therapeutic target for treating neurodegenerative and neuropsychiatric conditions.

Plain language summary

Article highlights.

• HDAC3 modulates cell type specific gene expression through histone and non-histone transcriptional regulators to control both neuroinflammation and neuroprotection.

• Within the brain, HDAC3 promotes neuroinflammation through the repression of inflammation-resolving pathways via histone deacetylation.

• HDAC3 promotes neuroprotective mechanisms through its interaction with non-histone proteins, such as transcription factors and signalling pathways that promote cell survival and repair

• HDAC3 inhibition reduces astrocyte reactivity and pro-inflammatory microglia and macrophages phenotypes, while promoting myelination, oligodendrocyte maturation and neural plasticity

• Inhibition of HDAC3 ameliorates various neuroinflammatory symptoms in animal models of disease states, including neurodegenerative disease, spinal cord injury and neuropsychiatric symptoms such as stress, anxiety and depression

1. Introduction

Histone deacetylase 3 (HDAC3) is a key epigenetic regulator of gene expression by deacetylating both histone tails and non-histone transcriptional regulators. Despite being the focus of study in non-brain cell types for decades [1], the role of HDAC3 in regulating gene expression in the brain is far from completely understood. Gene targets and functional impacts of HDAC3 differ by cell type and can produce complex impacts on gene regulation, including both repression and activation of gene expression [2]. HDAC3 maintains appropriate brain myelination, neuronal plasticity and differentiation through histone and non-histone protein deacetylation in neurons, astrocytes and microglia. This enzyme also has well-characterized roles in regulating immune cells [2]. New work in microglia, the resident central nervous system innate immune cells [3–7], supports HDAC3 as a critical regulator of neuroinflammation. Since HDAC3 controls expression of inflammation-resolving genes, inhibition of HDAC3 may be leveraged for treatment of brain injury, neurodegeneration and neuropsychiatric disorders presenting with neuroinflammation, such as major depressive disorder (MDD). Recent experimental use of HDAC3 inhibitors in rodent models facilitated recovery from brain injury [8–10], stroke [11,12] and post-stroke anxiety [6] as well as mitigated depression-like symptoms [13], neurotoxicity in Huntington's disease (HD) [14–16] and memory impairments in Alzheimer's disease (AD) [17–20]. Here, we outline the mechanisms of HDAC3 in different cell types and disease states and how pharmacological inhibition of HDAC3 can be leveraged to address molecular, neuroimmune and behavioral deficits in brain injury and disease. We highlight the dual aspect of HDAC3 in promoting both neuroinflammation and neuroprotection, with an emphasis on both histone and non-histone mediated regulation of cell type specific gene expression (Figure 1).

Figure 1.

Proposed regulation of inflammation by HDAC3 in microglia. Under inflammatory conditions HDAC3 activates expression of pro-inflammatory proliferation promoting genes, cGas and AIM2 by either deacetylation of NF-κB or direct interactions with NF-κB, PU.1 and potentially other activating transcription factors. HDAC3 also suppresses expression of Arg1 and other genes involved in resolution of inflammation. In the absence of HDAC3, both activating and repressing roles are lost, resulting in the suppression of inflammation and proliferation and the increased expression of Arg1 and pro-resolution genes. HDAC3 inhibitors have similar impacts but the activation of proliferative genes is hypothesized to be maintained, as this activating role of HDAC3 does not require its deacetylase activity. This deacetylase independent activating role is based in part on work in peripheral macrophages and remains to be fully elucidated in microglia in vivo.

2. HATs & HDACs regulate histone acetylation & gene expression

Within cells DNA is wrapped around a histone octamer, whose protein tails interact with DNA to regulate gene expression [21]. Posttranslational modifications to histone tails can alter histone-DNA interactions, either promoting or inhibiting transcription factor access to the underlying DNA. For example, histone acetylation is a key marker of active transcription, both at promoters (histone 3 lysine 9 acetylation; H3K9ac) and distal genomic regulator elements called enhancers (histone 3 lysine 27 acetylation; H3K27ac). These acetylation marks decrease interactions between DNA and histone tails, although this effect is modest [22]. Histone acetylation primarily serves as a permissive mark [23], that promotes transcription through recruitment of transcription factors and nucleosome remodelling complexes containing bromo-domains [24,25]. Acetylation can also recruit the basal transcription machinery [26] and itself can be dependent on new transcription [27].

Histone acetylation is regulated by two classes of opposing enzymes, histone acetyltransferases (HATs) and Histone deacetylases (HDACs). HATs add acetyl groups to histone tail lysines while HDACs remove histone acetylation. Indeed, HAT localization on the genome is positively correlated with histone acetylation levels, Polymerase II binding and gene expression [28]. The association between HATs and actively elongating RNA polymerase [28] could potentially facilitate transcription elongation or nucleosome stability [29]. In mammals there are 18 HDACs that are grouped into four classes: class I (HDAC 1, 2, 3 and 8), class II (HDAC 4, 5, 6, 7, 9 and 10), class III (SIRT 1, 2, 3, 4, 5, 6 and 7) and class IV (HDAC11). Class I HDACs are exclusively nuclear proteins, except for HDAC3 which shuttles back and forth between the nucleus and cytoplasm [30]. Different classes of HDACss have distinct roles in both normal physiology and disease contexts. For example, Class I HDACs, such as HDAC1 and HDAC2, are primarily involved in cell cycle control and neural development, while Class II HDACss are implicated in heart, skeleton and endothelial cell development [31]. HDAC3 is particularly noteworthy due to its unique dual role in both neuroinflammation and neuroprotection, making it a critical regulator in neurological disorders. Unlike other HDACs, HDAC3 has been shown to interact with a variety of cofactors and non-histone proteins, allowing it to fine-tune cell type specific gene expression [2]. This dual functionality of HDAC3, especially in the context of histone and non-histone mediated regulation, makes it a key focus of study, particularly in neurodegenerative diseases and brain injury where the balance between inflammation and protection is crucial. By exploring the specific role of HDAC3, we can gain deeper insights into its therapeutic potential in neurological disorders, where modulating its activity might influence disease progression.

When Class I HDACs were profiled across the genome of CD4⁺ Tcells, they were surprisingly bound at mostly actively transcribed genes and their occupancy correlated with histone acetylation levels [28]. This result supported a model by which HATs and HDACs compete for histone substrates, with HDACs removing histone acetylation after active transcription to regulate gene turn off and reset chromatin. To complicate matters further, both HATs and HDACs can have non-histone targets for acetylation and deacetylation. Many of these targets are transcription factors or transcription regulators, making manipulations of HATs and HDACs often difficult to interpret. Together, the literature supports complex roles for HATs and HDACS in transcriptional regulation through histone and non-histone targets.

Much of our understanding of the role of HDAC3 in different biological processes comes from the use of pharmacological inhibitors of all Class I HDACs or specifically HDAC3. These HDAC3 inhibitors have shown promise in preclinical studies for their potential therapeutic applications in various diseases, including neurodegenerative disorders and cancer. The specific HDAC3 inhibitor RGFP966 has been shown to enhance gene expression required for brain plasticity [1,32], modulate inflammatory gene expression in macrophages and microglia [2,33] and critically, cross the blood–brain barrier to enter the central nervous system [34]. HDAC3 inhibition with BRD3308 has improved symptoms in models of intraventricular hemorrhage by modulating microglial state [3]. Both RGFP966 and BRD3308 have been designed to specifically target HDAC3’s catalytic function without broadly disrupting its interactions within the co-repressive complex. Ensuring that these inhibitors only block HDAC3’s enzymatic activity without broadly affecting the NCoR/SMRT complexes remains a significant challenge. However, inhibition of HDAC3 has revealed its involvement in multiple mechanisms controlling gene regulation and underscores the significance of its deacetylase activity in various diseases.

3. HDAC3 as a regulator of histone & non-histone acetylation

HDAC3 is found in both the nucleus and the cytoplasm, with the ability translocate between them [30]. The deacetylase activity of HDAC3 is dependent on its interactions with the deacetylase activating domain of either the SMRT (silencing mediator for retinoid and thyroid receptors) or N-CoR (nuclear receptor corepressor) nuclear co-repressors [35,36]. HDAC3 does not contain a DNA binding domain but is instead targeted to specific genes as part of a larger repressor complex that includes N-CoR and SMRT [35,37,38]. Being the only HDAC found in the N-CoR/SMRT complex [39], HDAC3 serves as the catalytic component of the complex, providing a mechanistic link between histone deacetylation and transcriptional repression. Through their interactions with various transcription factors, the N-CoR/SMRT corepressors recruit HDAC3 to specific promoters, where the enzyme deacetylates histones and mediates silencing of the corresponding genes.

HDAC3 can deacetylate a number of non-histone proteins, including transcriptional regulators [40], which may lead to changes in transcriptional regulation. For instance, N-CoR [41] and HDAC3 [42] directly interact with CBP, and HDAC3 can deacetylate both P300 [43] and CBP [42], inhibiting their HAT functions. HDAC3 can also deacetylate NF-κB subunit RelA, promoting its nuclear export and termination of NF-κB signaling [44], as well as transcription factor myocyte enhancer factor 2 [43] and Stat3 [45]. Together, these non-histone targets of HDAC3 can mediate both transcription dependent and independent impacts on cell function.

Histone and non-histone regulation by HDAC3 are not mutually exclusive. For example, HDAC3 serves as a negative regulator of the nuclear receptor PPARγ by several mechanisms. At baseline, PPARγ activity is repressed by the corepressor complex containing HDAC3 and SMRT or NCoR. The corepressor is removed following ligand-dependent binding, activating adipocyte gene expression [46]. HDAC3 can also regulate suppression of PPARγ activity in response to TNF-α by non-histone mechanisms. Under basal conditions in adipocytes, HDAC3 is associated with IκBα in the cytoplasm. After IκBα degradation in response to TNF-α treatment, HDAC3 translocates into the nucleus and inhibits the transcriptional activity of PPARγ through deacetylation [47]. Inhibition of HDAC3 deacetylase activity has been shown to enhance PPARγ acetylation and induce target gene expression even in the absence of ligand in male mice [48], suggesting HDAC3 is sufficient to repress PPARγ activity by deacetylation in males.

4. HDAC3 regulates distinct targets & functions in different cell types

Total knockout of HDAC3 in mice results in embryonic lethality prior to embryonic day 8.5 [49–51]. However, conditional and cell type specific manipulations of HDAC3 have revealed novel roles for HDAC3 regulation of both histone and non-histone acetylation, which vary depending on age, disease, treatment and cell type. The majority of mechanistic work on HDAC3 has focused on macrophage regulation, which parallel some of the mechanisms identified in brain microglia. We describe impacts in each major brain cell type and specifically compare microglia to other macrophages populations.

4.1. Oligodendrocytes, schwann cells, & astrocytes

Conditional HDAC3 knockout in the developing central nervous system using Nestin-cre expression, severely disrupted brain cytoarchitecture resulting in lethality within 24 h of birth. Deficits included disruption of neuronal migration, increased astrocytes and reduced oligodendrocytes [52]. Further investigation revealed that HDAC3 directly targets and activates Olig2 expression, a major lineage determining transcription factor for oligodendrocyte differentiation. In the absence of HDAC3, oligodendrocyte progenitors differentiate ectopically into astrocytes, disrupting brain myelination. In mature oligodendrocytes, HDAC3 localized to P300-bound enhancers associated with genes elevated during oligodendrocyte maturation, suggesting HDAC3 serves as an activating signal for oligodendrocyte differentiation. In parallel, HDAC3 suppressed astrocyte differentiation genes by deacetylating STAT3, a key transcription factor required for astrocyte lineage differentiation [45]. Consequently, in the absence of HDAC3, Olig2 and other oligodendrocyte genes were not induced and the repression of astrocyte lineage removed, driving ectopic differentiation to astrocytes.

HDAC3 serves as a negative regulator of schwann cell differentiation. RGFP966 treatment during early postnatal development increased expression of myelination-associated genes, increased myelin thickness, as well as the percentage of myelinated axons of the sciatic nerve. Conditional deletion of HDAC3 in schwann cells during development resulted in over-myelination to the point of causing axonal damage by adulthood. Similar deletions in adulthood produced modest increases in myelination, consistent with lower expression of HDAC3 in adult schwann cells [53]. Together, this suggests that HDAC3 normally inhibits schwann cell maturation. Loss of HDAC3 in schwann cells led to increased H3K27ac at the enhancers of myelination-associated genes and increased expression of genes that promote myelination. Surprisingly, an additional subset of enhancers were bound by both HDAC3 and P300 that appear to positively regulate genes that are negative regulators of myelination. In the absence of HDAC3, these negative regulators are decreased in expression, thus promoting myelination [53].

HDAC3 plays a critical role in regulating astrocyte reactivity. Inhibition of HDAC3 deacetylase activity blocked the induction of reactive astrocytes by inhibiting pro-inflammatory gene expression [54]. HDAC3 inhibition led to genome wide increases in acetylation, however these changes were not correlated with changes in gene expression, suggesting additional non-histone mediated effects. HDAC3 inhibition increased RelA/p65 acetylation, driving nuclear export, alongside decreased NF-κB binding and decreased expression of downstream genes related to reactive astrocytes. HDAC3 inhibition also led to an increase in expression of genes associated with protective astrocytes states. In vivo, HDAC3 inhibition or astrocyte-specific conditional knockout of HDAC3 decreased the formation of pathological reactive astrocytes following Lipopolysaccharide (LPS) injection [54]. Reactive astrocytes are common to several brain disorders [55], therefore these findings support a potential therapeutic role for HDAC3 inhibition in dampening astrocyte reactivity in disease.

4.2. Neurons

Contrary to its proposed role as a transcriptional repressor, HDAC3 has been associated with increased gene expression during neuronal development [56]. Postnatal neuron-specific knockout of HDAC3 resulted in live animals that progressively developed hind limb paralysis, ataxia and death by 6 weeks of age due to degeneration of Purkinje neurons [52]. A similar neuron-specific conditional HDAC3 knockout mouse showed impaired coordination, sociability and long-term memory [56]. Interestingly, postnatal neuron-specific HDAC3 knockout did not produce significant increases in histone acetylation and HDAC3 occupancy was unexpectedly enriched at active gene promoters and the majority of differentially expressed genes in the knockout were decreased in expression. Among these were immediate early genes critical for neuronal development and plasticity, including Arc, Fos and Nr4a1. Furthermore, HDAC3 knockout impaired deacetylation of the FOXO3 transcription factor, preventing its recruitment to gene promoters and hence impairing activation of gene expression [56]. Together this work suggests that HDAC3 plays a role in promoting gene expression during neuronal development and that loss of HDAC3 severely impairs neuronal gene expression and proper brain function.

However, in adulthood, HDAC3 regulates neuronal plasticity and immediate early genes in the opposite direction. Impairing HDAC3 function in adulthood through conditional genetic deletion, pharmacological inhibition, or expression of a deacetylase dead mutant HDAC3 enhances learning-induced gene expression and memory formation in a wide variety of learning paradigms, brain regions and species [1,34,57–62]. This led Wood and colleagues to propose the “molecular brake-pad hypothesis” of HDAC3 function in adult neurons: HDAC3 is normally required to prevent gene expression and its removal is necessary for the induction of gene expression underlying the formation of long-term memories [1,57]. One interesting consequence of this hypothesis is that removing the HDAC3 “brake” is predicted to allow full activation of plasticity mechanisms that might not otherwise be engaged. In fact, conditional deletion or inhibition of HDAC3 in adulthood can transform an event that would otherwise be forgotten into a persistent memory [34,57,59,60,63]. For example, a hippocampal-dependent object location memory task, the enhancement of memory by HDAC3 deletion was dependent on the expression of the HDAC3 target gene Nr4a2 [57], a key regulator of plasticity and memory [64]. In the absence of HDAC3, Nr4a2 is hyper-induced even by a subthreshold stimulus, allowing for the engagement of the plasticity mechanisms required to transform short term information into long-term memory. Knockdown of Nr4a2 in the HDAC3 conditional deletion mice prevented this memory enhancement, supporting the role for HDAC3 as a negative regulator of neuronal plasticity-induced gene expression [57].

The contrasting roles of HDAC3 in neuronal development and adult plasticity are striking, as HDAC3 appears to regulate the same gene targets in opposing directions in adulthood and development. This may be due to differences in experimental design, such as long-term knockout across early postnatal development versus conditional adult deletion for several weeks, or brain wide versus region-specific manipulations. Alternatively, HDAC3 may play different roles in regulating neuronal gene expression at different stages of neuronal development or under different conditions. As discussed below, HDAC3 appears to have both activating and repressing functions in macrophages, suggesting that it may serve as more than a “brake-pad” depending on cellular context.

4.3. Macrophages

HDAC3 regulates a unique set of enhancer elements in different macrophage populations [65]. In response to IL4 stimulation, HDAC3 deacetylates a subset of enhancers bound by PU.1 [65], a key transcription factor in determining macrophage lineage. Loss of HDAC3 leads to hyperacetylation of thousands of PU.1 enhancers [66], clearly demonstrating a role for HDAC3 in controlling immune activity through enhancer acetylation. However, loss of HDAC3 produces complex impacts on macrophage gene expression. In one study by Mullican et al., HDAC3 knockout bone marrow-derived macrophage (BMDM) cultures showed an increase in baseline expression of genes involved in alternative macrophage activation and treatment with the alternative activating cytokines IL13 or IL4 further enhanced expression of alternative activation response genes such as Arg1 and Clec7a. Profiling of HDAC3 occupancy in BMDMs revealed HDAC3 localization to intergenic regions and introns. Loss of HDAC3 increased H3K9ac and H3K27ac in regions bound by HDAC3 in wildtype BMDM, supporting its role in repressing IL4 dependent genes by removing histone acetylation [65]. However, Chen et al. found that HDAC3 knockout in BMDM cultures dramatically changed histone H4 acetylation at enhancers after treatment with LPS, with half the sites increasing and half decreasing acetylation levels [66]. Surprisingly, LPS-induced gene expression was severely blunted in the HDAC3 knockouts without impacts on basal gene expression of the same genes [66]. Similarly, deletion of HDAC3 decreased STAT1/2 expression, impairing activation of the antiviral response [67]. Together, these studies indicate HDAC3-dependent mechanisms for suppressing and activating gene expression.

These two seemingly contradictory roles for HDAC3 were mechanistically clarified by Nguyen et al. Using a series of viral approaches to deliver either wildtype HDAC3 or a deacetylase dead HDAC3 mutant to HDAC3 knockout BMDMs, they demonstrated that HDAC3 has both deacetylase-dependent and independent roles in regulating gene expression. When these cultures were treated with LPS, about 1/3 of genes that increased in the WT BMDM failed to increase in the HDAC3 knockout cultures, but were rescued by the deacetylase dead HDAC3 mutant. This demonstrates that the gene-activating effects of HDAC3 in response to LPS treatment are independent of HDAC3’s deacetylase activity [2]. The specificity of HDAC3’s gene regulation appears to rely on key interacting partners. Upon LPS stimulation, HDAC3 was targeted to LPS-induced genes through an interaction with the transcription factor ATF2 and NF-κB. Disruption of HDAC3-ATF2 interaction impaired LPS induced gene expression. In contrast, HDAC3 was recruited by ATF3 and N-CoR1/2 to genes that were repressed upon LPS treatment. Together, this work shows that HDAC3 can switch between activation and repression of different target genes depending on the associated co-factors [2]. To date, the transcription factor dependent activating and suppressing properties of HDAC3 has not been explored in other cell types and so it remains to be seen if this is a conserved or unique role for HDAC3 in BMDMs.

4.4. Microglia

Microglia constitute ∼7% of non-neuronal cells in the mammalian brain [68] and have a conserved core gene program [69] from rodents to humans. Microglial function is regulated at the level of gene transcription [70,71] by utilizing distinct enhancers to adapt to the local brain environment, making their active enhancer repertoire unique from other resident tissue macrophages [72,73]. These enhancers are exquisitely sensitive to the local brain environment, becoming rapidly altered in disease states [74] and in response to in vitro culture conditions [71,75]. Microglial enhancers are specifically enriched for disease associated genetic variants linked to neurodegenerative and neuropsychiatric disorders [76–78], suggesting that disruption of acetylation at microglial enhancers could causally contribute to disease pathology.

There is growing evidence that HDAC3 is a regulator of microglial mediated inflammation. In primary mouse microglial cultures, LPS treatment increased HDAC3 protein levels and deacetylase activity [79]. In BV2 microglial cell cultures, RGFP966 enhanced histone acetylation and gene expression of pro- and anti- inflammatory cytokines at baseline and in response to LPS treatment [33]. This is consistent with the “brake pad” hypothesis observed in neurons. However, similar work in primary microglia cultures found that RGFP966 blunted LPS regulated protein expression [4], suggesting an activating role in protein regulation, potentially through non-histone mediated acetylation. Consistent with this finding, RGFP966 blunted LPS induced increases in Absent in Melanoma 2 (AIM2), a key regulator of the AIM2 inflammasome that controls the cleavage and secretion of IL-1β and IL-18. As expected, RGFP966 also blunted LPS induced IL-1β and IL-18 and shifts in microglial morphology [79]. RGFP966 significantly increased acetylation of STAT1, which AIM2 expression relies on and subsequently impaired AIM2 phosphorylation and activation [79]. Together, these culture findings support HDAC3 as a negative regulator of gene expression through histone acetylation and a positive regulator of protein regulation through non-histone acetylation.

HDAC3 regulation of microglial mediated inflammation has also been examined in vivo. In response to brain damage, translation of HDAC3 was upregulated in microglia, resulting in significant increases in expression within 48–72 h of immune activation in both cortical [79] and spinal cord microglia [80]. Following an ischemic stroke, HDAC3 was upregulated in IBA1+ cells after 24 h, peaked at 72 h and gradually decreased by 7 days post stroke [5,79]. HDAC3 protein in microglia and macrophages at the lesion site post spinal cord injury was similarly increased by 2 days, peaked at 7 days, but was still elevated at 28 days [80]. The damage-induced increase in HDAC3 protein occurs in a heterogenous subset of microglia that appear to confer pro-inflammatory functions [79,80]. The long-lasting increase in HDAC3 following brain and spinal injury suggest that HDAC3 serves a key role in regulating the inflammatory response to damage.

5. HDAC3 inhibition as a therapy for neuroinflammation

Inhibiting HDAC3 deacetylase activity is protective in numerous models of disease and injury where neuroinflammation drives increased HDAC3 expression and altered gene regulation. Here, we describe the different therapeutic applications of HDAC3 inhibition in the context of neuroinflammation across disorders and injury (Supplementary Table S1). Although underlying mechanisms of HDAC3 regulation are emphasized where known, there remains many cell- and context-specific mechanisms through which HDAC3 regulates neuroinflammation and neuroprotection that remain to be fully elucidated.

5.1. Spinal cord & brain injury: microglia & macrophages

HDAC3 protein levels increase in models of brain and spinal cord injury [79,80] and consequently there is growing interest in inhibiting HDAC3 as a potential therapeutic to mitigate inflammation induced brain damage. In a rat model of stroke, post-injury treatment with RGFP966 reduced infarct volume and apoptosis while improving functional recovery [11], neurological deficits and motor impairments [79]. Furthermore, RGFP966 decreased cerebral edema and blood–brain barrier leakage through increased expression of tight junction proteins and decreased expression of pro-inflammatory genes [12]. RGFP966 given acutely post spinal injury improved hindlimb locomotion recovery, spared axonal fibers in the lesion center [80], reduced spinal edema and improved motor recovery [81]. However, delaying RGFP966 treatment to 7 days after injury did not improve behavioral outcomes, suggesting that HDAC3 is important for early inflammatory events following spinal cord injury [82]. RGFP966 treatment before and following a surgically induced brain injury improved neurologic outcomes 24 hours later by repressing the injury induced increase in HDAC3 and increased expression of anti-oxidant proteins [8]. RGFP966 treatment during a Cuprizone diet induced demyelination model of Multiple Sclerosis found significant improvements in motor behaviors, reduced myelin loss and reduced markers of microglial inflammation [9]. Together, these findings support inhibition of HDAC3 as a viable therapeutic approach for mitigating neuroinflammtion.

There is growing evidence for a direct role of HDAC3 in microglial as a mediator of neuroinflammation in response to brain injury and stroke. In a model of traumatic brain injury, loss of microglial HDAC3 increased production of anti-inflammatory cytokines, reduced pro-inflammatory cytokines, reduced axonal injury and demyelination and improved long-term functional recovery of motor skills [10]. Conditional deletion of HDAC3 in microglia produced significantly small infarct volume and enhanced functional recovery of neurologic scores and cognitive performance following stroke. The animals with conditional HDAC3 deletion showed increased myelin and reduced axon degeneration a month post injury, further contributing to behavioral recovery [5]. HDAC3 microglial deletion also reduced infarct volume in ovariectomized female mice, indicating HDAC3 inhibitors may serve as potential therapeutics for women at increased risk for stroke after menopause [5].

Blocking HDAC3 activity or conditionally deleting HDAC3 appears to shift microglia away from a pro-inflammatory state and toward an inflammation-resolving state. Conditional deletion of microglial HDAC3 reduced the number of pro-inflammatory microglia and increased the number of inflammation-resolving microglia 3 days after traumatic brain injury [10]. Similarly, HDAC3 inhibition in models of ventricular hemorrhage significantly reduced damage-induced increases in C68+ microglia and increased the number of ARG1+ microglia, suggesting a shift toward an inflammation-resolving microglial state [83]. RGFP966 treatment significantly shifted gene expression in both microglia and macrophages in spinal cord tissue collected 5 days post injury. Microglia and macrophages shared only approximately 20% of the of HDAC3-dependent genes. Unique HDAC3-dependent genes in microglia included genes involved in synaptogenesis, synaptic long-term potentiation, phosphatidylinositol 3-kinase/AKT signaling and granulocyte-macrophage colony-stimulating factor signaling. In macrophages, HDAC3-dependent genes were involved in xenobiotic metabolism, leukocyte migration and cell survival [82]. Consistent with these finding, previous in vitro work in BV2 microglial cells demonstrated that RGFP966 increases Nos2 and Arg1 expression, suppressing the production of nitric oxide and enhancing phagocytosis activity [33], potentially to facilitate removal of dead and dying cells post injury.

Some impacts of HDAC3 inhibition on stroke outcomes are through non-histone targets. For example, RGFP966 failed to improve outcomes after stroke induced by middle cerebral artery occlusion (MCAO) in AIM2 knockout mice, suggesting the HDAC3 mediated benefits of RGFP966 were through AIM2 [79]. Similarly, HDAC3 upregulates cyclic GMP-AMP synthase (cGAS), a critical mediator of neuroinflammation following stroke, by deacetylating NF-κB, driving its localization to the cGAS promoter and activating Cgas expression. Conditional deletion of HDAC3 in microglia or treatment with RGFP966 significantly reduced cGAS in vivo and was protective following transient MCAO [84]. HDAC3 inhibition also drove an increase in PPARγ and decrease in inflammasome activation markers. Blocking PPARγ activity blocked the beneficial effects of HDAC3 inhibition on microglial state, neuronal death and functional recovery [83]. Together, these findings suggest that HDAC3 improves functional outcomes after stroke through deacetylation of several non-histone target proteins.

5.2. Stroke & injury: blood–brain barrier

Additional cell types may be impacted by systemic HDAC3 inhibition following injury [5]. In cultured human brain microvascular endothelial cells, oxygen glucose deprivation and reoxygenation (OGD/R) that induces an ischemia-like state, increased HDAC3 nuclear translocation and deacetylase activity. Inhibition of HDAC3 with RGFP966 prevented the ORD/R induced transendothelial permeability by increasing PPARγ protein acetylation and preventing downregulation of Claudin-5 expression. Blocking PPARγ activity, prevented the beneficial effects of HDAC3 inhibition, suggesting that HDAC3 is acting through PPARγ [85]. The importance of HDAC3 at the blood–brain barrier is further supported by mouse models of type 2 diabetes, where HDAC3 protein levels are increased in the brain. Inhibition of HDAC3 activity through RGFP966 ameliorated diabetes driven leakiness in the blood–brain barrier and increased tight junction protein expression [86]. HDAC3 inhibition also impacted gene expression in ependymal, oligodendrocytes, astrocytes and fibroblasts after injury, indicating that microglia and macrophages may only be contributing part of the protective effects of HDAC3 inhibition on functional recovery [82]. These findings suggest that systemic delivery of HDAC3 inhibitors may impact both immune and blood–brain barrier cells following brain damage.

5.3. Stress, anxiety & depression

The heritability of severe recurrent depression is estimated to be between 25–36% [87,88]. These levels are about half of the genetic risk of other neuropsychiatric disorders like schizophrenia (67%) and bipolar disorder (62%) [88], suggesting a significant role for environmental factors in MDD. Indeed, early life adversity consistently predicts the onset of MDD [89]. A causal relationship between environmental stressors and depression-like behavior has been further supported by rodent studies through chronic stress [90], social defeat [91] and maternal separation paradigms [92]. Emerging evidence identifies epigenetic differences among humans with MDD, which likely arises from an interaction between genetic risk factors and environmental stressors. Single nucleotide polymorphisms in HDAC3 have been observed in adolescents with high psychiatric disorder risk [93], which aligns with the observation that MDD patients express significantly more H3K14 acetylation in the nucleus accumbens, a key brain region implicated in MDD, relative to matched healthy controls [94]. Together, these data identify altered epigenetic regulation as a candidate mechanism for mediating the pathophysiology of MDD.

Alterations in histone acetylation may contribute to inflammatory symptoms observed in patients with depression who exhibit shifts in peripheral inflammatory cytokines, such as increases in TNF-α, IL-6, IL-13 [95–97] and decreases in IL1-β and IL-2 [96]. Several rodent models of depression demonstrate both altered histone acetylation and signatures of neuroinflammation. For example, the chronic unpredictable stress (CUS) rodent model of depression produces increased CD11b+ microglia, peripheral immune cells [98] and IBA1 density [99]. Within one hour of the defeat stress session, H3 acetylation is decreased in the nucleus accumbens. This decrease in histone acetylation was transient and followed by a compensatory increase that lasted for at least two weeks, similar to the increase observed in humans with MDD. The transient decrease was proposed to contribute to the observed depressive behaviors, as treatment with an HDAC inhibitor rescued the deficits [94]. A similar study found that rats with less resilience to defeat stress showed increased acetylation at multiple histone sites in the dorsal raphe nucleus, medial prefrontal cortex and ventral hippocampus 24 hours after the last stress session. The impacts were brain region specific, with basolateral amygdala, locus coeruleus, paraventricular thalamus and dorsal hippocampus showing no stress dependent impacts on histone acetylation [100]. Both studies observed no differences in mRNA levels of Hdac3 with defeat stress [94,100]. However, HDAC3 nuclear protein expression increased only in resilient animals relative to all other groups [101], suggesting regulation at the protein level similar to that observed in injury models. The seemingly contradictory findings in resilient animals of increased HDAC3 protein and increased histone acetylation may be due to differences in timing of tissue collection, brain regions analyzed and in the behavioral paradigms examined. However, the ability of Class I (HDACs 1, 2 and 3) inhibitors to exert antidepressant-like effects [94,102,103] warrants further investigation into the mechanisms of HDAC regulation in MDD and animal models of depression.

Neuroinflammatory symptoms co-occurring with depressive symptoms can be investigated by modelling the inflammatory aspects of MDD in rodents through repeated exposure to LPS. Repeated LPS administration can result in the onset of depression-like behavioral endophenotypes [104,105], deficits in hippocampal long-term potentiation (LTP) [106,107], cognition [108] and increased hippocampal microglial activation, which has been correlated with behavioral deficits [105]. Similar to the pattern of MDD onset in humans, depression-like symptoms are inducible in rodents during adulthood [104] and development [109,110] and persist beyond LPS induced sickness. Pre-treating mice with RGFP966 prior to LPS injection rescued LPS-induced depression-like endophenotypes in forced swim and open field task [13]. RGFP966 also attenuated LPS induced microglial IBA1 and IL-1β increases [13], suggesting that targeting microglial HDAC3 may alleviate brain and behavior deficits in immune induced depression models.

After stroke recovery, a subset of patients develop phobic anxiety [111]. A mouse model of post stroke phobic anxiety that combined photothrombotic stroke with restraint stress showed that the combination produced anxiety-like behaviors, microglial inflammation and increased local prostaglandin E2 (PGE₂) production. Interestingly, HDAC3 protein levels were increased in microglia in the damaged cortex and inhibiting HDAC3 deacetylase activity during the restraint stress reduced anxiety-like behaviors, reversed increases in microglial CD68 and PGE₂. Mechanistically, HDAC3 inhibition increased NF-κB acetylation and nuclear export, driving decreases in PGE₂ production and lower anxiety-like behaviors [6]. Further evidence for the role of HDAC3 in driving depression-like behaviors comes from studies on esketamine, an isomer of ketamine. When administered during the post-stroke restraint stress period, esketamine rescued anxiety behaviors and blunted changes in microglial morphology and pro-inflammatory cytokines. Additionally, esketamine treatment blunted increases in HDAC3, NF-κB acetylation, COX1 and PGE₂ expression in the stroke + restraint condition. Further in vitro experiments support that the impacts of esketamine in vivo were mediated by repression of HDAC3 in microglia [7].

Rodent studies have further investigated the use of HDAC3 inhibition in tandem with selective serotonin reuptake inhibitors (SSRIs). Cotreatment with the HDAC inhibitor sodium butyrate enhanced the antidepressant and anxiolytic effect of fluoxetine in the forced swim task [112]. Similarly, cotreatment with the selective HDAC 1 and 3 inhibitor MS-275 and the broad HDAC inhibitor Trichostatin A, eliminated depression-like behavior in a maternal separation mouse model of depression [112]. Direct inhibition of Class I HDACs exerted both antidepressant-like effects [94,102,103] and anti-inflammatory effects [13] in other mouse models of depression. Ultimately, human and rodent studies converge to suggest that HDAC3 inhibition may successfully alleviate or augment the treatment of behavioral and mood deficits in MDD.

5.4. Neurodegeneration

There is growing, but often mixed, evidence to support the use of HDAC inhibitors in neurodegeneration. HDAC3 appears to promote disease development and progression and knockdown or suppression of HDAC3 activity improves disease phenotypes and pathologies. For example, over-expression of HDAC3 in neuronal cell cultures induced cell death and HDAC3 knock-down was protective against several neurotoxic treatments [113]. In Huntington's disease (HD), Alzheimer's disease (AD) and Parkinson's disease (PD) there is growing evidence that HDAC3 promotes disease progression, but the findings are not unanimous nor conclusive on if HDAC3 inhibitors have significant therapeutic benefits for treating these diseases.

HD arises from an expansion of polyglutamine repeats in the huntingtin (HTT) protein. The disease is attributed to both the loss of normal HTT function and the acquisition of toxic properties by the mutated polyglutamine-expanded HTT protein. One of the functions of wildtype HTT is to sequester HDAC3 in the cytoplasm. The HTT-HDAC3 interaction was greatly reduced for mutant HTT and expression of mutant HTT out competed wildtype, releasing HDAC3 to enter the nucleus [114], potentially contributing to epigenetic misregulation of gene expression in HD [115]. Indeed, the R6/2 HD mouse model exhibited HDAC1 and 3 accumulation in the nucleus and depletion from the cytoplasm, suggesting a shift in abundance that may drive misregulation of gene expression in HD [116]. In line with this hypothesis, knockdown of HDAC3 blunted mutant HTT neurotoxicity [114]. Treatment of the R6/2 mice with an HDAC1/3 inhibitor attenuated striatal atrophy, improved motor performance and reversed histone hypo-acetylation and alterations in gene expression across several brain regions [14]. Furthermore, RGFP966 improved motor deficits and reduced negative impacts on striatal volume in the N171–82Q transgenic mouse model of HD [15]. Early chronic RGFP966 treatment of Hdh^Q111 knock-in mice, another HD model, prevented long-term memory impairments and corticostriatal-dependent motor learning deficits. RGFP966 also partially rescued striatal protein marker expression and reduced accumulation of mutant HTT oligomers [16]. However, crossing the R6/2 mouse to HDAC3 heterozygous deletion mice did not ameliorate molecular, physiological or behavioral phenotypes [50], suggesting that more complete inhibition of HDAC3 activity is required for HD treatment.

AD is characterized by the progressive accumulation of amyloid (Aβ) plaques and hyper-phosphorylated tau tangles, which trigger loss of neuronal synapses, neuronal death and memory deficits. HDAC3 protein, but not HDAC1 or 2, is increased in the frontal cortex of AD post-mortem human brain samples [117]. In vitro, addition of soluble Aβ oligomers to cultured neurons was sufficient to increase HDAC3 protein levels [118] and HDAC3 can bind to the amyloid precursor protein promoter and suppress its transcription [117]. In vivo, the transgenic AD mouse model APPswe/PS1dE9 (APP/PS1) showed increased nuclear HDAC3 in the hippocampus at 6 and 9 months of age [119]. Hippocampus-specific HDAC3 knockdown in these mice attenuated spatial memory deficits and decreased Aβ brain levels. The HDAC3 knockdown also attenuated hippocampal IBA1 expression and improved dendritic spine density in 9-month-old APP/PS1mice [119]. In 6-month-old APP/PSI mice, knockdown of HDAC3 reduced markers of lipid peroxidation, protein oxidation, DNA/RNA oxidation and decreased apoptosis [120]. In comparison, HDAC3 over-expression in the hippocampus of APP/PS1 mice further impaired spatial memory deficits and increased Aβ levels and IBA1 expression, while decreasing dendritic spine density [119]. Together, these conditional deletion and over-expression studies suggest a key role for HDAC3 in AD.

Pharmacological inhibitors of HDAC3 have also been utilized in AD mouse models. Inhibition of HDAC1 and 3 with RG2833 in the TgF344-AD mouse model of AD mitigated spatial memory deficits, while increasing synaptic plasticity and immediate early gene expression in females [17]. HDAC3 inhibition with RGFP966 attenuated impaired long-term potentiation induced by Aβ_1–42 oligomers [18] and reversed age-induced impairments in synaptic plasticity through activation of NF-κB [19]. RGFP966 improved spatial memory in the triple transgenic AD mouse model (3xTg-AD) while reversing tau hyper-phosphorylation and decreasing Aβ_1–42 protein levels. RGFP966 also decreased Aβ_1–42, tau phosphorylation in neurons derived from induced pluripotent stem cells from AD patients [20]. RGFP966 rescued changes in microglial and astrocyte morphology and density in organotypic brain cultures from 5xFAD AD model mice. In the 5xFAD cultures, HDAC3 inhibition increased expression of genes promoting synapse function and nervous system development, driving the recovery of synapse deficits [117]. Together, the majority of these findings support positive therapeutic potential for HDAC3 inhibitors in mitigating both AD pathology and memory impairments. However, RGFP966 treatment in 6 month old APP/PS1mice did not improve long-term fear memory [121], suggesting that the effects of HDAC3 inhibition on AD memory deficits may be specific to brain region or memory task.

PD is defined by the gradual and progressive degeneration of dopaminergic neurons located in the substantia nigra. There are several known genetic risk genes for PD including PINK1 and LRRK2, both of which interact with HDAC3 and phosphorylate Ser424 of HDAC3 to enhance its deacetylase activity [122,123]. However, the impacts of the enhanced HDAC3 activity appear to have opposing effects. PINK1-mediated phosphorylation of HDAC3 in the cytoplasm prevented HDAC3 cleavage and degradation in response to oxidative damage. PINK1 phosphorylation of HDAC3 also increased HDAC3 mediated deacetylation of the transcription factor p53, reducing the ability of p53 to bind and induce expression of pro-apoptotic genes. The net result of the PINK1-HDAC3 interaction promoted neuronal survival by attenuating oxidative stress-induced damage and induction of apoptosis. PD mutant PINK fails to phosphorylate HDAC3-Ser424, increasing vulnerability to p53-dependent neuronal apoptosis [122]. These findings suggest that augmenting, not inhibiting, HDAC3 deacetylase activity may be protective for PD.

However, findings with LRRK2 directly oppose the potential therapeutic benefits of enhancing HDAC3 activity in PD. LRRK2 also phosphorylates HDAC3 at Ser-424, increasing HDAC3 nuclear localization and deacetylase activity. The PD G2019S mutation that increases LRRK2 activity further enhanced HDAC3 nuclear translocation. There were also elevated levels of phosphorylated HDAC3-S424 and decreased histone acetylation in the brains of 12–16 month-old transgenic mice expressing LRRK2-G2019S. Overexpression of phosphorylation-resistant HDAC3 or treatment with a pan-HDAC inhibitor was protective from LRRK2-induced cellular toxicity in neuronal cells. Together, the data on LRRK2 support a model in which the hyperactive PD mutant LRRK2 promotes HDAC3 phosphorylation and activity leading to decreased histone acetylation and increased vulnerability to cytotoxicity [123]. However, the gene targets mediating this effect were not examined in this study nor was a specific HDAC3 inhibitor tested in the PD mouse model. Together, the PINK1-HDAC3 interaction in the cytoplasm and LRRK2-HDAC3 interactions driving HDAC3 nuclear translocation, appear to engage distinct mechanisms of non-histone and histone deacetylation respectively. Resolving the conflicting findings on HDAC3 in promoting or protecting neurons from PD pathology in different PD risk gene models will be required before moving HDAC3 inhibitors or activators forward in the clinic to treat PD patients.

6. Conclusion

HDAC3 has multiple roles in regulating gene expression, including deacetylation of histone and non-histone proteins, with its functional impact varying across different brain cell types. Intriguingly, HDAC3 inhibition has been shown to promote the resolution of inflammation across various models of injury and disease. Specifically, pharmacological inhibition or conditional deletion of HDAC3 in microglia facilitates a shift toward an inflammation-resolving state. This inhibition also shows promise in addressing pathological and behavioral symptoms in neuropsychiatric and neurodegenerative disorders where HDAC3 is implicated in their onset and progression. However, it should be noted that many behavioral studies on HDAC3 inhibition have been conducted exclusively in male rodents, thus limiting the current generalizability of these results.

Regardless, it is evident that HDAC3 plays a multifaceted role of in regulating both neuroinflammation and neuroprotection, illustrating how it can promote seemingly opposing processes within the central nervous system. This dual function is highlighted by its ability to modulate cell type specific gene expression through both histone- and non-histone-mediated mechanisms. On the one hand, HDAC3 contributes to neuroinflammation by deacetylating histones, leading to the repression of inflammation resolving pathways. On the other hand, HDAC3 also plays a neuroprotective role by interacting with non-histone proteins, influencing transcription factors and signaling pathways that promote cell survival and repair. By exerting influence on both histone modifications and non-histone protein functions, HDAC3 coordinates a complex balance between promoting inflammation and supporting neuroprotection, depending on the context and cell type. This duality underscores HDAC3's critical regulatory function in the brain's response to injury and disease.

7. Future Perspective

RGFP966, a selective HDAC3 inhibitor, has demonstrated efficacy in ameliorating depression-like behaviors and post-stroke anxiety-like symptoms in rodent models. Additionally, RGFP966 has been employed in HD, PD and AD neurodegeneration models with the majority of work showing positive impacts on pathology and behavioral deficits. Despite promising results, HDAC3 inhibitors remain underexplored in the context of brain disorders, especially in female animal models. Preliminary evidence suggests that HDAC3 inhibition could have significant therapeutic potential, particularly in MDD and stress-related disorders. Further research is required to fully understand the potential of HDAC3 inhibitors as a therapeutic strategy in these conditions. With continued investigation, HDAC3 inhibition could become a cornerstone in the development of epigenetic therapeutics and treatment of a wide range of neurological, psychiatric and neurodegenerative disorders.

Funding Statement

This work was funded by the Canadian Institutes of Health Research CGS-M to C Rosete; Canadian Institutes of Health Research (CRC-RS 950–232402, AWD-025853, AWD-025854 to AV Ciernia); Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-04450 to AV Ciernia); Michael Smith Health Research Foundation (AWD-005509 to AV Ciernia); and the Brain Foundation to AV Ciernia.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17501911.2024.2419357

Author contributions

C Rosete and AV Ciernia contributed to the writing and editing of the manuscript together.

Financial disclosure

This work was funded by the Canadian Institutes of Health Research CGS-M to C Rosete; Canadian Institutes of Health Research (CRC-RS 950–232402, AWD-025853, AWD-025854 to AV Ciernia); Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-04450 to AV Ciernia); Michael Smith Health Research Foundation (AWD-005509 to AV Ciernia); and the Brain Foundation to AV Ciernia. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

AI based tools (Chat GPT Version 4.0) were used to identify and summarize research findings in preparation for this manuscript.