Elsevier

Neurobiology of Disease

Volume 36, Issue 2, November 2009, Pages 269-279
Neurobiology of Disease

Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo

https://doi.org/10.1016/j.nbd.2009.07.019Get rights and content

Abstract

Histone deacetylase inhibitors (HDACi) are emerging tools for epigenetic modulation of gene expression and suppress the inflammatory response in models of systemic immune activation. Yet, their effects within the brain are still controversial. Also, whether HDACs are expressed in astrocytes or microglia is unclear. Here, we report the identification of transcripts for HDAC 1–11 in cultured mouse glial cells. Two HDACi such as SAHA and ITF2357 induce dramatic increase of histone acetylation without causing cytotoxicity of cultured cells. Of note, the two compounds inhibit expression of pro-inflammatory mediators by LPS-challenged glial cultures, and potentiate immunosuppression triggered by dexamethasone in vitro. The anti-inflammatory effect is not due to HDACi-induced transcription of immunosuppressant proteins, (including SOCS-1/3) or microRNA-146. Rather, it is accompanied by direct alteration of transcription factor DNA binding and ensuing transcriptional activation. Indeed, both HDACi impair NFκB-dependent IκBα resynthesis in glial cells exposed to LPS, and, among various AP1 subunits and NFκB p65, affect the DNA binding activity of c-FOS, c-JUN and FRA2. Importantly, ITF2357 reduces the expression of pro-inflammatory mediators in the striatum of mice iontophoretically injected with LPS. Data demonstrate that mouse glial cells have ongoing HDAC activity, and its inhibition suppresses the neuroinflammatory response because of a direct impairment of the transcriptional machinery.

Introduction

Dynamics of chromatin remodeling are crucial for transcriptional activation and gene expression. Among the various mechanisms underpinning chromatin unravelling, histone acetylation is one of the better characterized at the molecular level (Jenuwein and Allis, 2001). A complex network of signaling pathways converges on histone acetyl transferases leading to transfer of acetyl groups originating from acetyl-CoA to lysine residues of histone tails, thereby prompting electrostatic repulsion from nucleosome histones and DNA, resulting in chromatin decondensation. This allows the binding of transcription-regulating factors and RNA pol-II activation. Different repressors of gene transcription, in turn, recruit histone deacetylases (HDACs) to promoters, prompting chromatin compaction and gene silencing. Of note, HATs and HDACs also target transcription-regulating proteins different from histones such as the specific transcription factors NFκB, p53, Sp1 and others (Dokmanovic and Marks, 2005, Glozak et al., 2005c).

Epigenetic pharmacology witnessed a real explosion of interest in the development of inhibitors of HDAC (HDACi) (Yoo and Jones, 2006). These are potent compounds able to unbalance cellular acetylation toward a hyperacetylation status, thereby affecting overall gene expression profiles (Xu et al., 2007). Interestingly, HDACi are currently evaluated in several clinical trials for neoplastic disorders (Minucci and Pelicci, 2006). In this light, the potent HDACi suberoylanilide hydroxamic acid (SAHA) has significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients, and has been recently approved for the treatment of cutaneous T-cell lymphoma (Marks, 2007). Preclinical and clinical evidence indicates that HDACi are also of potential therapeutic relevance to disorders of the central nervous system (Kazantsev and Thompson, 2008). Indeed, perturbation in acetylation homeostasis is emerging as a central event in the processes leading to neuronal death [see (Langley et al., 2005, Saha and Pahan, 2006, Abel and Zukin, 2008, Hahnen et al., 2008) for comprehensive reviews]. In keeping with this, recent studies demonstrate that HDACi of various chemical classes afford protection in models of Huntington's disease (Gardian et al., 2005, Ferrante et al., 2003, Hockly et al., 2003), spinal muscular atrophy (Chang et al., 2001), amyotrophic lateral sclerosis (Corcoran et al., 2004, Ryu et al., 2005, Petri et al., 2006), experimental autoimmune encephalomyelitis (Camelo et al., 2005) and stroke (Ren et al., 2004, Faraco et al., 2006, Kim et al., 2007). Experimental evidence indicates that inhibition of HDAC significantly affects immune cell activation (Blanchard and Chipoy, 2005), an effect that might underlie the neuroprotective effects of HDACi. However, the mechanisms responsible for HDACi-dependent immunosuppression have not been understood, and both potentiating and suppressing effects by HDACi on neuroimmune activation have been reported (Suuronen et al., 2005, Suuronen et al., 2006, Suuronen et al., 2003, Zhang et al., 2008, Chen et al., 2007, Kim et al., 2007).

Hence, in the present study we sought to determine the pharmacodynamic effects of the potent HDACi suberoylanilide hydroxamic acid (SAHA) and its structural analog ITF2357 on acetylation levels and immune activation of glial cells in vitro and in vivo. We also investigated the mechanisms through which HDACi modulate the inflammatory glial response.

Section snippets

Mouse primary glial cultures

Primary mixed cultures of astrocytes and microglia (referred as “glial cells”) were prepared from post natal day 1 mice as previously described (Chiarugi and Moskowitz, 2003) and grown in DMEM + 10% fetal bovine serum. Briefly, cortices were isolated in cold phosphate-buffered saline (PBS) and then incubated 10 min at 37 °C in phosphate-buffered saline containing 0.25% trypsin and 0.05% DNase. After blocking enzymatic digestion with the addition of 10% heat-inactivated fetal bovine serum,

Effects of different HDACi on HDAC activity, viability and histone H3 acetylation levels in glial cells

The brain distribution of the eleven HDACs has been recently reported in the rat (Broide et al., 2007). Surprisingly, HDAC isoforms are expressed primarily in neurons, with a subset also found in oligodendrocytes. No expression is detected in astrocytes or microglia (Broide et al., 2007). To confirm these findings, we analyzed transcripts for the different HDAC isoforms in primary mixed glial cell cultures. As shown in Fig. 1A, mRNAs for all the known HDACs belonging to Classes I (1–3, 8), II

Discussion

It is now well appreciated that glia activation and ensuing production of inflammatory mediators can be detrimental for neural cell functioning and survival. Hence, a great deal of effort has been directed at the development of pharmacological strategies targeting the inflammatory response within the brain (Allan and Rothwell, 2001). Still, with the exception of corticosteroid, efficacious drugs that are able to counteract acute as well as chronic neuroinflammation are still an unmet need. In

Acknowledgments

This study was supported by the University of Florence, the Italian Ministry of University and Scientific and Technological Research PRIN 2007, Associazione Italiana Sclerosi Multipla, Ente Cassa di Risparmio di Firenze and Italfarmaco SpA.

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