Neuroprotection by Histone Deacetylase-Related Protein<sup><a href="#fn1" rid="fn1" class=" fn">†</a></sup>

Brad Morrison

Nazanin Majdzadeh

Xiaoguang Zhang

Aaron Lyles

Rhonda Bassel-Duby

Eric Olson

Santosh D'Mello

Department of Molecular and Cell Biology, University of Texas at Dallas, 2601 N. Floyd Rd., Richardson, Texas 75083,^{Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390}²

^{Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Texas at Dallas, 2601 N. Floyd Road, Richardson, TX 75083. Phone: (972) 883-2520. Fax: (972) 883-2409. E-mail:}ude.salladtu@ollemd.

Received 2005 Jul 27; Revised 2005 Sep 10; Accepted 2006 Feb 7.

Abstract

The expression of histone deacetylase-related protein (HDRP) is reduced in neurons undergoing apoptosis. Forced reduction of HDRP expression in healthy neurons by treatment with antisense oligonucleotides also induces cell death. Likewise, neurons cultured from mice lacking HDRP are more vulnerable to cell death. Adenovirally mediated expression of HDRP prevents neuronal death, showing that HDRP is a neuroprotective protein. Neuroprotection by forced expression of HDRP is not accompanied by activation of the phosphatidylinositol 3-kinase-Akt or Raf-MEK-ERK signaling pathway, and treatment with pharmacological inhibitors of these pathways fails to inhibit the neuroprotection by HDRP. Stimulation of c-Jun phosphorylation and expression, an essential feature of neuronal death, is prevented by HDRP. We found that HDRP associates with c-Jun N-terminal kinase (JNK) and inhibits its activity, thus explaining the inhibition of c-Jun phosphorylation by HDRP. HDRP also interacts with histone deacetylase 1 (HDAC1) and recruits it to the c-Jun gene promoter, resulting in an inhibition of histone H3 acetylation at the c-Jun promoter. Although HDRP lacks intrinsic deacetylase activity, treatment with pharmacological inhibitors of histone deacetylases induces apoptosis even in the presence of ectopically expressed HDRP, underscoring the importance of c-Jun promoter deacetylation by HDRP-HDAC1 in HDRP-mediated neuroprotection. Our results suggest that neuroprotection by HDRP is mediated by the inhibition of c-Jun through its interaction with JNK and HDAC1.

Abstract

Apoptosis is an essential aspect of normal nervous system development, but when aberrantly activated, apoptosis leads to undesirable neuronal death, and such inappropriate neuronal loss is the hallmark of a variety of neurodegenerative diseases and neurological conditions, such as stroke or traumatic brain injury. The mechanisms underlying the regulation of apoptosis are beginning to be understood. Among the molecules that have recently been implicated are the histone deacetylases (HDACs). HDACs are the catalytic subunits of multiprotein complexes that deacetylate histones (11, 42). The action of HDACs is opposed by histone acetyltransferases (HATs) such as CREB-binding protein and p300, which catalyze the transfer of an acetyl moiety from acetyl-coenzyme A to specific lysine residues of histones (25). Acetylation of histones relaxes the chromatin structure to a state that is transcriptionally active, while histone deacetylation transforms chromatin to a transcriptionally repressed state (25). Hence, gene expression is regulated, in part, by the balance of HDAC and HAT activities. Although best studied for their effects on histones and transcriptional activity, it is now known that HDACs and HATs regulate the acetylation of a number of other nonhistone proteins, such as p53, p65/RelA, E2F1, GATA1, and MyoD, suggesting complex functions of HDACs in different cellular processes (11, 42). Precisely which cellular functions are involved is currently the subject of intense investigation.

Vertebrates express at least 18 distinct HDACs, which have been grouped into three classes based on their similarities with Saccharomyces cerevisiae HDACs (11, 42). Class I HDACs (HDACs 1, 2, 3, 8, and 11) are homologous to yeast Rpd3. Class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to yeast Hda1. The highly conserved C termini of class I and class II HDACs contain a catalytic domain, which associates with transcriptional corepressors such as N-CoR, SMRT, and B-CoR within the nucleus. The third class of deacetylases, called sirtuins (SIRT1-7), have catalytic domains similar to that of the yeast NAD^{-dependent deacetylase Sir2 (}8). While serving as HDACs in yeast, in mammalian cells SIRTs are involved in the deacetylation of other proteins, rather than histones, and hence are not considered classical HDACs.

Class I HDACs consist of little more than a deacetylase domain and function as transcriptional repressors. They generally are nuclear proteins expressed in most tissue and cell types (11, 42). On the other hand, members of the class II HDAC subfamily display cell type-restricted patterns of expression and contain a large extended N-terminal extension with which a variety of signaling proteins interact, including MEF2, HP1α, Bcl6, CtBP, calmodulin, and 14-3-3 (11, 42). Phosphorylation of conserved serine residues in class II HDACs by calcium/calmodulin-dependent kinase (CaMK) or protein kinase D in response to specific stimuli creates docking sites for the 14-3-3 family of protein chaperones (11, 28, 31, 42). Binding of 14-3-3 results in the export of these HDACs from the nucleus and disrupts their interactions with transcriptional corepressor proteins, resulting in derepression of their target genes.

Several classes of small-molecule HDAC inhibitors have been identified (11, 29). Treatment of cultured cells with such HDAC inhibitors has a variety of effects, including transformation, differentiation, cell survival, and cell death, implicating HDACs in many different biological processes (11, 29). Because of their ability to induce the death of transformed cells, HDAC inhibitors are in clinical trials for the treatment of cancers. It is noteworthy, however, that while there are small differences in the sensitivities of individual class I and class II HDACs to different inhibitors, most of the commonly used inhibitors inhibit all HDACs efficiently. The significance of individual HDACs in any biological effect has thus been difficult to ascertain using inhibitors. Despite this limitation, a number of laboratories have used such pharmacological inhibitors to investigate the involvement of HDACs in the regulation of neuronal survival both in culture and in animal models of neurological disease (24). These studies have provided conflicting results. For example, the administration of HDAC inhibitors reduced neuronal loss in a Drosophila and a mouse model of Huntington's disease (13, 19). Treatment of cultured cortical neurons with HDAC inhibitors has also been reported to have a protective effect (36). While neuroprotection by HDAC inhibitors in experimental systems has prompted their consideration as therapeutic agents in the treatment of neurological diseases, it has been reported that treatment of cultured cerebellar granule neurons (CGNs) with HDAC inhibitors actively promotes cell death (4, 5, 37).

In this study, we have investigated the role of HDACs in the regulation of neuronal survival, using cultured cerebellar granule neurons. We report that an alternatively spliced form of a class II HDAC lacking intrinsic enzymatic activity, histone deacetylase-related protein (HDRP)/MITR, has neuroprotective functions. We provide evidence indicating that HDRP acquires deacetylase activity by the recruitment of HDAC1. Together, HDRP and HDAC1 inhibit low-potassium-medium (LK medium)-induced acetylation of the c-Jun gene promoter, thus inhibiting apoptosis-associated c-Jun expression. Furthermore, HDRP interacts with c-Jun N-terminal kinase (JNK) and inhibits its ability to phosphorylate and activate c-Jun.

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Acknowledgments

The research described in this report was funded by NIH-NINDS grants NS40408 and NS047201 to S.R.D. This work was also supported by funds from the Department of Defense (DNMD 17-99-1-9566) to S.R.D.

We thank Asligul Yalcin and Megan Kong for preliminary work that contributed to the development of this project.

Acknowledgments

Footnotes

^{Supplemental material for this article may be found at}http://mcb.asm.org/.

Footnotes

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