Gavin R. Sangrey

Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease

We have previously demonstrated amelioration of Huntingtons disease (HD)-related phenotypes in R6/2 transgenic mice in response to treatment with the novel histone deacetylase (HDAC) inhibitor 4b. Here we have measured the selectivity profiles of 4b and related compounds against class I and class II HDACs and have tested their ability to restore altered expression of genes related to HD pathology in mice and to rescue disease effects in cell culture and Drosophila models of HD. R6/2 transgenic and wild-type (wt) mice received daily injections of HDAC inhibitors for 3 days followed by real-time PCR analysis to detect expression differences for 13 HD-related genes. We find that HDACi 4b and 136, two compounds showing high potency for inhibiting HDAC3 were most effective in reversing the expression of genes relevant to HD, including Ppp1r1b, which encodes DARPP-32, a marker for medium spiny striatal neurons. In contrast, compounds targeting HDAC1 were less effective at correcting gene expression abnormalities in R6/2 transgenic mice, but did cause significant increases in the expression of selected genes. An additional panel of 4b-related compounds was tested in a Drosophila model of HD and in STHdhQ111 striatal cells to further distinguish HDAC selectivity. Significant improvement in huntingtin-elicited Drosophila eye neurodegeneration in the fly was observed in response to treatment with compounds targeting human HDAC1 and/or HDAC3. In STHdhQ111 striatal cells, the ability of HDAC inhibitors to improve huntingtin-elicited metabolic deficits correlated with the potency at inhibiting HDAC1 and HDAC3, although the IC50 values for HDAC1 inhibition were typically 10-fold higher than for inhibition of HDAC3. Assessment of HDAC protein localization in brain tissue by Western blot analysis revealed accumulation of HDAC1 and HDAC3 in the nucleus of HD transgenic mice compared to wt mice, with a concurrent decrease in cytoplasmic localization, suggesting that these HDACs contribute to a repressive chromatin environment in HD. No differences were detected in the localization of HDAC2, HDAC4 or HDAC7. These results suggest that inhibition of HDACs 1 and 3 can relieve HD-like phenotypes in model systems and that HDAC inhibitors targeting these isotypes might show therapeutic benefit in human HD.

Increased brain‐derived neurotrophic factor (BDNF) expression in the ventral tegmental area during cocaine abstinence is associated with increased histone acetylation at BDNF exon I‐containing promoters

J. Neurochem. (2012) 120, 202–209.

Genome-Wide Histone Acetylation Is Altered in a Transgenic Mouse Model of Huntington's Disease

In Huntingtons disease (HD; MIM ID #143100), a fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature. Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (AcH3; H3K9K14ac), and downregulated gene expression. However, the question remains whether changes in histone modifications correlate with transcriptional abnormalities across the entire transcriptome. Using chromatin immunoprecipitation paired with microarray hybridization (ChIP-chip), we interrogated AcH3-gene interactions genome-wide in striata of 12-week old wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation.

Cocaine Alters BDNF Expression and Neuronal Migration in the Embryonic Mouse Forebrain

Prenatal cocaine exposure impairs brain development and produces lasting alterations in cognitive function. In a prenatal cocaine exposure mouse model, we found that tangential migration of GABA neurons from the basal to the dorsal forebrain and radial neuron migration within the dorsal forebrain were significantly decreased during the embryonic period. The decrease in the tangential migration occurred early in gestation and normalized by late gestation, despite ongoing cocaine exposure. The decrease in radial migration was associated with altered laminar positioning of neurons in the medial prefrontal cortex. The cocaine exposure led to transient decreases in the expression of Tbr2 and Tbr1, transcription factors associated with intermediate progenitor cells and newborn neurons of the dorsal forebrain, respectively, although neurogenesis was not significantly altered. Since cocaine can modulate brain derived neurotrophic factor (BDNF) expression in the mature brain, we examined whether cocaine can alter BDNF expression in the embryonic brain. We found a transient decrease in BDNF protein expression in the cocaine-exposed embryonic forebrain early in gestation. By late gestation, the BDNF expression recovered to control levels, despite ongoing cocaine exposure. In basal forebrain explants from cocaine-exposed embryos, cell migration was significantly decreased, corroborating the in vivo data on tangential GABA neuron migration. Since BDNF can influence tangential neuronal migration, we added BDNF to the culture medium and observed increased cell migration. Our data suggest that cocaine can alter tangential and radial neuronal migration as well as BDNF expression in the embryonic brain and that decreased BDNF may mediate cocaines effects on neuronal migration.

MeCP2: a novel Huntingtin interactor

Transcriptional dysregulation has been proposed to play a major role in the pathology of Huntingtons disease (HD). However, the mechanisms that cause selective downregulation of target genes remain unknown. Previous studies have shown that mutant huntingtin (Htt) protein interacts with a number of transcription factors thereby altering transcription. Here we report that Htt directly interacts with methyl-CpG binding protein 2 (MeCP2) in mouse and cellular models of HD using complimentary biochemical and Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer approaches. Htt-MeCP2 interactions are enhanced in the presence of the expanded polyglutamine (polyQ) tract and are stronger in the nucleus compared with the cytoplasm. Furthermore, we find increased binding of MeCP2 to the promoter of brain-derived neurotrophic factor (BDNF), a gene that is downregulated in HD, in the presence of mutant Htt. Finally, decreasing MeCP2 levels in mutant Htt-expressing cells using siRNA increases BDNF levels, suggesting that MeCP2 downregulates BDNF expression in HD. Taken together, these findings suggest that aberrant interactions between Htt and MeCP2 contribute to transcriptional dysregulation in HD.

ADAR2-dependent GluA2 editing regulates cocaine seeking

Activation of AMPA receptors (AMPARs) in the nucleus accumbens is necessary for the reinstatement of cocaine-seeking behavior, an animal model of drug craving and relapse. AMPARs are tetrameric protein complexes that consist of GluA1–4 subunits, of which GluA2 imparts calcium permeability. Adenosine deaminase acting on RNA 2 (ADAR2) is a nuclear enzyme that is essential for editing GluA2 pre-mRNA at Q/R site 607. Unedited GluA2(Q) subunits form calcium-permeable AMPARs (CP-AMPARs), whereas edited GluA2(R) subunits form calcium-impermeable channels (CI-AMPARs). Emerging evidence suggests that the reinstatement of cocaine seeking is associated with increased synaptic expression of CP-AMPARs in the nucleus accumbens. However, the role of GluA2 Q/R site editing and ADAR2 in cocaine seeking is unclear. In the present study, we investigated the effects of forced cocaine abstinence on GluA2 Q/R site editing and ADAR2 expression in the nucleus accumbens. Our results demonstrate that 7 days of cocaine abstinence is associated with decreased GluA2 Q/R site editing and reduced ADAR2 expression in the accumbens shell, but not core, of cocaine-experienced rats compared with yoked saline controls. To examine the functional significance of ADAR2 and GluA2 Q/R site editing in cocaine seeking, we used viral-mediated gene delivery to overexpress ADAR2b in the accumbens shell. Increased ADAR2b expression in the shell attenuated cocaine priming-induced reinstatement of drug seeking and was associated with increased GluA2 Q/R site editing and surface expression of GluA2-containing AMPARs. Taken together, these findings support the novel hypothesis that an increased contribution of accumbens shell CP-AMPARs containing unedited GluA2(Q) promotes cocaine seeking. Therefore, CP-AMPARs containing unedited GluA2(Q) represent a novel target for cocaine addiction pharmacotherapies.

Alterations in brain-derived neurotrophic factor in the mouse hippocampus following acute but not repeated benzodiazepine treatment.

Benzodiazepines (BZs) are safe drugs for treating anxiety, sleep, and seizure disorders, but their use also results in unwanted effects including memory impairment, abuse, and dependence. The present study aimed to reveal the molecular mechanisms that may contribute to the effects of BZs in the hippocampus (HIP), an area involved in drug-related plasticity, by investigating the regulation of immediate early genes following BZ administration. Previous studies have demonstrated that both brain derived neurotrophic factor (BDNF) and c-Fos contribute to memory- and abuse-related processes that occur within the HIP, and their expression is altered in response to BZ exposure. In the current study, mice received acute or repeated administration of BZs and HIP tissue was analyzed for alterations in BDNF and c-Fos expression. Although no significant changes in BDNF or c-Fos were observed in response to twice-daily intraperitoneal (i.p.) injections of diazepam (10 mg/kg + 5 mg/kg) or zolpidem (ZP; 2.5 mg/kg + 2.5 mg/kg), acute i.p. administration of both triazolam (0.03 mg/kg) and ZP (1.0 mg/kg) decreased BDNF protein levels within the HIP relative to vehicle, without any effect on c-Fos. ZP specifically reduced exon IV-containing BDNF transcripts with a concomitant increase in the association of methyl-CpG binding protein 2 (MeCP2) with BDNF promoter IV, suggesting that MeCP2 activity at this promoter may represent a ZP-specific mechanism for reducing BDNF expression. ZP also increased the association of phosphorylated cAMP response element binding protein (pCREB) with BDNF promoter I. Future work should examine the interaction between ZP and DNA as the cause for altered gene expression in the HIP, given that BZs can enter the nucleus and intercalate into DNA directly.

Genome-Wide Increase in Histone H2A Ubiquitylation in a Mouse Model of Huntington's Disease

BACKGROUND Huntingtons disease (HD) is a neurodegenerative disorder with selective vulnerability of striatal neurons and involves extensive transcriptional dysregulation early in the disease process. Previous work in cell and mouse models has shown that histone modifications are altered in HD. Specifically, monoubiquitylated histone H2A (uH2A) is present at the promoters of downregulated genes which led to the hypothesis that uH2A plays a role in transcriptional silencing in HD. OBJECTIVE To broaden our view of uH2A function in transcription in HD, we examined genome-wide binding sites of uH2A in 12-week old striatal tissue from R6/2 transgenic HD mouse model. METHODS We used chromatin immunoprecipitation followed by genomic promoter microarray hybridization (ChIP-chip) and then interrogated how these binding sites correlate with transcribed genes. RESULTS Our analysis reveals that, while uH2A levels are globally increased at the genome in the transgenic (TG) striatum, uH2A localization at a gene did not strongly correlate with the absence of its transcript. Furthermore, analysis of differential ubiquitylation in wild-type (WT) and TG striata did not reveal the expected enrichment of uH2A at genes with decreased expression in the TG striatum. CONCLUSIONS This first description of genome-wide localization of uH2A in an HD model reveals that monoubiquitylation of histone H2A may not function at the level of the individual gene but may rather influence transcription through global chromatin structure.

Increased BDNF Expression in the Ventral Tegmental Area During Cocaine Abstinence is Associated with Increased Histone Acetylation at BDNF Exon I-Containing Promoters

J. Neurochem. (2012) 120, 202–209.

Increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area during cocaine abstinence is associated with increased histone acetylation at BDNF exon I-containing promoters: Cocaine and chromatin remodeling of the BDNF promoter

J. Neurochem. (2012) 120, 202–209.