Epigenetic modulation through BET bromodomain inhibitors as a novel therapeutic strategy for progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is a debilitating neurodegenerative disorder with currently no disease-modifying treatment options available. Mutations in GRN are one of the most common genetic causes of FTD, near ubiquitously resulting in progranulin (PGRN) haploinsufficiency. Small molecules that can restore PGRN protein to healthy levels in individuals bearing a heterozygous GRN mutation may thus have therapeutic value. Here, we show that epigenetic modulation through bromodomain and extra-terminal domain (BET) inhibitors (BETi) potently enhance PGRN protein levels, both intracellularly and secreted forms, in human central nervous system (CNS)-relevant cell types, including in microglia-like cells. In terms of potential for disease modification, we show BETi treatment effectively restores PGRN levels in neural cells with a GRN mutation known to cause PGRN haploinsufficiency and FTD. We demonstrate that BETi can rapidly and durably enhance PGRN in neural progenitor cells (NPCs) in a manner dependent upon BET protein expression, suggesting a gain-of-function mechanism. We further describe a CNS-optimized BETi chemotype that potently engages endogenous BRD4 and enhances PGRN expression in neuronal cells. Our results reveal a new epigenetic target for treating PGRN-deficient forms of FTD and provide mechanistic insight to aid in translating this discovery into therapeutics.

WNT/ß-Catenin Pathway and Epigenetic Mechanisms Regulate the Pitt-Hopkins Syndrome and Schizophrenia Risk Gene TCF4.

Genetic variation within the transcription factor TCF4 locus can cause the intellectual disability and developmental disorder Pitt-Hopkins syndrome (PTHS), whereas single-nucleotide polymorphisms within noncoding regions are associated with schizophrenia. These genetic findings position TCF4 as a link between transcription and cognition; however, the neurobiology of TCF4 remains poorly understood. Here, we quantitated multiple distinct TCF4 transcript levels in human induced pluripotent stem cell-derived neural progenitors and differentiated neurons, and PTHS patient fibroblasts. We identify two classes of pharmacological treatments that regulate TCF4 expression: WNT pathway activation and inhibition of class I histone deacetylases. In PTHS fibroblasts, both of these perturbations upregulate a subset of TCF4 transcripts. Finally, using chromatin immunoprecipitation sequencing in conjunction with genome-wide transcriptome analysis, we identified TCF4 target genes that may mediate the effect of TCF4 loss on neuroplasticity. Our studies identify new pharmacological assays, tools, and targets for the development of therapeutics for cognitive disorders.

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests.

Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary--albeit often ineffective--treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.

Crebinostat: a novel cognitive enhancer that inhibits histone deacetylase activity and modulates chromatin-mediated neuroplasticity.

Long-term memory formation is known to be critically dependent upon de novo gene expression in the brain. As a consequence, pharmacological enhancement of the transcriptional processes mediating long-term memory formation provides a potential therapeutic strategy for cognitive disorders involving aberrant neuroplasticity. Here we focus on the identification and characterization of small molecule inhibitors of histone deacetylases (HDACs) as enhancers of CREB (cAMP response element-binding protein)-regulated transcription and modulators of chromatin-mediated neuroplasticity. Using a CREB reporter gene cell line, we screened a library of small molecules structurally related to known HDAC inhibitors leading to the identification of a probe we termed crebinostat that produced robust activation of CREB-mediated transcription. Further characterization of crebinostat revealed its potent inhibition of the deacetylase activity of recombinant class I HDACs 1, 2, 3, and class IIb HDAC6, with weaker inhibition of the class I HDAC8 and no significant inhibition of the class IIa HDACs 4, 5, 7, and 9. In cultured mouse primary neurons, crebinostat potently induced acetylation of both histone H3 and histone H4 as well as enhanced the expression of the CREB target gene Egr1 (early growth response 1). Using a hippocampus-dependent, contextual fear conditioning paradigm, mice systemically administered crebinostat for a ten day time period exhibited enhanced memory. To gain insight into the molecular mechanisms of memory enhancement by HDAC inhibitors, whole genome transcriptome profiling of cultured mouse primary neurons treated with crebinostat, combined with bioinformatic analyses of CREB-target genes, was performed revealing a highly connected protein-protein interaction network reflecting modules of genes important to synaptic structure and plasticity. Consistent with these findings, crebinostat treatment increased the density of synapsin-1 punctae along dendrites in cultured neurons. Finally, crebinostat treatment of cultured mouse primary neurons was found to upregulate Bdnf (brain-derived neurotrophic factor) and Grn (granulin) and downregulate Mapt (tau) gene expression-genes implicated in aging-related cognitive decline and cognitive disorders. Taken together, these results demonstrate that crebinostat provides a novel probe to modulate chromatin-mediated neuroplasticity and further suggests that pharmacological optimization of selective of HDAC inhibitors may provide an effective therapeutic approach for human cognitive disorders. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

An epigenetic blockade of cognitive functions in the neurodegenerating brain.

Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer's disease. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge. Here we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase 2, which is increased by Alzheimer's-disease-related neurotoxic insults in vitro, in two mouse models of neurodegeneration and in patients with Alzheimer's disease. Histone deacetylase 2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the build-up of histone deacetylase 2 by short-hairpin-RNA-mediated knockdown unlocks the repression of these genes, reinstates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of selective inhibitors of histone deacetylase 2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade.

Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by cognitive impairments that progress to dementia and death. The earliest symptoms of AD present as a relatively pure deficit in memory retrieval. Therefore, drug treatments that intervene in the early stages of AD by rescuing memory deficits could be promising therapies to slow, or even reverse progression of the disease. In this study, we tested the potential of systemic histone deacetylase inhibitor (HDACi) treatment to rescue cognitive deficits in a mouse model of AD. APPswe/PS1dE9 mice showed pronounced contextual memory impairments beginning at 6 months of age. Chronic HDACi injections (2-3 weeks) did not alter contextual memory formation in normal mice, but had profound effects in transgenic animals. Injections of sodium valproate, sodium butyrate, or vorinostat (suberoylanilide hydroxamic acid; Zolinza) completely restored contextual memory in these mutant mice. Further behavioral testing of the HDACi-treated transgenic mice showed that the newly consolidated memories were stably maintained over a 2-week period. Measurement of the HDAC isoform selectivity profile of sodium valproate, sodium butyrate, and vorinostat revealed the common inhibition of class I HDACs (HDAC1, 2, 3, 8) with little effect on the class IIa HDAC family members (HDAC4, 5, 7, 9) and inhibition of HDAC6 only by vorinostat. These preclinical results indicate that targeted inhibition of class I HDAC isoforms is a promising avenue for treating the cognitive deficits associated with early stage AD.

TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth.

Target of rapamycin (TOR) is a central regulator of cellular and organismal growth in response to nutrient conditions. In a genetic screen for novel TOR interactors in Drosophila melanogaster, we have identified the clathrin-uncoating ATPase Hsc70-4, which is a key regulator of endocytosis. We present genetic evidence that TOR signaling stimulates bulk endocytic uptake and inhibits the targeted endocytic degradation of the amino acid importer Slimfast. Thus, TOR simultaneously down-regulates aspects of endocytosis that inhibit growth and up-regulates potential growth-promoting functions of endocytosis. In addition, we find that disruption of endocytosis leads to changes in TOR and phosphatidylinositol-3 kinase activity, affecting cell growth, autophagy, and rapamycin sensitivity. Our data indicate that endocytosis acts both as an effector function downstream of TOR and as a physiologically relevant regulator of TOR signaling.