Hongxin Dong - US grants

DESCRIPTION (provided by applicant): Antipsychotic drugs are widely prescribed to elderly patients for the treatment of a variety of psycho- pathological conditions, including psychosis and behavioral disturbances associated with cognitive impairment. However, the current treatment strategy for elderly individuals is often ineffective, with an increased incidence of sid effects. The factors contributing to reduced antipsychotic efficacy in the elderly population are not yet full understood. Induction of immediate-early genes such as c-fos has been shown to affect antipsychotic drug activity in the CNS. Both typical and atypical antipsychotics induce c Fos expression in specific brain regions, including the striatum and prefrontal cortex. Our preliminary data shows lower levels of antipsychotic induced c-Fos expression in the nucleus accumbens of aged mice, as well decreased acetylation of histone H3 lysine residue 27 (H3K27) on the c-fos promoter. Co-treatment with valproic acid (VPA), a histone deacetylase HDAC inhibitor, was shown to restore antipsychotic induced c-Fos induction and improve behavioral performance in aged mice. Our preliminary data suggests that an epigenetic mechanism may play a key role in the reduced drug efficacy seen in elderly individuals. In this study, we hypothesize that age-associated decreases in antipsychotic efficacy are the result of epigenetic changes in the brain that can be ameliorated by co-treatment with antipsychotics and HDAC inhibitors. To test our hypotheses, young (3-month old) and aged (24-month old) mice will be treated with haloperidol (HAL, a typical) or clozapine (CLZ, an atypical) alone or in combination with the HDAC1-specific inhibitor entinostat (MS-275) or pan-HDAC inhibitor VPA for 14 days. First, we will investigate the relationship between the acetylation of histone H3 lysine residue 27 (H3K27) on the c-fos promoter and antipsychotic induced c-Fos induction in the brains of aged mice using chromatin immunoprecipitation (ChIP) assays and real-time PCR. We will then examine whether increased c-Fos expression following HDAC inhibitor/antipsychotic co-treatment is specific to dopaminergic or serotoninergic neurons using immunofluorescence double labeling. Using behavioral tests relevant to memory and motor function, we will then investigate whether MS-275 treatment results in cognitive improvements similar to those seen with VPA treatment. This study will shed light on the interactions between aging, antipsychotic drug efficacy, and epigenetic regulation. By advancing our understanding of the epigenetic mechanisms of drug efficacy, it will be possible to develop new psychotropic treatment strategies that maximize benefits while minimizing side effects.

? DESCRIPTION (provided by applicant): a common class of medication prescribed to the elderly for the treatment of psychiatric disorders and behavioral and psychological symptoms of dementia (BPSD). However, in aged patients, the incidence and severity of the side effects such as experiencing extrapyramidal motor symptoms induced by APDs is increased. Although aged-induced changes in pharmacokinetics may contribute to the increased sensitivity to the side effects of APDs in the elderly, age-related pharmacodynamic changes at the target receptor level likely play a key role in the increased sensitivity to the side effects of APDs. However, the mechanisms underlying these age-related declines in receptor function are not well understood. Recently, we identified that histone modifications alter the therapeutic actions of a typical APD, haloperidol, in aged mice. In addition, our preliminary data in this application demonstrates that increases in the severity of extrapyramidal symptom-like side effects (motor side effects) in aged mice can be related to histone hypoacetylation of the dopamine 2 receptor (D2R) gene (Drd2) promoter that in turn decreases the expression of striatal D2Rs. Co- treatment with histone deacetylase inhibitors (HDACis) valproic acid (VPA) or entinostat (MS-275) restored the expression of striatal D2Rs and reduced age-related increases in the motor side effects of haloperidol. Our findings and preliminary results suggest that age-related histone modifications at the gene promoters of target receptors could affect APD action. In this proposal, we seek to confirm the novel epigenetic mechanisms underlying the regulation of APD action during aging and determine whether HDACis could be a candidate to improve APD treatment in the elderly. Our central hypothesis is that age-related increases in the motor side effects of APDs are due to histone hypoacetylation on their targeted receptor genes and that these epigenetic changes and their functional consequences can be reversed by co-treatment with HDACis. To test our hypotheses, first, we will verify that age-related histone modifications are one of the mechanisms underlying increased sensitivity to side effects induced by APDs. Then, we will identify the HDAC subtype(s) that contribute to histone modification and increase in the severity of APD-induced side effects in aged mice. Finally, we will evaluate the therapeutic benefits of HDACi and APD co-treatment that could reduce the severity of APD-induced side effects in aged mice and in Tg2576 mice, an animal model of Alzheimer's disease also being considered as a model of BPSD. The proposed study will advance our understanding of the mechanisms of age-related epigenetic alterations and their effects on APD action. This mechanistic concept will have implications not only for neuropsychiatric medication but also for other medications in geriatrics. Our study will serve as a guide to investigate epigenetic mechanisms on drug action with ultimate benefiting for the aged population.

Summary Women have a higher risk for developing Alzheimer's disease (AD) than men, and 70% of the 5.4 million Americans that have been diagnosed with AD are women. Despite the clear epidemiological evidence for this sex-specific risk increase, the neurobiological basis for this sexual dimorphism is unclear. Convergent findings from our group and others indicate that stress contributes to the pathogenesis of AD through its effects on corticotropin releasing factor (CRF) transmission. Sexual dimorphism exists at the second messenger level for CRF receptor 1 (CRF1) transmission, in that CRF1-Gs signaling is favored in females while CRF1-?-arrestin-2 signaling is favored in males. This sex bias in CRF1 signaling likely results in different phosphorylation patterns among downstream targets associated with AD neuropathy, and so provide a mechanistic explanation for the difference in the risk of AD between men and women. Consistent with this mechanism, our preliminary data demonstrate that amyloid plaque development is much greater in female than male transgenic mice (APP+/CRF+/tTA+; or TT), in which both human APP and CRF are overexpressed in the forebrain. We now propose to extend these findings by investigating the hypothesis that chronic stress increases the risk of AD neuropathology in female transgenic mice due to sexual dimorphism in CRF1 signaling pathways and sex-specific protein phosphorylation patterns. We will utilize two stress-related AD mouse models (TT mice and isolation-stressed Tg2576 mice) to test our hypothesis. First, we will confirm the increase of amyloid plaque and cognitive deficits in females compared to males determine the degree to which plaque development and cognitive deficits can be prevented in female and male mice by suppressing CRF release or blocking CRF1 transmission. Then, we will interrogate CRF1's downstream effectors in primary cortical and hippocampal neuronal cultures derived from male and female mice, to investigate the degree to which Gs-PKA signaling selectively promotes the development of AD neuropathy during administration of exogenous CRF, CRF1 antagonists, and PKA inhibitors. Further, we will demonstrate the sufficiency of increased Gs-PKA signaling to promote AD pathology using the two mouse models and a virally-mediated DREADD (Designer Receptors Exclusively Activated by Designer Drugs) that is specific for Gs signaling (AAV-CaMKIIa-HArM3D-IRES-mCitrine). Finally, we will perform a quantitative phosphoproteomic assay to identify the sexually dimorphic phosphorylation patterns associated with increased in AD-like neuropathy and CRF1 signaling in both TT mice and stressed Tg2576 mice. This study would be the first to demonstrate the plausibility of a mechanism that could explain the increased risk of AD in women, and thus provide a mechanistic framework and novel targets for treatments that are sex- specific.

Summary Clinical studies indicate that Alzheimer's disease (AD) disproportionately affects women more than men, but the biological mechanisms underlying this sexual divergence are not well understood. Convergent findings from our group and others indicate that stress contributes to the pathogenesis of AD through its effects on corticotrophin releasing factor (CRF) transmission. Recently we found that during stress, CRF coverts its function by triggering second messenger signaling through the CRF receptor 1 (CRF1) favoring Gs-PKA signaling in females, while ?-arrestin2 signaling is favored in males. This sex bias in CRF1 signaling likely results in different phosphorylation patterns among downstream targets known to drive AD neuropathology and so provides one mechanistic explanation for the difference in AD risk by sex. Consistent with this mechanism, our preliminary data demonstrate that amyloid plaque development is much greater in female than male transgenic mice, in which human APP with Tg2576 mice background and forebrain CRF are overexpressed (named TT mice). We hypothesize that chronic stress increases the risk of AD neuropathology in female transgenic mice due to sexual dimorphism in downstream CRF1 signaling pathways and resultant AD related- protein phosphorylation, which may be reversible with specific CRF1 antagonists or PKA inhibitors. To test our hypothesis, we will utilize a transgenic mouse model of AD that displays no sex-specific difference in AD neuropathology under non-stressed conditions (tTA:APPsi mouse strain). First, using two independent stress paradigms, chronic unpredictable stress to tTA:APPsi mice and genetically forebrain-restricted CRF overexpression by creating tTA/APPsi/CRF mice, we will compare that amyloid pathology and cognitive deficits are increased in females compared to males during stress and that antagonism of CRF1 signaling is able to reduce these pathologies in a sex-dependent manner. Then, we will interrogate CRF1's downstream effectors in primary neuronal cultures derived from male and female AD transgenic mice to investigate the degree to which Gs-PKA signaling selectively promotes the development of AD-like neuropathology after administration of exogenous CRF, CRF1 antagonists, and PKA inhibitors. Further, we will demonstrate the sufficiency of increased Gs-PKA signaling to promote AD-like pathology using a virally mediated Designer Receptors Exclusively Activated by Designer Drugs (DREADD) approach, which specifically increases Gs signaling (AAV- CaMKIIa-HArM3D-IRES-mCitrine), in both male and female tTA:APPsi mice. The similar approach will be used in tTA/APPsi/CRF mice to demonstrate that male-biased ?-arrestin2 signaling is not neuroprotective. Finally, we will perform a quantitative phosphoproteomic assay to identify the sexually dimorphic phosphorylation motifs associated with increased AD-like neuropathology and CRF1 signaling in stressed tTA:APPsi and tTA/APPsi/CRF mice. This study will demonstrate plausible mechanisms that could explain the increased risk of AD in women, and thus provide a mechanistic framework and novel targets for treatments of AD.

Summary Over 90% of Alzheimer's disease (AD) patients suffer from behavioral and psychological symptoms of dementia (BPSD) including agitation, aggression, depression, apathy and psychosis. BPSD can present at almost any stage of AD, and in some patients, these symptoms can even appear before dementia develops. The severity of BPSD increases significantly with disease progression, and affects the quality of life of both patients and their caregivers. In many patients, BPSD is the main reason for institutionalization. However, the mechanisms underlying BPSD are not known, and there is no specific treatment strategy available. Although BPSD presents differently in each patient, the presence of certain symptoms in a patient make the co-occurrence of other symptoms more likely. In an ongoing collaboration with Rush Alzheimer's Disease Center, we have developed a method for clustering the symptoms of BPSD into four domains (affective, hyperactivity/disinhibition, psychosis and apathy). Based on these domains, we then conducted an RNA-seq and found different gene expression profiles in AD patients with and without BPSD. This evidence supports the notion that distinct molecular pathways may be involved in the appearance of BPSD. In this proposal, we hypothesize that individual BPSD domains in patients with AD are due to definable perturbations in molecular pathways and that these pathways can be analogized in AD mouse models, allowing for a causal investigation of the relationship between specific pathway alterations and domain behaviors. We will test this hypothesis through both human study and animal work. For the human study, 1) we will expand on our behavioral analyses by increasing subjects for pre-mortem clinical assessments and defining BPSD trends over time in AD patients. 2) Within each behavioral domain, we will employ RNA-seq to investigate gene expression patterns in different brain sub-regions that are unique to each BPSD domain and the gene expression pattern will be compared across normal, MCI and AD subjects. 3) Finally, we will identify which pathways are most clearly associated with each of the BPSD domains using bioinformatics and biochemical analyses. For the animal model work, 1) we will characterize how mouse behaviors analogous to human BPSD symptoms evolve during AD-like neuropathgenesis progression 2) We will identify the most promising molecular candidates for intervention from our RNA-seq findings using these AD/BPSD models. 3) Finally, we will determine whether altering these pathways leads to changes in BPSD-like behavior using virally mediated genetic manipulations (AAV9/CRISPR-Cas9). Overall, this project will establish a translational pipeline by associating BPSD symptom domains with molecular alterations in human AD patients, and by demonstrating that manipulations of these pathways can cause BPSD-like behaviors in transgenic mouse models of AD. These data-driven approaches will lead to a better understanding of the molecular mechanisms that underlie BPSD in AD and potentially identify novel targets for future therapeutic interventions.