Kazuko Sakata - US grants

DESCRIPTION (provided by applicant): A more effective prevention/treatment is needed for major depressive disorder (MDD), a leading disease burden. Enriched environment treatment (EET), which includes physical exercise, mental stimulation, and social interactions, is a potential intervention to prevent/treat MDD. The neurotrophic effects of EET have been extensively studied; however, its antidepressant effects and underlying biological mechanisms are unclear. In particular, its age-dependent effects and (epi-)genetic mechanisms remain to be established. Identifying the life period for maximal EET effects and its antidepressive mechanisms is important in helping develop strategies for a more effective prevention/treatment of MDD. Our long-term goal is to clarify age x (epi-)genetic interference of the antidepressive effects of EET. This project will specifically aim to clarify the antidepressive effects of early-lfe EET, focusing on long-lasting expression changes of brain-derived neurotrophic factor (BDNF; a major neuronal growth factor in the brain related to MDD) and depression-related genes. BDNF deficiency, particularly, Bdnf promoter IV deficiency caused by epigenetic regulation, has been observed in MDD patients and stressed animals. Early-life maltreatment of infants results in DNA methylation of the promoter IV-controlled exons and reduces BDNF expression throughout life. Abusive maternal behavior and previously acquired DNA methylation patterns then transmit perpetually from generation to generation. We recently showed causal evidence whereby Bdnf promoter IV deficiency leads to depression-like behavior. Further, using our promoter IV-deficient depression-model mice (KIV), we showed that chronic EET, but not chronic antidepressant treatment, was able to compensate for the reduced BDNF levels caused by promoter IV deficiency through multiple promoter-driven BDNF, which paralleled antidepressive behavioral effects of EET. Since early-life experiences involve dynamic gene expression regulations, we hypothesize that the antidepressive effects of EET and Bdnf compensation mechanisms may be maximized when EET is provided during early-life development and that these effects will endure in later life due to long-lasting expression changes of Bdnf and Bdnf-/depression-related genes. We will test these hypotheses with two aims: Aim 1) to determine antidepressant, Bdnf, and gene effects of early-life EET, and Aim 2) to examine whether these effects of EET endure when EET is provided during early-life development. In Aim 1, we will determine the effectiveness of EET across ages-during early-life and at two (young or old) adult stages-in both normal and depressed (KIV) mice, by measuring i) depression-like behavior, ii) BDNF levels, and iii) expression changes in Bdnf-/depression-related genes in the brain regions related to MDD. We will use a novel high-throughput gene analysis. In Aim 2, we will determine prolonged effects of 8 weeks of EET after a subsequent 4 weeks of standard condition treatment, by measuring i-iii as in Aim1. Once this project is completed, we will further clarify te precise critical period for effective EET and the gene mechanisms underlying depression resistance.

DESCRIPTION (provided by applicant): Inflexible learning, the inability to change from one course of action to another by learning from a behavioral consequence, is a common symptom of many psychiatric disorders. Its biological mechanisms are largely unknown, but one important epigenetic cause is brain-derived neurotrophic factor (BDNF), a major neuronal growth factor in the brain. BDNF deficiency in the hippocampus (HIP) and medial prefrontal cortex (mPFC) causes inflexible learning. Stress reduces BDNF levels in these regions via epigenetic inactivation of promoter IV, a major activity-dependent BDNF promoter. Reduced BDNF levels and inactive promoter IV are observed in psychiatric patients. Extensive studies have elucidated the mechanisms underlying BDNF deficiency within specific brain regions. However, the neural mechanisms underlying BDNF deficiency between different brain regions remain unknown. In particular, we still do not know how BDNF deficiency affects signal processing between the HIP and mPFC during flexible learning. This knowledge gap may be attributable to the technical difficulties of manipulating BDNF in multiple brain regions and in measuring neural functions across brain regions. We have addressed these issues by generating mutant mice (KIV) that lack promoter IV-driven BDNF and show inflexible learning. We also have developed an in vivo electrophysiological system that allows simultaneous recording and stimulation of multiple brain regions in mice behaving in a smell-taste flexible learning test. Our long-term goal is to elucidat the neural mechanisms of inflexible learning caused by BDNF promoter IV deficiency. Meeting this goal will help to explain the pathophysiology underlying many psychiatric disorders arising from stress that inactivates promoter IV. We recently found that BDNF promoter IV deficiency reduces long-term potentiation (LTP), a cellular form of memory, in the HIP, but enhances LTP in the mPFC. How do these opposing effects of BDNF deficiency affect the signal processing between the HIP and mPFC, and how does this relate to flexible learning? From our preliminary results, we hypothesize that BDNF deficiency impairs the normal suppression of mPFC responses to input from the HIP during breaks between flexible learning tasks, which reflects inflexible behavior. To explain how this occurs, we also propose a novel model: neuronal synchrony acts as a gate to control the timing of the HIP-mPFC signals in a frequency-dependent manner; BDNF deficiency reduces neuronal synchrony and thus impairs timing controls of HIP-mPFC signals. We will test these hypotheses by determining the effects of BDNF deficiency on Aim 1) HIP-mPFC signals/LTP and Aim 2) neuronal synchrony during flexible learning, and by determining timing relations among HIP-mPFC signals, neuronal synchrony, and correct behavioral responses. Successful completion of this project will elucidate the timing-dependent neural mechanisms of inflexible learning, and will expand the understanding of learning mechanisms from synaptic plasticity within single brain regions to timing-dependent signaling across brain regions.

Induction of heat-shock proteins (Hsp), such as via Hsp90 inhibition, is being investigated as a treatment option for neurodegenerative diseases such as Alzheimer's disease (AD). We have demonstrated that a central nervous system-permeable Hsp90 inhibitor, OS-20, shows potent efficacy for ameliorating memory deficit in various acute and chronic AD mouse models. Further, we have found that the synaptic effect of OS- 20 is largely dependent on heat-shock transcription factor, HSF1, a major stress-responsive transcriptional factor. HSF1 induces transcriptions of genes that have heat shock elements (HSE) upon stress (e.g., heat shock and oxidative stress) and protect cells from cell death. HSF1 function has been most studied in relation to cancer. However, its function in neurons remains largely unknown. We recently found that activation of HSF1 by OS-20 increased hippocampal levels of brain-derived neurotrophic factor (BDNF), a critical factor for neuroprotection and learning/memory. Thus, activation of HSF1 by Hsp90 inhibition likely increases neuroprotection, in part, by inducing BDNF. What remains unknown is how HSF1 controls transcription of the BDNF gene. Nine promoters control BDNF gene transcription, leading to different effects in neural functions and behavior. Much is known about BDNF promoter regulation by neuronal activity such as via cAMP- response element-binding protein (CREB). By contrast, limited information is available for BDNF promoter regulation for resilience upon cellular stress. Our sequence search identified 12 putative HSEs in most of the BDNF promoters. Interestingly, some HSEs are located in proximity to cAMP-response element-binding protein (CREB) sites. These findings led us to hypothesize that HSF1 mediates OS-20- or stress-induced BDNF transcription by activating specific BDNF promoters, interacting with CREB pathways. Our idea of HSF1 controlling BDNF induction and its interaction with CREB is novel. Our goal is to test this hypothesis, by determining HSF1 controls of promoter-specific BDNF transcription upon Hsp90 inhibition (Aim 1) and its interplay with CREB (Aim 2). In Aim 1, we will determine HSF1 kinetics, HSF1 binding to individual BDNF promoters, and promoter-specific BDNF transcription induced by OS-20 treatment and shock stress both in vitro (cultured neuron) and in vivo (mice). In Aim 2, we will investigate the interplay of HSF1 and CREB in regulating BDNF transcription. We will assess their temporal and spatial regulation of BDNF promoter activity under OS-20 treatment or stress both in vitro and in vivo. We will also identify gene populations bound to HSF1 and CREB by using comparative ChIP-sequencing analysis and those activated by OS-20 by using RNA- sequencing. Successful outcomes will elucidate transcriptional mechanisms of HSF1 and validate HSF1- promoter interaction as an important and novel AD therapeutic target. This study is significant because the verified HSE sites will be targets for developing drugs that directly activate the HSE sites to induce BDNF and for identifying any defects at the promoter sites for AD susceptibility (e.g. SNPs, epigenetic controls).