Zhaolan Zhou, Ph.D. - US grants

PROJECT SUMMARY (See instructions): Rett Syndrome (RTT) is one of the Autism Spectrum Disorders (ASDs) with a known genetic cause and represents one of the leading causes of mental retardation in fernales. RTT is caused by mutations in the Xlinked gene encoding methyl-CpG-binding protein 2 (MeCP2). The onset of RTT after normal early postnatal development and the precipitous loss of learned language and motor skills suggest a hypothesis that the clinical features of RTT result from a failure of activity-dependent neuronal development Recently we discovered that MeCP2 supresses Brain Derived Neurotrophic Factor (BDNF) expression in the absence of neuronal stimuli. In the presence of stimuli, MeCP2 undergpesCaMKlI-mediated phosphorylation and releases repression to Bdnf We have characterized a phosphorylatioii site (S421) on MeCP2 that selectively senses neuronal activity to control Bdnf transcription, modulate dendritic outgi-ovrth and spine maturation. Our findings challenged the canonical view of MeCP2 as a global transcriptiorial repressdrand implicated MeCP2 in the molecular program controlling experience-dependent neuronal development. To uiiderstand the molecular mechanisms by which activity-dependent phosphoryaltion of MeCP2 modulates its functioh and to assess the role of MeCP2 phosphoryailtion on neuronal development in vivo, we have identified a second phosphorylation event on MeCP2 at Threonine 158 (T158) and developed a knock-in mouse model in which T158 is mutated to alanine during the mentcred phase of this award (K99), Notably, T158 is located at the methyl-CpG binding domain of MeCP2 and is one of the most frequently mutated residue found in RTT patients. Thus, we plan to continue our research as initially proposed. The updated specific aims are 1) To characterize the nature of T158 phosphorylation and the mechanisms by which MeCP2 function is regulated by T158 phosphorylation;2) To characterize the function of MeCP2 T158 phosphorylation in neuronal development by analyzing the molecular and cellular phenotypes of T158A knock-in mouse model.

DESCRIPTION (provided by applicant): The genetic underpinnings of mental health disorders are highly complex, involving multifaceted interactions between risk genes, the environment, and experiential factors. It is well known that adverse early life events confer significantly greater susceptibility to psychiatric conditions in later life. However, the epigenetic mechanisms by which environmental factors interact with genetic programs in the nervous system remain poorly understood. This is partially due to the complex heterogeneity of neuronal cell types and the limitations of existing techniques. Here we propose to investigate the epigenetic modifications such as DNA methylation and chromatin organization induced by early-life stress with conceptually and technically innovative approaches. We plan to map the stress-induced DNA methylation changes by directly sequencing the methylated DNA and by directly examining the dynamic association of methyl- CpG binding proteins (MBPs) with methylated DNA. To achieve this, we will generate two transgenic mouse lines: one line expresses biotin ligase (BirA) and GFP in a spatially and temporally controlled manner;while the other line carries an endogenous MBP tagged with a biotinylation signal sequence. In the resulting progeny of these two mouse lines, MBP will be specifically biotinylated in a defined population of GFP-positive neurons. Following experimental treatment of these transgenic mice, the genome-wide DNA methylation loci in specific neuronal populations will be mapped by high throughput sequencing MeDIP-seq and bioMBP-ChIP-seq. In addition, the chromatin complexes associated with each MBP will be characterized by systematic mass spectrometry bioMBP-ChIP-MS/MS to investigate the molecular mechanisms underlying epigenetic modifications. With the combined genomic and proteomic approaches, we hope to gain an insight into the epigenetic mechanisms through which early-life stress interacts with susceptibility genes and confers risks to mental illness. Our proposed study will also allow greater understanding of the underlying causes of mental health disorders and provide the necessary foundation for improved diagnosis and interventions. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop an innovative strategy to investigate the epigenetic mechanisms by which environmental factors such as early life stress interact with genetics, and how these interactions increase the risk of mental illness. We plan to generate novel genetically modified mouse lines to tag methyl-CpG binding proteins specifically in a defined population of neurons. We will then investigate the epigenetic changes associated with environmental cues in the brain with both genomic and proteomic approaches. The proposed studies will be of significance not only in understanding the epigenetic control of experience-dependent brain development, but also in understanding the molecular and cellular basis of mental disorders. It is tempting to argue that our research may identify potential molecular, cellular, and circuit targets to intervene and/or prevent mental illness.

DESCRIPTION (provided by applicant): Mutations in the methyl-CpG binding protein 2 (MECP2) gene cause the autism spectrum disorder Rett Syndrome (RTT). To understand the pathogenesis of RTT, we previously developed and characterized a mouse model recapitulating an RTT-associated missense mutation, MeCP2 T158A. We found that mice with T158A mutation show similar RTT-like phenotypes to that of Mecp2-null mice. T158A mutation decreases the binding of MeCP2 to methylated DNA and reduces MeCP2 protein stability. Mice with MeCP2 dysfunction also show age-dependent impairment of neuronal event-related potentials (ERPs) indicative of disrupted neural circuitry. Moreover, our ongoing work supports a role of MeCP2 in modulation of gene transcription and dendritic development in a cell-type specific manner. Together, these findings lead to a new series of questions pertaining to the pathogenic mechanisms of RTT. We propose to address them in the following specific aims: 1) To define the role of methyl-DNA binding of MeCP2 in the etiology of RTT-like phenotypes; 2) To dissect the role of MeCP2 in different neuronal cells regulating information processing; and 3) To investigate the molecular mechanisms by which MeCP2 modulates cell type-specific neuronal function. With the combined genetic, genomic, behavioral and neurophysiological approaches, we hope to not only reveal novel insight into the pathogenic mechanisms underlying RTT, but also to expedite the development of mechanism-based therapeutics that are focused on MeCP2 methyl-DNA binding and specific neuronal types. Moreover, our proposed study will provide the research community at large with innovative tools and resources to investigate the epigenetic mechanisms underlying a variety of biological processes and diseases.

Abstract Epidemiological studies provide mounting evidence supporting that environmental and experiential influences, such as stressful life events, interact with genetic variations and compound the risks for neuropsychiatric disorders, such as major depressive disorder (MDD). MDD afflicts about 6.7% of the United States adults, but treatment options for MDD are limited and not effective. The objective of this proposal is to define the global epigenetic landscape associated with stress experience and assess the effect of locus-specific epigenetic changes on the manifestation of depression-like phenotypes. We propose to first expose both male and female mice to chronic unpredictable stress paradigms. We will then take a genomic approach to identify the stress responsive epigenetic code in targeted neuronal cell types in the brain. Finally, we will modify and apply CRISPR/Cas9 technology to address the causal relationship between the identified epigenetic code to the expression of depression-like behaviors. With currently available state-of-the-art technologies and our newly developed, genetically modified mouse tools, we hope to gain an insight into the epigenetic mechanisms through which stress interacts with susceptibility genes and confers increased risk to MDD. Our proposed study will also allow greater understanding of the underlying causes of stress-related neuropsychiatric disorders and provide the necessary foundation for improved diagnosis and intervention.

Title Pathogenic Studies of CDKL5 Disorder Abstract Mutations in the X-linked gene encoding cyclin-dependent kinase-like 5 cause CDKL5 disorder, an infantile epileptic encephalopathy sharing features with intellectual disability and autism. To understand the biological function of CDKL5 in vivo and the pathogenic mechanisms underlying CDKL5 disorder, we previously developed and characterized a knockout mouse model incorporating a CDKL5 patient-associated genetic defect. We found that mice with CDKL5 dysfunction develop behavioral phenotypes mimicking key symptoms of CDKL5 disorder. These mice also show deficits in neural circuit communication and alterations in multiple signal transduction pathways. Given that CDKL5 expression is highly enriched in the forebrain, we also employed a conditional knockout approach and ablated CDKL5 expression in different neuronal populations of the forebrain. We found that mice lacking CDKL5 from different neuronal cells show distinct behavioral phenotypes, mimicking intellectual disability-like and autism-like features of CDKL5 disorder. These findings raise a hypothesis that CDKL5 regulates cell type-specific signal transduction pathways and different neural circuit mechanisms underlying intellectual disability and autistic features of CDKL5 disorder. We therefore propose to develop an innovative mouse line to characterize cell type-specific functions of CDKL5, and take a combined biochemical, genetic, behavioral, and neurophysiological approach to investigate the molecular and cellular basis of CDKL5 disorder using knockout and conditional knockout mice in both male and females. Together, we expect to uncover new aspects of CDKL5 function, develop a framework for testing therapeutics, and ultimately reveal new opportunities for therapeutic development to alleviate symptoms associated with CDKL5 disorder, as well as other related disorders such as syndromic intellectual disability and autism.

Congenital disorders of glycosylation (CDG) are a group of neurometabolic disorders characterized by genetic defects in the highly conserved cellular glycosylation machinery. A majority of CDG patients have biallelic mutations in PMM2, a gene encoding the protein phosphomannomutase 2 required to activate mannose monosaccharides for N-linked protein glycosylation. PMM2-CDG patients suffer from multi-systemic involvement, and all patients uniformly suffer from neurological impairment that is prominent, progressive, and produces lifelong intellectual disability, ataxia and often seizures. Based on the genetic basis of CDG, we propose to establish and characterize a novel mouse model of PMM2-CDG to specifically investigate the function of PMM2 in neuronal and glial cells. We will also investigate the role of PMM2 in cerebellum development and function. With combined genetic, molecular, and behavioral approaches, we hope to not only reveal novel insight into the pathogenic mechanisms of CDG, but also to expedite the development of mechanism-based therapeutics to improve treatment. Moreover, our proposed study will provide the research community at large with innovative tools and resources to investigate the pathophysiology underlying a variety of glycosylation deficit-related disorders.