Jan Krzysztof Blusztajn, Ph.D. - US grants

biomedical equipment purchase;

Degeneration or malfunction of brain cholinergic neurons underlies the pathophysiology of a variety of mental disorders: Alzheimer's disease (AD) attacks the most human of all brain functions; cognition, language and social awareness; tardive dyskinesia, a chronic neurological syndrome, is a common side-effect of neuroleptic drugs, which are used to treat mental illness; Korsakoff's dementia develops as a side-effect of alcoholism. Other diseases characterized by cholinergic dysfunction include Motor Neuron Disorders, Huntington's Chorea, and Familial Dysautonomias. Crucial to the development of experimental approaches to study these diseases, and to design treatment strategies, is the establishment of a homogeneous cell preparation which expresses most aspects of the cholinergic phenotype. The goal of the studies described in this application is to characterize the cholinergic properties of a cell line (SN56.B5.G4) developed by hybridization of neuroblastoma cells with mouse brain septal neurons (the neuronal population which is important in memory processing and which degenerates in AD). The activity of choline acetyltransferase, choline uptake, acetylcholine (ACh) storage in vesicles and ACh release in these cells will be studied. The effects of growth factors on the development of the cholinergic phenotype in these cells as well as the effects of pharmacologic agents affecting ACh turnover will be examined. Further, the studies will explore a metabolic peculiarity of cholinergic neurons that might make them especially vulnerable to certain disease processes; their ability of use membrane choline-containing phospholipids as a precursor for ACh. If this process is activated for a prolonged period of time, pathological changes in neuronal membranes might occur, impairing neuronal function or viability. We have previously shown that membrane phosphatidylcholine (PC) is a source of choline for ACh synthesis in human neuroblastoma (LA-N-2) cells and we have obtained preliminary evidence that the choline is liberated from PC by phospholipase D (PLD). Because this enzyme is stimulated by muscarinic receptor activation, we postulate that, in cholinergic synapses, where these receptors are localized, PLD may be activated by ACh, leading to generation of choline which may be taken up by the presynaptic terminals and converted into ACh. We will investigate the regulation of this pathway by a) neuronal firing rate, b) extracellular choline concentration, c) growth factors, d) pharmacological agents that affect ACh synthesis and release, and e) agents that effect phospholipid metabolism. Most experiments will be performed using radiolabeled precursors of ACh and of choline-containing phospholipids and tracing the metabolic fate of the labeled precursors by purifying their metabolites with high performance liquid chromatography. The proposed studies will establish new experimental approaches (which do not involve experimental animals) to study cholinergic functions and will provide new information on the mechanisms regulating ACh synthesis and release.

The overall goal of this study is to characterize the neurochemical correlates of the improved memory performance in rats treated perinatally with supplemental choline. Choline is a precursor of the neurotransmitter acetylcholine (ACh) in cholinergic neurons and cholinergic mechanisms are important in memory processes. In addition, choline is a precursor of phosphatidylcholine (PC), sphingomyelin and plasmenylcholine; phospholipids which are collectively the most abundant components of all biological membranes. Thus choline administration may alter either cholinergic neurotransmission or membrane events or both. Our studies to date indicate that perinatal choline supplementation alters the development of brain cholinergic system. We propose to vary the supply of choline (using choline-sufficient; and choline-supplemented treatment protocols) to male and female rats during defined periods of brain development [embryonic (E) days E12-17, postnatal (P) days P1-P15, days P16-P30, and days E12-P30] and to measure indices of cholinergic neurotransmission and of membrane turnover as a function development [from embryonic day 17 to late adulthood (30 months of age)]. Specifically we shall determine: 1) the developmental changes in the concentrations of water-soluble and lipid-soluble metabolites of choline in brain regions: 2) ACh release in vivo using the microdialysis technique; 3) ACh synthesis and release using in vitro brain preparations; 4) the ACh-evoked phosphatidylinositol turnover; and 5) PC turnover. The studies will provide new information on the cholinergic system and on membrane phospholipid turnover during brain development and aging. The biochemical correlates of memory enhancement in aged rats associated with perinatal choline will be established. This information will help to develop perinatal nutritional strategies which could enhance memory in aged humans.

An important goal of biomedical science is the design of treatments which prevent memory loss associated with aging. We have observed that administration of choline to rats during two specific perinatal periods results in enhanced performance on memory tests which persists throughout animals' lifetime. These behavioral changes are accompanied by neuroanatomical and neurochemical modifications of the brain. We have also described several mechanisms which lead to maintenance of high plasma choline concentrations early in the development of animals and humans. We hypothesize that, during critical perinatal periods, availability of choline influences the anatomical and biochemical organization of developing brain. These organizational changes influence memory performance in adult and aged animals. Choline administration may alter either cholinergic neurotransmission or membrane events or both. Choline is a precursor of a neurotransmitter, acetylcholine, in cholinergic neurons and cholinergic mechanisms are important in memory processes. Choline is also a precursor of phosphatidylcholine, sphingomyelin and plasmenylcholine; phospholipids which collectively are the most abundant components of all biological membranes. In addition, after oxidation to betaine, choline is a methyl donor, and thus its availability influencess the metabolism of methionine and folic acid. Our studies to date indicate that perinatal choline supplementation alters the biosynthesis of brain phosphatidylcholine and affects the development of brain cholinergic system. The overall goal of this study is to characterize the physiological processes which underlie the long- term memory enhancement associated with perinatal choline status and to characterize further the factors which influence choline availability to brain during the perinatal period. We propose to vary the supply of choline (using choline-deficient; choline-sufficient; and choline- supplemented treatment protocols) to male and female rats during defined periods of brain development [embryonic (E) days E12-17, postnatal (P) days P1-P15, days P16-P30, and days E12-P30] and to correlate behavioral measurements with: changes in the metabolism of choline; function of cholinergic neurons and membrane turnover; and changes in the anatomy of the brain. Since males and females respond differently to choline supplementation, we propose to investigate the effects of genetic sex and of gonadal steroids on these parameters. In order to characterize fully these processes throughout animals' life, these measurements will be made in animals between the embryonic day 17 and 30 months of age. The studies will provide new information on brain development and aging. The biochemical physiological and anatomical correlates of memory enhancement in aged rats associated with perinatal choline will be established. The ultimate goal of our studies is to relate our results to the age- associated changes in memory in humans, and to develop perinatal nutritional strategies which could benefit people.

The overall goal of Project 1 is to determine the molecular mechanisms involved in brain reorganization governed by prenatal availability of choline or folic acid and by apolipoprotein E (apoE) genotype. We have found that the availability of choline during the second half of gestation in rats causes biochemical, structural and electrophysiologic changes in brain as well as profound behavioral modifications.. In general, young adult and aged rats supplemented prenatally with choline and improved performance relative to control and prenatally-deficient animals in tasks measuring memory and attention. In contrast,, prenatally deficient animals were impaired in attentional tasks measuring memory and attention. In contrast, prenatally deficient animals were impaired in attentional tasks but somewhat improved in memory tasks. Studies performed to data indicated that prenatal availability of choline may affect the development of multiple synaptic signaling pathways in brain. Specifically prenatal choline availability modifies hippocampal long-term potential,.acetylcholine (ACh) turnover, phospholipase D activity, and indices of nerve growth factor signaling. We will determine the effects of prenatal choline availability and folate availability on signal transduction systems in brain during development, adulthood and aging. Studies performed to date show that prenatal availability of choline alters the patterns of mitosis and apoptosis in developing brain as well as patterns of expression of several proteins. These data are consistent with our hypothesis that prenatal availability of essential nutrients causes multiple changes in brain organization. We propose to determine the developmental patterns of expression of brain genes using hybridization to high density oligonucleotide arrays and reverse Northern analysis followed by in situ hybridization assays of thus identified genes. The metabolism of folate and choline are highly interrelated; therefore the effects of folate availability on ACh turnover in brain will be examined. Within the brain, choline may be redistribut4ed between cells by a mechanism involving apolipoprotein A-mediated transport of a choline- containing lipid. phosphatidylcholine (PC). We will determine if brain ACh turnover is altered in ApoE-/- mice. We will investigate the possibility that dietary choline will modify ACh turnover in folate deficient and ApoE-/- animals. In addition we propose to determine if apoE-containing lipoproteins can supply PC to cholinergic neurons using a cull culture model.

The overall goal of the proposed studies is to determine the mechanisms by which the availability of choline and folic acid during the prenatal period modifies brain structure and function in development, adulthood and old age. Our major premise is that the development of the brain during critical periods in embryogenesis is sensitive to changes in maternal diet, and specifically, is influenced by changes in the intake of choline and folic acid. We found that rats treated with choline during specific perinatal periods inhibited improved memory function which lasted throughout their lifespan, i.e. supplementation with choline in development prevented age-related deterioration in learning and memory. Moreover, variations in maternal choline intake during the second half of pregnancy caused biochemical, structural, and electrophysiological changes in the brains of the offspring. We also found that memory performance in rats was improved by prenatal supplementation with folic acid. The proposed studies will be conducted using a unified experimental design common to all projects. Dr. Blusztajn will determine the molecular mechanisms involved in the brain reorganization that is governed by choline and folate availability by studying signal transduction pathways and developmental patterns of gene expression in brain. Dr. Swartzwelder will measure synaptic function and plasticity (long-term potentiation in hippocampus of rats exposed to varying levels of choline or folate in utero. Dr. Meck will examine age-related changes in conditioned stimulus processing (attention) as a function of the prenatal availability of choline and folate. Dr. Williams will determine if supplementation with folate in early development leads to life long changes in spatial memory, brain anatomy, and neurochemistry, as has been documented for choline supplementation, and will investigate whether choline supplementation with folate in early development leads to lifelong changes in spatial memory, brain anatomy, and neurochemistry, as has been documented for choline supplementation, and will investigate whether choline supplementation either prenatal or across the lifespan ameliorates behavioral, anatomical, and biochemical deficits seen in mice lacking the gene for apolipoprotein E-a molecular involved in the transport of phosphatidylcholine within brain. Dr. Zeisel will study metabolic interrelationships between folate and choline in order to determine if they share a common mechanism of action on brain organization. He will determine the effects of choline and folate on patterns of fetal brain cell division and apoptosis, and will investigate the mechanism by which choline deficiency causes apoptosis. Dr. Kowall will provide neuroanatomy core services for the five projects. The ultimate goal of our studies is to related our results to age-related changes in memory in humans, and to develop perinatal nutritional strategies which will benefit people.

The overall goal of the proposed studies is to determine the mechanisms by which the availability of choline and folic acid during the prenatal period modifies brain structure and function in development, adulthood and old age. Our major premise is that the development of the brain during critical periods in embryogenesis is sensitive to changes in maternal diet, and specifically, is influenced by changes in the intake of choline and folic acid. We found that rats treated with choline during specific perinatal periods inhibited improved memory function which lasted throughout their lifespan, i.e. supplementation with choline in development prevented age-related deterioration in learning and memory. Moreover, variations in maternal choline intake during the second half of pregnancy caused biochemical, structural, and electrophysiological changes in the brains of the offspring. We also found that memory performance in rats was improved by prenatal supplementation with folic acid. The proposed studies will be conducted using a unified experimental design common to all projects. Dr. Blusztajn will determine the molecular mechanisms involved in the brain reorganization that is governed by choline and folate availability by studying signal transduction pathways and developmental patterns of gene expression in brain. Dr. Swartzwelder will measure synaptic function and plasticity (long-term potentiation in hippocampus of rats exposed to varying levels of choline or folate in utero. Dr. Meck will examine age-related changes in conditioned stimulus processing (attention) as a function of the prenatal availability of choline and folate. Dr. Williams will determine if supplementation with folate in early development leads to life long changes in spatial memory, brain anatomy, and neurochemistry, as has been documented for choline supplementation, and will investigate whether choline supplementation with folate in early development leads to lifelong changes in spatial memory, brain anatomy, and neurochemistry, as has been documented for choline supplementation, and will investigate whether choline supplementation either prenatal or across the lifespan ameliorates behavioral, anatomical, and biochemical deficits seen in mice lacking the gene for apolipoprotein E-a molecular involved in the transport of phosphatidylcholine within brain. Dr. Zeisel will study metabolic interrelationships between folate and choline in order to determine if they share a common mechanism of action on brain organization. He will determine the effects of choline and folate on patterns of fetal brain cell division and apoptosis, and will investigate the mechanism by which choline deficiency causes apoptosis. Dr. Kowall will provide neuroanatomy core services for the five projects. The ultimate goal of our studies is to related our results to age-related changes in memory in humans, and to develop perinatal nutritional strategies which will benefit people.

DESCRIPTION (provided by applicant): Degeneration or malfunction of central and peripheral cholinergic neurons underlies and/or contributes to the pathophysiology of a variety of disorders including Alzheimer's Disease, Motor Neuron Disorders, and Familial Dysautonomias, and possibly memory loss associated with aging. Understanding the cholinergic phenotype and the mechanisms regulating it is crucial to the development of experimental approaches to study these diseases, and to the design of treatment strategies. We have focused on the elucidation of mechanisms that regulate the cholinergic neurotransmitter phenotype and our data show that specific bone morphogenetic proteins (BMPs) dramatically upregulate the expression of this phenotype in primary cultures of mouse central nervous system, and in the brain in vivo. The cellular and molecular mechanisms of action of BMPs in inducing and maintaining the neuronal cholinergic phenotype remain unknown and will be the subject of the current proposal. Specifically we propose: 1. To identify the intracellular signaling molecules that mediate the induction of the cholinergic phenotype by BMPs, and 2. To delineate the promoter regions within the cholinergic gene locus that respond to BMPs and to identify protein transcription factors that bind to those BMP-responsive regions. The studies will be performed on primary neuronal cultures obtained from wild type mice and from animals with targeted mutations of genes encoding candidate transcription factors that may mediate the effects of BMPs on the cholinergic phenotype. Investigations will focus on the signaling functions of Smad proteins and other transcription regulators, expression of cholinergic-specific promoters in reporter constructs, and DNA binding of candidate transcription factors. In addition, we propose animal studies to test the hypothesis that administration of BMPs in vivo can correct septo-hippocampal lesion evoked downregulation of cholinergic function using neuroanatomical tools. Taken together this research will provide basic and preclinical data on the mechanisms of action of BMPs in the brain and the potential for their use in treating neurological disorders affecting cholinergic neurons.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of our studies is to determine the mechanisms by which prenatal availability of choline, and of methyl groups derived from folic acid, modify brain structure and function during development, adulthood and old age. Our principal hypothesis is that the development of the brain during critical periods in embryogenesis is sensitive to changes in maternal diet, specifically to changes in the intake of the essential nutrients, choline and folic acid. We found that rats and mice treated with choline during specific prenatal periods exhibited improvements in memory and attention which lasted throughout their entire lifespan, i.e. supplementation with choline in development prevented age-related memory deterioration. Moreover, variations in maternal choline intake during the second half of pregnancy caused biochemical, structural, and electrophysiologic changes in brain of the offspring. The proposed studies will be conducted with a unified experimental design common to all projects, including the use of mice with targeted mutations in key genes of choline and methyl group metabolism (i.e. apolipoprotein E, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase, and choline dehydrogenase) as models of human conditions associated with polymorphisms in these genes. Dr. Blusztajn (Project 1) will determine if choline availability in utero regulates patterns of gene expression by altering brain DNA methylation and if it alters development and aging of selected neuronal populations identified by gene expression patterns. He will identify the molecular mechanisms of choline action using mutant mouse models. Dr. Meck (Project 2) will determine if altered memory performance in animals exposed to prenatal choline supplementation or deficiency is correlated with changes in sleep patterns, and altered hippocampal ensemble activity patterns. Dr. Williams (Project 3) will determine if prenatal availability of choline influences adult neurogenesis and neuronal survival and will assess behavior in mutant mouse models. Dr. Zeisel (Project 4) will determine the mechanisms by which choline availability alters the patterns of gene expression, cell cycling, migration, apoptosis, and differentiation during fetal development and will study these processes in mutant mouse models. Dr. Kowall (Core B) will provide qualitative and quantitative neuroanatomy analyses using immunohistochemistry and image analysis, and will provide genotyping services for the five projects. The ultimate goal of our studies is to relate our results to the age-associated changes in memory in humans, and to develop perinatal nutritional strategies which could benefit people. [unreadable] [unreadable] [unreadable]

The overall goal of Project 0001 is to determine the molecular mechanisms involved in brain reorganization governed by prenatal availability of choline. Aim 1 is to test the hypothesis that prenatal choline availability modulates developmental patterns of gene expression by altering DNA methylation of the regulatory elements of genes whose expression is controlled by 5-methylcytosine content, based on the evidence showing that choline, via its action as a donor of methyl groups, can alter DNA methylation profiles and gene expression in vivo. We will study DNA methylation of selected genes known to be regulated by DNA methylation and whose expression in brain is modulated by prenatal availability of choline (e.g. insulin-like growth factor II, lgf2). Aim 2 is to test the hypothesis that prenatal choline availability alters the development and aging of selected neuronal populations that can be identified by gene expression profiles. Using oligonucleotide microarrays, we found that the expression pattern of multiple hippocampal and cerebral cortical genes, including receptor ligands, receptors, protein kinases, and transcription factors (e.g. Igf2, GABABR1, TrkB, Camkl, Camkllbeta, PKCbeta2, and Zif268), is modulated by the prenatal availability of choline. We will map the expression of these proteins using immunoblotting, in situ hybridization, and immunohistochemistry (with the Neuroanatomy Core) in order to identify the relevant neuronal populations. Aim 3 is to test the hypothesis that individual requirements for choline depend on genotype. Several genes encoding proteins involved in the metabolism of choline and of methyl groups display potymorphism in humans and cause metabolic abnormalities that, in some cases, can be successfully treated with nutritional strategies. Genetic mouse models of these conditions will be used to obtain information on the mechanisms by which alterations in the supply of choline can modulate the phenotype of these animals. Four models will be studied, including mice with targeted mutations in apolipoprotein E (Apoe), phosphatidylethanoamine N-methyltransferease, methylenetetrahydrofolate reductase, and choline dehydrogenase. Project 3 has shown that cognitive defects observed in Apoe null mice can be rescued by choline supplementation throughout gestation, thus providing the experimental paradigm for these studies. In the four mouse models, we will determine gene expression patterns and signal transduction mechanisms, sensitive to prenatal choline availability, including MAPK and CREB phosphorylation and acetylcholine turnover, and the effects of dietary choline on their phenotypes.

The goal of the proposed studies is to test the hypothesis that prenatal availability of choline modulates the risk of development of mammary carcinoma. This notion is based on evidence that prenatal availability of choline has long-lasting biological effects persisting into adulthood and old age. Choline is an essential nutrient for humans. Because fetal development constitutes a period of increased demand for choline, US dietary guidelines call for increased choline intake by women during pregnancy. In our previous studies, that focused on the effects of prenatal availability of choline on brain development and cognitive functions, we found that rats supplemented with choline during the second half of gestation exhibited improvements in memory which lasted throughout their entire lifespan. These effects correlated with molecular, biochemical, and structural changes in brain. In adult rats, lack of dietary choline causes hepatocarcinoma and is the only nutritional deficiency that leads to cancer in the absence of any known carcinogens. We found that choline deficiency in adulthood increased mammary cancer risk while choline supplementation reduced it in rats treated with procarbazine as a carcinogen. Aim 1 is designed to test the hypothesis that prenatal choline availability (deficiency, sufficiency, and supplementation) modulates the development of the mammary gland in female rats as monitored by histological techniques and patterns of gene expression determined by microarrays. To assess the mechanisms of the latter changes we will test the hypothesis that prenatal availability of choline alters DMA methylation of the regulatory elements of genes, based on the evidence that choline, via its action as a donor of methyl groups, can alter DNA methylation and gene expression in vivo. We will study DNA methylation of selected genes that are regulated by this process and whose expression we found to be modulated by prenatal availability of choline (e.g.insulin-like growth factor II, Igf2). Aim 2 is designed to test the hypothesis that prenatal availability of choline modulates mammary carcinogenesis using as a model Sprague Dawley rats treated with 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogen that causes tumors similar to human breast cancers. Tumor incidence and tumor development will be determined. Premalignant as well as malignant changes in mammary tissues from these animals will be evaluated using histological and microarray techniques. In a pilot study of this kind we observed trends for a slowed disease progression in prenatally choline-supplemented rats and an acceleration of tumor growth by prenatal choline deficiency. The mammary tumors from prenatally choline- supplemented rats had higher expression of cyclin D1, STATS, and prolactin receptor and lower expression of STATS mRNA as compared to the tumors from the control- and prenatally choline-deficient subjects. This proposal, prepared in response to an NIH Program Announcement, uses the R21 Exploratory Developmental grant mechanism designed to provide support for the early stages of research development such as projects to assess the feasibility of a novel area of investigation. The ultimate goal of our studies is to relate our results to humans and to develop perinatal nutritional strategies which could benefit people.

DESCRIPTION (provided by applicant): Aging is associated with a decline of multiple cognitive functions. In particular, the ability to form memories of recent events and learn new complex information tends to diminish. The hippocampus is critical for the establishment of new memories, and there is evidence from studies in animals and humans that advanced age causes structural and neurochemical changes in the hippocampus that result in its functional impairment, leading to cognitive deficits. One of the key components of the hippocampal neuronal circuitry that is necessary for learning and memory is its innervation by the septo-hippocampal pathway that provides a modulatory input mediated by the neurotransmitter, acetylcholine (ACh). This cholinergic innervations develops during the first two postnatal weeks in rats and mice but its function declines in aging brain. Unlike most other brain regions, the hippocampus is characterized by a life-long capacity for neurogenesis - an outcome of divisions of the dentate gyrus neuronal precursor cells whose progeny can differentiate into functional granule neurons integrated into the granule cell layer. This neurogenic activity is high in the young animal but declines with age. While the role of adult neurogenesis remains to be understood, there is evidence that it is necessary for normal hippocampal function and that impaired neurogenesis in aged brain correlates with cognitive decline. Hippocampal structure and function are regulated by trophic and differentiating factors. These molecules modulate the establishment and maintenance of the septohippocampal cholinergic pathway and hippocampal neurogenic activity under normal conditions and in response to disease and/or injury. The expression of some of these trophic factors is particularly high during early postnatal maturation of the hippocampus and low in the adult. We have identified two such juvenile factors, bone morphogenetic protein 9 (BMP9) and insulin-like growth factor 2 (IGF2). The overall goal of the proposed studies is to test the hypothesis that administration of these putative juvenile protective factors (BMP9 or IGF2) to the brain of aging Fischer 344 rats will prevent the decline or cause a recovery of hippocampal function as determined by assays of cholinergic markers, neurogenesis, and cognitive performance. We will 1) verify that BMP9 and IGF2 function as juvenile trophic factors in postnatal development of the hippocampus by reducing their levels during the critical postnatal periods with intracerebroventricular (icv) administration of neutralizing antibodies against these factors, and 2) determine if the icv administration of BMP9 or IGF2 to aged rats improves hippocampal function. The ultimate goal of our studies is to relate our results to the age-associated changes in memory in humans, and to develop juvenile trophic-factor replacement strategies which could benefit people. Aging is associated with a decline of multiple cognitive functions. We have identified two proteins, bone morphogenetic protein 9 (BMP9) and insulin-like growth factor 2 (IGF2) that are abundant in juvenile brain but decline with brain's maturation and we propose that administration of these putative juvenile protective factors (BMP9 or IGF2) to the brain will prevent the decline or cause a recovery of brain function associated with aging, using old rats as a model. The ultimate goal of our studies is to relate our results to the age-associated changes in memory in humans, and to develop juvenile growth-factor replacement strategies that could benefit people.

DESCRIPTION (provided by applicant): Accumulating evidence indicates that epigenetic mechanisms are vital for normal brain development including modulation of generation of neurons and glia, neuronal cell-type specification and differentiation, circuit and synapse formation, and myelination. These events - that occur during a critical period that spans gestation and early postnatal time - are highly sensitive to the supply of essential nutrients and in particular to the supply of metabolic methyl group donors including choline. The central hypothesis to be tested by the studies described in this proposal is that alterations in the availability of choline during this critical period modulates the differentiation and phenotype of basal forebrain cholinergic neurons (BFCN) via epigenetic mechanisms involving changes in DNA and histone methylation. BFCN constitute a key component of the neuronal circuitry that is necessary for the processes of learning, memory, attention, and sleep. We have developed a method to purify BFCN throughout lifespan by fluorescence-activated cell sorting using transgenic mice that that express enhanced green fluorescent protein (eGFP) under the control of the regulatory elements of the choline acetyltransferase gene. Using purified BFCN from mice at various developmental stages between embryonic day 16 and postnatal day 30 we will determine: 1) global transcriptome profile using microarrays;2) DNA methylation patterns with methylated DNA immunoprecipitation (MeDIP) followed by microarray analysis and 3) chromatin immunoprecipitation (ChIP) with antibodies against different methylated epitopes on specific lysine residues of histone 3 (e.g. H3K9me2;H3K4me1;H3K4me3) followed microarray analysis (ChIP-chip). These data will constitute the first characterization of the epigenome and its correlation with the transcriptome of a developing pure neuronal population. We will perform an analogous study on the fetuses and offspring of mice whose mothers consume diets containing choline-deficient, choline-sufficient, or choline-supplemented diets throughout pregnancy until weaning. The results of the investigations will provide information on epigenetic processes that are associated with the completion of developmental milestones of BFCN and on the modulation of these processes by gestation and postnatal nutritional choline intake. PUBLIC HEALTH RELEVANCE: The basal forebrain cholinergic neurons (BFCN) constitute a key component of the neuronal circuitry that is necessary for the processes of learning, memory, attention, and sleep. BFCN development is modulated by the perinatal availability of the methyl-group-donating essential nutrient, choline, and changes of choline intake in pregnancy cause dramatic modifications in DNA and histone methylation patterns in fetal brain. We will perform the first of its kind study on murine BFCN purified by FACS throughout development (fetal to adulthood) by measuring the effects of choline intake on the pattern of BFCN gene expression in correlation with global DNA and histone methylation patterns.

DESCRIPTION (provided by applicant): This R21 application was prepared in response to the RFA entitled Epigenomic Modifications in Neurodevelopment. As described in the RFA, our studies are broadly designed to characterize epigenetic events involved in neurodevelopmental processes and to identify epigenome-wide marks associated with vulnerability to mental disorders. The proposed studies will address cell specificity in the brain across development, and focus on how several epigenetic modifications contribute to the development and maturation of a defined population of brain cells known as the locus coeruleus noradrenergic neurons (LCNN). LCNN constitute a critical component of the circuitry that is necessary for the processes of learning, memory, attention, sleep, arousal, anxiety, and responses to stress. Abnormalities of the LCNN are thought to contribute to the symptoms of attention deficit hyperactivity disorder, depression, Parkinson's and Alzheimer's diseases, and sleep disorders. Our studies focus on the key periods of development of LCNN from the completion of neurogenesis in the fetus through postnatal maturation and the establishment of functional synaptic connections, and conclude with adulthood in both male and female subjects. We have developed a method to purify defined neuronal populations throughout lifespan by fluorescence-activated cell sorting using transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the regulatory elements of genes expressed exclusively in these populations. In this study we will use transgenic mice (designated Dbh- EGFP) that express EGFP under the control of the regulatory elements of the dopamine 2-hydroxylase (Dbh) gene - a marker of noradrenergic neurons. By combining several state-of-the-art experimental approaches, we will generate the first comprehensive characterization of the genome-wide developmental pattern of the LCNN transcriptome [for both messenger RNA (mRNA) and microRNA (miRNA)], the DNA CpG methylome, and genome-wide maps of methylated histone H3 (e.g. H3K4me1, H3K4me2, H3K4me3, H3K9me3, H3K27me3). We will integrate this novel information into a framework defined by chromatin state maps and developmental gene expression profiles of LCNN. We will develop interactive bioinformatics tools designed to manage, visualize and integrate these data. This study, supported by an R21 mechanism that focuses on a single population of neurons highly relevant to mental health (the LCNN), will serve as a proof of concept for our long- term goal: to establish a comprehensive atlas of developmental epigenomic events that occur in key neuronal populations purified from specific brain regions and identified by their neurotransmitter (e.g. GABA, dopamine, serotonin, acetylcholine) or neuropeptide (e.g. galanin, NPY, VIP, substance P) phenotype. A large collection of the relevant EGFP-expressing transgenic mouse lines has been generated and is available at the NIH- supported GENSAT resource.

DESCRIPTION: Aging is frequently associated with a decline in multiple cognitive functions. In particular, the ability to form memories of recent events and assimilate new and complex information tends to diminish. Moreover, these cognitive defects are hallmarks of devastating, age-associated dementias such as Alzheimer's disease (AD). Due to their high prevalence and the lack of any effective therapies, the development of prevention measures and treatment strategies for these conditions constitutes one of the highest priorities of the biomedical sciences. The concept of utilizing juvenile protective factors for this purpose is an attractive on - however, it presents two central challenges: 1) the identification and characterization of a candidate factor, and 2) the utilization of its potential for therapeutic benefit. The proposed studies focus on a compelling candidate molecule - growth and differentiation factor 2 (GDF2), more commonly referred to as bone morphogenetic protein 9 (BMP9), and its actions on critical neuronal systems that underlie cognition. One of the key components of the neuronal circuitry necessary for learning, memory and attention is the innervation of the hippocampus and cerebral cortex by basal forebrain cholinergic neurons (BFCN), which provide modulatory input mediated by the neurotransmitter, acetylcholine (ACh). A decline in BFCN function and diminished cholinergic marker expression is apparent in aged humans and animal, in AD patients, and in animal models of AD. Thus, it has been postulated that dysfunction and/or degeneration of BFCN contributes to the memory deficits seen in advanced age and in AD. We have obtained evidence that BMP9 is a key differentiating factor for BFCN during development and, when infused intracerebroventricularly in mice with experimental injury to these neurons, prevents BFCN loss. Moreover, our preliminary data show that BMP9 infusion reverses the downregulation of BFCN markers seen in a transgenic mouse model of AD and ameliorates amyloidosis. These data indicate that BMP9 is sufficient to support BFCN differentiation and function in the adult brain; however we do not yet know to what extent BMP9 is necessary for cholinergic neuron biology. In aim 1 this central question will be addressed by loss-of-function studies on Bmp9 knockout mice. In aim 2 we will test the utility of BMP9 as a therapeutic agent for age-associated cognitive and BFCN dysfunction, with the focus on AD, using transgenic mouse models. In aim 3, we will explore the hypothesis that BMP signaling may be abnormal in the brains of aging humans and AD patients, using post-mortem brain samples from a unique collection of cases with a thorough cognitive and histopathological assessment, available through the Framingham Heart Study.

Studies proposed in this application are designed to develop an understanding of the molecular and cellular basis for age-related changes in lipid metabolism that impact the development of Alzheimer's disease (AD). To achieve this goal we will use a cross-disciplinary approach that will combine unique sets of clinical data and human biospecimens together with mechanistic hypotheses testing using a mouse model of AD with a relevant targeted genetic abnormality in phospholipid metabolism. There is strong evidence that phospholipid metabo- lism is abnormal in AD brain and there are data indicating that prodromal and frank clinical AD have correlates in the blood plasma lipidome that include apparent abnormalities of the turnover of phosphatidylcholine (PC) ? a major constituent of all biological membranes, including those in neurons and glial cells. This is reflected by reduced plasma levels of certain molecular species of PC containing eicosapentaenoic- [EPA, 20:5n-3] and docosahexaenoic acid [DHA, 22:6n-3] in AD. Together, the data suggest the hypothesis that AD pathophysiol- ogy may be characterized by lipidomic abnormalities that occur early in the disease and encompass both the periphery (as reflected by plasma lipidomic changes) and the brain. However, heretofore it has not been possi- ble to address this idea directly due to the lack of an appropriately optimized scientific and clinical infrastructure that incorporates blood and brain samples. We propose to test this hypothesis by interdisciplinary approaches that will employ sets of data and biorepositories of plasma and postmortem brain tissue derived from the par- ticipants of the Framingham Heart Study (FHS) population. The FHS has epidemiology of dementia and brain donation programs (over 200 brains available), resulting in longitudinal clinical and neuropsychological data for subjects whose diagnoses range from cognitively intact to mild cognitive impairment to dementia. The neuro- pathological diagnoses of these subjects range from normal to advanced AD. The FHS has food frequency questionnaire data and has collected plasma samples longitudinally over time from these subjects, making it an optimal resource to identify abnormalities in the metabolism of lipids, associated with cognitive impairment and AD pathology, that may serve as biomarkers and targets for therapy. The polyunsaturated species of PC (such as PC-DHA) are primarily synthesized by the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). PEMT abnormalities reduce plasma PC-DHA concentrations in humans and have been associated with increased risk of AD. Therefore, abnormal PEMT activity may contribute to AD pathophysiology. We pro- pose the following aims: 1) To identify lipidomic- and PEMT activity changes associated with age and AD de- velopment in brain tissue; 2) To determine lipidomic profiles of serial, stored plasma samples obtained in life from the above subjects, and correlate the plasma lipidomic data with the information obtained in Aim 1 and 3) To determine the effect of PEMT activity and PC-DHA levels on the development of AD-like pathology in AD model mice with 2, 1, or 0 copies of the Pemt gene, for mechanistic hypothesis testing.

The high prevalence of age-related dementias including Alzheimer's disease (AD), and the lack of effective therapies, makes the development of preventive measures and treatments for these conditions a high priority for biomedical sciences. We found that an increased supply of the essential nutrient, choline, during early life prevents age-related cognitive decline and ameliorates neuropathologic hallmarks of AD in animal models. Be- cause choline serves as a donor of metabolic methyl groups, we hypothesized that the long-term effects of choline availability during development on adult brain function may be related to altered patterns of DNA meth- ylation (DNAm) ? an epigenetic mechanism for regulation of gene expression. Our data are consistent with this notion. Recent studies provided evidence that methylation of a specific set of DNA CpG sites varies with an in- dividual's age, allowing estimates of aging rates in various tissues, and giving rise to the terms ?DNAm age,? ?DNAm clock? and ?epigenetic clock?. DNAm age may also be altered by disease and environment, including nutrition. The discovery of the DNAm clock as a potential biomarker of aging has provided an intellectual framework for testing of hypotheses that link the epigenome to age-related phenotypes. We propose that the brain epigenetic clock is accelerated by the pathophysiologic mechanisms of AD, and slowed by high choline intake during development. Our aim is to determine the effect of high choline nutrition during development on hippocampal DNAm epigenetic aging in wild type and AD-model (APP.PS1), mice and to correlate these pa- rameters with gene expression patterns, cognitive function, and measures of neuropathology. We will use male and female WT and APP.PS1 mice raised by mothers who consumed diets containing standard or high amounts of choline during gestation and nursing. At 6, 12, and 18 months of age we will assess learning and memory of the mice, and subsequently perform high-resolution DNAm analyses using reduced representation bisulfite sequencing (RRBS) and global gene expression analyses using RNA sequencing (RNA-Seq) in their hippocampi. Using state-of-the-art bioinformatics and statistics tools we will a) Determine DNAm patterns, epi- genetic clock DNAm patterns as well as RNA expression patterns as a function of age, sex, AD-model, and developmental choline intake; b) Correlate differential DNAm patterns within the genomic regions of differen- tially-expressed RNAs in order to identify possible cis-acting regulatory sites; and c) Correlate the DNAm- and the RNA expression changes with cognition and neuropathology. Our comprehensive temporal data sets and analyses will provide novel information on the plasticity of the DNA methylome, including the DNAm clock, in WT and AD-model mice in response to availability of choline in early life, and will characterize the relationships of these epigenomic events to transcriptomic, cognitive, and neuropathological measures. The demonstration that the epigenetic clock predicts the trajectory of cognitive aging, and that timely intervention can alter that tra- jectory, will be invaluable in the design of therapeutic strategies to delay age-associated dementias.