Project description:The complexity of events associated with age-related memory loss (ARML) cannot be overestimated. The problem is further complicated by the enormous diversity of neurons in the CNS and even synapses of one neuron within a neural circuit. Large-scale single-neuron analysis is not only challenging but mostly impractical for any model currently used in ARML. We simply do not know: do all neurons and synapses age differently or are some neurons (or synapses) more resistant to aging than others? What is happening in any given neuron while it undergoes “normal” aging? What are the genomic changes that make aging apparently irreversible? What would be the balance between neuron-specific vs global genome-wide changes in aging? In the proposed paper we address these questions and develop a new model to study the entire scope of genomic and epigenomic regulation in aging at the resolution of single functionally characterized cells and even cell compartments. In particular, the mollusc Aplysia californica has been implemented as a powerful paradigm in addressing fundamental questions of the neurobiology of aging. The proposed manuscript will consist of four parts. First, we will provide an introduction to Aplysia as a representative of the largest superclade of bilaterian animals (Lophotrochozoa). Aplysia has a short lifespan of 220-300 days with a well-characterized life cycle and characterized phenomenology of aging. Most importantly, Aplysia possess the largest nerve cells in the entire animal kingdom (only eggs are larger); these cells can be uniquely identified and mapped in terms of their well-defined interactions with other neurons forming relatively simpler neural circuits underlying several stereotypic and learned behaviors. Second, we have identified in Aplysia more that a hundred neurological- and age-related genes that were lost in other established invertebrate models (such as Drosophila and C. elegans). The proposed long-term regulatory age-related mechanisms include a high level of conservation among many epigenetic processes known to be lost in nematodes and flies with extremely short lifecycles and particularly derived genomes. We also identify and cloned more than 30 evolutionarily conserved homologs of genes involved in Alzheimer’s, Parkinson’s and Huntington’s diseases as well as age-related hormones. Third, we performed genome-wide analysis of expression patterns of more than 55,000 unique transcripts by comparing two different identified cholinergic neurons (R2 and LPl1) among young and aged animals. This direct single neuron genomic analysis indicates that there are significant cell-specific changes in gene-expression profiles as a function of aging. We estimated that only ~10-20% of genes that are differently expressed in the aging brain are common for all neuronal types - the remaining 80% are neuron-specific (i.e. found in aging neurons of one but not another type). The list of “common aging genes” includes components of insulin growth factor pathways, cell bioenergetics, telomerase-associated proteins, antioxidant enzymes, water channels and estrogen receptors. The rest were neuron-specific gene products (including apoptosis-related proteins, Alzheimer-related genes, growth factors and their receptors, ionic channels, transcription factors and more than 120 identified proteins known to be involved in neurodevelopment and synaptogenesis). Surprisingly, even two different identified cholinergic motoneurons age differently and each of them has a unique subset of genes differentially expressed in older animals. Fourth, we showed that the activity of the entire genome and associated epigenomic modifications (e.g. DNA methylation, histone dynamics) can be efficiently monitored within a single Aplysia neuron and can be modified as a function of aging in a neuron-specific manner including selective histones and histone-modifying enzymes and DNA methylation-related enzymes. This genome-wide analysis of aging allows us to propose novel mechanisms of active DNA demethylation and cell-specific methylation as well as regional relocation of RNAs as three key processes underlying age-related memory loss. These mechanisms tune the dynamics of long-term chromatin remodeling, control weakening and the loss of synaptic connections in aging. At the same time, our genomic tests revealed evolutionarily conserved gene clusters in the Aplysia genome associated with senescence and regeneration (e.g. apoptosis- and redox- dependent processes, insulin signaling, etc.). This is a reference design experiment with all samples being compared to one CNS from Aplysia. Two cholinergic neurons (R2 and LPl1), two ages (young and old), two arrays (AAA and DAA), three biological replicates each sample type. Two direct comparison experiments were also performed. One with young and old abdominal ganglion and the other with young and old R2.

Project description:The marine opisthobranch mollusk, Aplysia californica, is a powerful experimental system in cellular, molecular, and behavioral neuroscience as well as cell and evolutionary biology because of the distinctive organization of its nervous system, which makes it advantageous for cellular and comparative analysis of a variety of behaviors and learning and memory. Aplysia's large neurons allow examination of neuronal architecture of instinctive and learned behaviors at the level of single characterized cells and defined cellular compartments (e.g., synapses). As a result, many fundamental problems in neurobiology can be analyzed more effectively in Aplysias than in Drosophila, C. elegans, and vertebrates. In a larger sense work on Aplysia is synergistic with these other experimental systems. Here, we are sequencing the genome of Aplysia both to gain access to genomic mechanisms of basic neuronal and other functions and to study these mechanisms in real physiological time with single-neuron resolution. The distinction of Aplysia as a neurobiological and cellular model system is due to the following: (1) Its nervous system has a relatively small number of nerve cells. (2) Many of these cells are large (sometimes gigantic, up to 1 mm in diameter). (3) As a result of their size, pigmentation, and position in the nervous system, many cells can easily be uniquely identified at the single cell level and can be reliably linked to the animal's behavior. (4) The cells provide enough messenger RNA to generate cDNA libraries from single cells. (5) These neurons can be isolated and cultured in vitro and they form circuits which can be explored in detail at molecular and cellular levels. (6) The animal generates a variety of behaviors, many of which can be specified in terms of their underlying circuitry. (7) Some of these behaviors can be modified by different forms of learning. Aplysia are easy to rear in the laboratory from fertilized ova to mature adults and it is possible to obtain an inbred stock of reproducing animals. In 1995, the NIH established a National Resource Center for Aplysia at the University of Miami to meet the growing needs of the biomedical community. This Center supplies over 20,000 cultured Aplysia at all developmental stages annually to the research community throughout the world. This community consists of more than 100 laboratories and over a thousand investigators with overall research budgets of tens of millions of dollars annually. In addition to its value to the neurobiological community, Aplysia is also of interest from a comparative biological perspective. Aplysia is a free-living representative of Mollusca, the second largest animal phylum (after Arthropoda). Members of this phylum have received relatively little genetic study, even though they are of considerable significance for evolutionary and developmental biology and for basic and applied biomedical studies. Aplysia has a stable diploid genome consisting of 17 haploid chromosomes with highly polyploid central neurons. The genome size of about 850 million base pairs is typical for many molluscan species. Expressed sequence tags (ESTs) have already been sequenced, including more than 50,000 unique sequences representing several thousand genes, from the central nervous system of Aplysia – see Moroz et al (2006) . These ESTs have revealed many signaling molecules and pathways, including their key receptors, kinases and substrate proteins, but many rare yet key genes (e.g., ionic channels) have escaped detection so far. To achieve a complete understanding of the biologically important genes and their regulatory elements, a careful annotation and analysis of the sequenced genome is analysis is required. The Aplysia genome project will serve two major functions. First, it will be important for neuroscience by providing a critical resource for facilitating: (a) identification of all genes and regulatory regions of this valuable organism, (b) assembling microarrays and cDNA libraries for global expression studies, including those based on single neurons and their processes, and (c) gene function analysis by RNA interference screens. Second, the Aplysia genome will be important for comparative, developmental and evolutionary biology. Aplysia will serve as a complementary invertebrate/molluscan genomic model from the most diverse and second largest clade of bilaterally symmetrical animals. The Aplysia genome will promote studies of metazoan evolution, developmental biology, neuroscience, and human health, including identifying and validating therapeutic targets for human disorders that are related to aging, cancer, the central nervous system, as well as learning and memory mechanisms.

Project description:The complexity of events associated with age-related memory loss (ARML) cannot be overestimated. The problem is further complicated by the enormous diversity of neurons in the CNS and even synapses of one neuron within a neural circuit. Large-scale single-neuron analysis is not only challenging but mostly impractical for any model currently used in ARML. We simply do not know: do all neurons and synapses age differently or are some neurons (or synapses) more resistant to aging than others? What is happening in any given neuron while it undergoes “normal” aging? What are the genomic changes that make aging apparently irreversible? What would be the balance between neuron-specific vs global genome-wide changes in aging? In the proposed paper we address these questions and develop a new model to study the entire scope of genomic and epigenomic regulation in aging at the resolution of single functionally characterized cells and even cell compartments. In particular, the mollusc Aplysia californica has been implemented as a powerful paradigm in addressing fundamental questions of the neurobiology of aging. The proposed manuscript will consist of four parts. First, we will provide an introduction to Aplysia as a representative of the largest superclade of bilaterian animals (Lophotrochozoa). Aplysia has a short lifespan of 220-300 days with a well-characterized life cycle and characterized phenomenology of aging. Most importantly, Aplysia possess the largest nerve cells in the entire animal kingdom (only eggs are larger); these cells can be uniquely identified and mapped in terms of their well-defined interactions with other neurons forming relatively simpler neural circuits underlying several stereotypic and learned behaviors. Second, we have identified in Aplysia more that a hundred neurological- and age-related genes that were lost in other established invertebrate models (such as Drosophila and C. elegans). The proposed long-term regulatory age-related mechanisms include a high level of conservation among many epigenetic processes known to be lost in nematodes and flies with extremely short lifecycles and particularly derived genomes. We also identify and cloned more than 30 evolutionarily conserved homologs of genes involved in Alzheimer’s, Parkinson’s and Huntington’s diseases as well as age-related hormones. Third, we performed genome-wide analysis of expression patterns of more than 55,000 unique transcripts by comparing two different identified cholinergic neurons (R2 and LPl1) among young and aged animals. This direct single neuron genomic analysis indicates that there are significant cell-specific changes in gene-expression profiles as a function of aging. We estimated that only ~10-20% of genes that are differently expressed in the aging brain are common for all neuronal types - the remaining 80% are neuron-specific (i.e. found in aging neurons of one but not another type). The list of “common aging genes” includes components of insulin growth factor pathways, cell bioenergetics, telomerase-associated proteins, antioxidant enzymes, water channels and estrogen receptors. The rest were neuron-specific gene products (including apoptosis-related proteins, Alzheimer-related genes, growth factors and their receptors, ionic channels, transcription factors and more than 120 identified proteins known to be involved in neurodevelopment and synaptogenesis). Surprisingly, even two different identified cholinergic motoneurons age differently and each of them has a unique subset of genes differentially expressed in older animals. Fourth, we showed that the activity of the entire genome and associated epigenomic modifications (e.g. DNA methylation, histone dynamics) can be efficiently monitored within a single Aplysia neuron and can be modified as a function of aging in a neuron-specific manner including selective histones and histone-modifying enzymes and DNA methylation-related enzymes. This genome-wide analysis of aging allows us to propose novel mechanisms of active DNA demethylation and cell-specific methylation as well as regional relocation of RNAs as three key processes underlying age-related memory loss. These mechanisms tune the dynamics of long-term chromatin remodeling, control weakening and the loss of synaptic connections in aging. At the same time, our genomic tests revealed evolutionarily conserved gene clusters in the Aplysia genome associated with senescence and regeneration (e.g. apoptosis- and redox- dependent processes, insulin signaling, etc.).

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Age-associated bidirectional modulation of gene expression in single identified R15 neuron of Aplysia

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