Toward synaptic transcriptomes: Direct sequencing and identification of RNAs actively transported by the kinesin complex from the cell body to synapses in Aplysia neurons
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ABSTRACT:
ORGANISM(S): Aplysia californica
SUBMITTER: Andrea Kohn
PROVIDER: E-GEOD-30440 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
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Project description:Specific mRNAs are transported from the cell body to synapses where their translation can modify communication of pre-existing synapses and induce formation of new synaptic connections in response to learning. Little is known, however, about the identity of the RNAs that are actively transported and when and how these RNAs are utilized during learning. By focusing on RNAs that are associated with kinesin, a motor protein that transports gene products from the cell body to synapses, we have now applied microarrays and 454 sequencing to identify actively transported RNAs from the Aplysia central nervous system. Using a library prepared from the kinesin complex immunoprecipitated from the central nervous system (CNS), we have identified thousands of unique transcripts, of which ~600 mRNAs were annotated.
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Project description:Toward synaptic transcriptomes: Direct sequencing and identification of RNAs actively transported by the kinesin complex from the cell body to synapses in Aplysia neurons
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Project description:Understanding cell-type-specific epigenetic codes on a global level is a major challenge after the sequencing of the human genome has been completed. Here we applied methyl-CpG immunoprecipitation (MCIp) to obtain comparative methylation profiles of coding and noncoding genes in three human tissues, testis, brain, and monocytes. Forty-four mainly testis-specific promoters were independently validated using bisulfite sequencing or single-gene MCIp, confirming the results obtained by the MCIp microarray approach. We demonstrate the previously unknown somatic hypermethylation at many CpG-rich, testis-specific gene promoters, in particular in ampliconic areas of the Y chromosome. We also identify a number of miRNA genes showing tissue-specific methylation patterns. The comparison of the obtained tissue methylation profiles with corresponding gene expression data indicates a significant association between tissue-specific promoter methylation and gene expression, not only in CpG-rich promoters. In summary, our study highlights the exceptional epigenetic status of germ-line cells in testis and provides a global insight into tissue-specific DNA methylation patterns. Keywords: MCIp-on-Chip The promoter hypomethylation profiles of the two somatic tissues (monocytes and brain) were compared to human testis (reference). The set includes two hybridisations with independent testis samples for each comparison. Each comparison uses a set of two microarrays.
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