Project description:This study examines a co-culture model of human iPSC-derived cholinergic neurons with glioblastoma (GBM) cells derived from GBM organoids (GBOs; UP-10072). To examine transcriptional regulation of GBM cells by cholinergic neurons, we performed scRNAseq of a set of distinct conditions: 1) cholinergic neurons only; 2) GBM cells only; 3) GBM cells treated with conditioned medium from neurons; 4) direct GBM cell-cholinergic neuron co-cultures.
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:We performed ATAC-seq on iPSC-derived hypothalamic arcuate-like neuron cells to identify putative regulatory elements. All samples were derived from the same individual and from the same differentiation/cell line but ATAC-seq was performed in 3 separate experiments (3 technical replicates).
Project description:The results provide significant insights into the role of Grin2D in regulating the secretion of neurotrophic factors that promote neuritogenesis. Transcriptomic analysis of orthotopically transplanted PDAC cancer cells with a knockout of the NMDA receptor subunit Grin2D, along with dorsal root ganglia (T8–T12) innervating the pancreas, strongly supports this conclusion. Furthermore, RNA-Seq analysis of tumor and ganglion biopsies from PDAC patients was performed to validate the identified gene candidates in a human context. This study tested the hypothesis that neuronal glutamate drives pancreatic cancer progression via glutamate-mediated GluN2D signaling at the cancer-neuron pseudo-synapses.
Project description:The nervous system plays an increasingly appreciated role in the regulation of cancer. In gliomas, neuronal activity drives tumor progression through paracrine signaling factors such as neuroligin-3 and brain-derived neurotrophic factor (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. The consequent glioma cell membrane depolarization drives tumor proliferation. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity and strength. Here, we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signaling through the receptor TrkB (tropomyosin receptor kinase B), BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity that contributes to memory and learning in the healthy brain. BDNF-TrkB signaling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of TrkB in human glioma cells robustly inhibits tumor progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of pediatric glioblastoma and diffuse intrinsic pontine glioma (DIPG). Taken together, these findings indicate that BDNF-TrkB signaling promotes malignant synaptic plasticity and augments tumor progression.
Project description:The nervous system plays an increasingly appreciated role in the regulation of cancer. In gliomas, neuronal activity drives tumor progression through paracrine signaling factors such as neuroligin-3 and brain-derived neurotrophic factor (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. The consequent glioma cell membrane depolarization drives tumor proliferation. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity and strength. Here, we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signaling through the receptor TrkB (tropomyosin receptor kinase B), BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity that contributes to memory and learning in the healthy brain. BDNF-TrkB signaling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of TrkB in human glioma cells robustly inhibits tumor progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of pediatric glioblastoma and diffuse intrinsic pontine glioma (DIPG). Taken together, these findings indicate that BDNF-TrkB signaling promotes malignant synaptic plasticity and augments tumor progression.
Project description:Proteostasis involves a dynamic network of biological pathways that regulate protein synthesis, maintenance, and degradation. As postmitotic cells, neurons are particularly sensitive to environmental changes, and dysfunction in cellular proteostasis can lead to an accumulation of aggregated and misfolded proteins. However, how proteins turnover on a global scale in human neurons is not well understood. In this study, we systematically improved a dynamic SILAC proteomic approach to enable a deep and accurate measurement of protein turnover in human induced pluripotent stem cell (iPSC)-derived cholinergic spinal motor and glutamatergic cortical neurons. Furthermore, we applied this deep proteome turnover method to evaluate how inhibiting the ubiquitin-proteasome and lysosome-autophagy pathway impacts proteostasis in iPSC-derived neurons. Using these datasets, we developed a freely available resource called Neuron Profile, an interactive website for visualizing and querying protein turnover in subcellular locations in human neurons.
Project description:We developed a targeted single nucleus RNA sequencing approach to enrich and transcriptomically characterize cholinergic neurons of the adult mouse spinal cord. Our data expose markers for known classes of cholinergic neurons and their extremely rich diversity. Visceral/preganglionic motor neurons could be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons was also reflected in our analysis with alpha, gamma, and a third subtype clearly distinguished. We further identified previously unrecognized subtypes of cholinergic interneurons.