Project description:During neurogenesis, expression of the basic Helix-Loop-Helix NeuroD6/Nex1/MATH-2 transcription factor parallels neuronal differentiation, while maintaining the differentiated state in the mature nervous system. To further dissect NeuroD6 differentiation properties, we previously generated a NeuroD6-overexpressing stable PC12 cell line, PC12-ND6, which displays a neuronal phenotype characterized by spontaneous neuritogenesis, accelerated NGF-induced differentiation, and increased regenerative capacity. Furthermore, we reported that NeuroD6 promotes long-term neuronal survival upon oxidative stress triggered by serum deprivation. In this study, we identified the NeuroD6-mediated transcriptional regulatory pathways linking neuronal differentiation to survival, by conducting a genome-wide microarray analysis using PC12-ND6 cells and serum deprivation as a stress paradigm. Through a series of filtering steps and a gene-ontology analysis, we found that NeuroD6 promotes distinct but overlapping gene networks, consistent with the differentiation, regeneration, and survival properties of PC12-ND6 cells. Using a gene set enrichment analysis, we provide the first evidence of a compelling link between NeuroD6 and a set of heat shock proteins in the absence of stress, which may be instrumental to confer stress tolerance to PC12-ND6 cells. Immunocytochemistry results showed that HSP27 and HSP70 interact with cytoskeletal elements, consistent with their roles in neuritogenesis and preserving cellular integrity. HSP70 also colocalizes with mitochondria located in the soma, growing neurites and growth cones of PC12-ND6 cells prior to and upon stress stimulus, consistent with its neuroprotective functions. Collectively, our findings support the notion that NeuroD6 links neuronal differentiation to survival via the network of molecular chaperones and endows the cells with increased stress tolerance. Experiment Overall Design: The experimental design involved six replicates of serum-grown PC12 cells (control), serum-grown PC12-ND6 cells (t=0), and serum-deprived PC12-ND6 cells (t=48 hrs).

Project description:Multiple sclerosis (MS) begins as an inflammatory/demyelinating disease but axon injury and neurodegeneration underlie chronic progression. Extracellular matrix (ECM) heparan sulfate proteoglycans (HSPG) are catabolized in acute inflammatory MS lesions and HSPG catabolic products may contribute to failure of neuron repair. Neurite outgrowth and axon elongation of PC12 cells are inhibited when these cells are grown on heparanase-digested HSPG. This inhibition of differentiation is counteracted by nerve growth factor (NGF) but failure to completely reverse inhibition may be due to loss of other growth factors affected by HSPG breakdown. To understand effects of the digested ECM in this model, expression of genes that control differentiation in PC12 cells grown on digested and undigested HSPG will be analyzed. This will permit identification of new potential therapeutic targets for specifically counteracting inhibitory effects of ECM breakdown on neuron regrowth/regeneration in MS. We will compare gene expression patterns of PC12 cells grown on undigested and digested HSPG in the presence and in the absence of NGF over a two week time course to identify the alterations induced by the catabolized HSPG on expression of genes involved in their acquisition of a neuronal phenotype and to determine which genes are and are not targeted by NGF. We are studying ECM HSPG breakdown in human and experimental demyelinating diseases and have developed an in vitro model of ECM catabolism-induced inhibition of neuronal differentiation. Our data indicate that: 1) the numbers of PC12 cells that exhibit neurite outgrowth are decreased when the ECM contains digested, as opposed to intact HSPG; and 2) in the presence of NGF, neurite outgrowth inhibition is counteracted, but complete axonal growth/elongation is not achieved. We will use gene microarray analysis to identify the specific gene expression alterations that digested ECM HSPG induces in PC12 cells in this model of inhibition of neural differentiation. We hypothesize that the genes that are critical for PC12 cell differentiation will be altered by the pathologically altered ECM. Rat pheochromocytoma PC12 cell lines are widely used for analyzing neuronal differentiation. Microarray analyses have identified gene expression patterns associated with their differentiation and response to growth factors. We developed a model of catabolized HSPG inhibition of PC12 cell differentiation that mimics the ECM in MS lesions. HSPG coated on tissue culture dishes is digested with heparanases prior to plating of the PC12 cells. Cells grown on digested ECM without NGF show inhibition of neuronal differentiation (decreased numbers with neurite outgrowth) from days 7 to 14. NGF can partially, but not completely counteract the inhibition, suggesting that signaling pathways in addition to those affected by NGF may be affected by the ECM HSPG digestion. Therefore, we will compare gene expression patterns in samples of cells grown on digested and undigested HSPG ECM both in the presence and absence of NGF. The cells are plated on washed, undigested or digested HSPG-coated 100 mm Petri dishes and grown with or without NGF. Total RNA will be extracted from cells grown in the 4 conditions (w/without digestion; w/without NGF) on 100mm Petri dishes using the RNeasy extraction kit (Qiagen). We propose: 2 duplicate samples/culture condition (8 samples) per time point X 5 time points (days 1, 5, 7, 10, and 14 of culture) = 40 samples/experiment. The RNA will be sent to the Microarray Consortium center for QC check and microarray analysis. Previous analyses of PC12 cells have used the NIA Mouse 15K cDNA clone set scanned with the Agilent Oligo array platform (Vaudry, 2002; Grumolato, 2003). Therefore, this methodology will be useful for comparisons to published studies. (Number of experiments to be determined). Vaudry D, et al, 2002. J Neurochem 83:1272-1284 Grumolato L, et al, 2003. Endocrinology 144:2368-2379

Project description:Multiple sclerosis (MS) begins as an inflammatory/demyelinating disease but axon injury and neurodegeneration underlie chronic progression. Extracellular matrix (ECM) heparan sulfate proteoglycans (HSPG) are catabolized in acute inflammatory MS lesions and HSPG catabolic products may contribute to failure of neuron repair. Neurite outgrowth and axon elongation of PC12 cells are inhibited when these cells are grown on heparanase-digested HSPG. This inhibition of differentiation is counteracted by nerve growth factor (NGF) but failure to completely reverse inhibition may be due to loss of other growth factors affected by HSPG breakdown. To understand effects of the digested ECM in this model, expression of genes that control differentiation in PC12 cells grown on digested and undigested HSPG will be analyzed. This will permit identification of new potential therapeutic targets for specifically counteracting inhibitory effects of ECM breakdown on neuron regrowth/regeneration in MS. We will compare gene expression patterns of PC12 cells grown on undigested and digested HSPG in the presence and in the absence of NGF over a two week time course to identify the alterations induced by the catabolized HSPG on expression of genes involved in their acquisition of a neuronal phenotype and to determine which genes are and are not targeted by NGF. We are studying ECM HSPG breakdown in human and experimental demyelinating diseases and have developed an in vitro model of ECM catabolism-induced inhibition of neuronal differentiation. Our data indicate that: 1) the numbers of PC12 cells that exhibit neurite outgrowth are decreased when the ECM contains digested, as opposed to intact HSPG; and 2) in the presence of NGF, neurite outgrowth inhibition is counteracted, but complete axonal growth/elongation is not achieved. We will use gene microarray analysis to identify the specific gene expression alterations that digested ECM HSPG induces in PC12 cells in this model of inhibition of neural differentiation. We hypothesize that the genes that are critical for PC12 cell differentiation will be altered by the pathologically altered ECM. Rat pheochromocytoma PC12 cell lines are widely used for analyzing neuronal differentiation. Microarray analyses have identified gene expression patterns associated with their differentiation and response to growth factors. We developed a model of catabolized HSPG inhibition of PC12 cell differentiation that mimics the ECM in MS lesions. HSPG coated on tissue culture dishes is digested with heparanases prior to plating of the PC12 cells. Cells grown on digested ECM without NGF show inhibition of neuronal differentiation (decreased numbers with neurite outgrowth) from days 7 to 14. NGF can partially, but not completely counteract the inhibition, suggesting that signaling pathways in addition to those affected by NGF may be affected by the ECM HSPG digestion. Therefore, we will compare gene expression patterns in samples of cells grown on digested and undigested HSPG ECM both in the presence and absence of NGF. The cells are plated on washed, undigested or digested HSPG-coated 100 mm Petri dishes and grown with or without NGF. Total RNA will be extracted from cells grown in the 4 conditions (w/without digestion; w/without NGF) on 100mm Petri dishes using the RNeasy extraction kit (Qiagen). We propose: 2 duplicate samples/culture condition (8 samples) per time point X 5 time points (days 1, 5, 7, 10, and 14 of culture) = 40 samples/experiment. The RNA will be sent to the Microarray Consortium center for QC check and microarray analysis. Previous analyses of PC12 cells have used the NIA Mouse 15K cDNA clone set scanned with the Agilent Oligo array platform (Vaudry, 2002; Grumolato, 2003). Therefore, this methodology will be useful for comparisons to published studies. (Number of experiments to be determined). Vaudry D, et al, 2002. J Neurochem 83:1272-1284 Grumolato L, et al, 2003. Endocrinology 144:2368-2379 Keywords: time-course

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Genomic Studies upon NeuroD6 overexpression in PC12 cells, in the presence or absence of an apoptotic stimulus

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