Dataset Information

Dystrophin isoform Dp71eΔ71 is involved in the neurite outgrowth and neuronal differentiation of PC12 cells

ABSTRACT: The Dp71 protein is the most abundant dystrophin in the central nervous system (CNS). Several dystrophin Dp71 isoforms have been described and are classified into three groups, each with a different C- terminal end. However, the functions of Dp71 isoforms remain unknown. In the present study, we analyse the effect of Dp71eΔ71 overexpression on the neuronal differentiation of PC12 Tet-On cells. Overexpression of the dystrophin Dp71eΔ71 stimulates neuronal differentiation, increasing the percentage of cells with neurites and the neurite length. According to the 2-DE analysis, Dp71eΔ71 overexpression modified the protein expression profile of PC12 Tet-On cells that had been treated with neuronal growth factor (NGF) for nine days. Interestingly, all differentially expressed proteins were up-regulated compared to the control. The proteomic analysis showed that Dp71eΔ71 increases the expression of proteins with important roles in the differentiation process, such as the HspB1, S100A6, and K8 proteins involved in the cytoskeletal structure and the HCNP protein involved in neurotransmitter synthesis. The expression of the neuronal marker TH was also up- regulated.

INSTRUMENT(S): 4800 Proteomics Analyzer

ORGANISM(S): Rattus Norvegicus (rat)

DISEASE(S): Duchenne Muscular Dystrophy

SUBMITTER: Juan Pablo Reyes Grajeda

LAB HEAD: Juan Pablo Reyes Grajeda

PROVIDER: PXD009114 | Pride | 2018-04-09

REPOSITORIES: Pride

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			Action	DRS
	(Spot ID 10).mzid_(Spot_ID_10).pride.mgf.gz	Mzid
	(Spot ID 10).pride.mztab.gz	Mztab
	(Spot ID 12).mzid_(Spot_ID_12).pride.mgf.gz	Mzid
	(Spot ID 12).pride.mztab.gz	Mztab
	(Spot ID 3).mzid_(Spot_ID_3).pride.mgf.gz	Mzid

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The dystrophin isoform Dp71eΔ71 is involved in neurite outgrowth and neuronal differentiation of PC12 cells.

García-Cruz César C Merino-Jiménez Candelaria C Ceja Víctor V Aragón Jorge J Siqueiros-Márquez Lourdes L Reyes-Grajeda Juan Pablo JP Montañez Cecilia C

Journal of proteomics 20180404

The Dp71 protein is the most abundant dystrophin in the central nervous system (CNS). Several dystrophin Dp71 isoforms have been described and are classified into three groups, each with a different C-terminal end. However, the functions of Dp71 isoforms remain unknown. In the present study, we analysed the effect of Dp71eΔ71 overexpression on neuronal differentiation of PC12 Tet-On cells. Overexpression of dystrophin Dp71eΔ71 stimulates neuronal differentiation, increasing ...[more]

PMID: 29625189

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Project description:Dystrophin Dp71 is the main product of the DMD (Duchenne muscular dystrophy) gene in the brain. Dystrophin Dp71 is essential for the development of the nervous system and its alteration is associated with intellectual disability. Different isoforms of Dp71, with different subcellular locations, are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand their functions. PC12 cells stably transfected with pTRE2pur-Myc/Dp71dΔ71 or the empty vector (EV, pTRE2pur-Myc) were analyzed by immunoprecipitation coupled with quantitative proteomics. Samples were resolved and digested in gel, and peptides were separated using a UHPLC ACQUITY M-Class. Spectral data were acquired in an MS with electrospray ionization and ion mobility separation Synapt G2-Si operated with data-independent acquisition and ion mobility spectrometry using the high-definition multiplexed MS/MS mode. We used the Hi3 method to quantify absolutely every protein detected. A total of 121 proteins were identified and absolutely quantified using the internal standard and the intensity of the most abundant peptides per protein with Progenesis QI software and the database UP000002494. Seven new proteins associated with Dp71dΔ71 were selected with at least 2-fold quantity between immunoprecipitated proteins of PC12 Myc/Dp71dΔ71 versus PC12 empty vector cells. Proteins included β-tubulin, S-adenosylmethionine synthase isoform type-2, adapter molecule crk, helicase with zinc finger 2, WD repeat domain 93, cyclin-L2 and myosin-10. Immunoprecipitation followed by quantitative proteomics revealed new proteins that interact with Dp71dΔ71. These proteins are related to cell migration or cell growth, suggesting that Dp71dΔ71 and its associated proteins are involved in these cell functions. The results lay the foundation for future research on the relationship between these proteins and dystrophin Dp71 isoforms

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:The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (ncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of DMD symptomatic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD-specific lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by selective targeting and down-modulation of dystrophin promoter transcriptional activity. We tiled the entire DMD gene, in both sense and antisense directions, using the web-based Agilent eArray database, Version 4.5 (Agilent Technologies), with 60-mer oligos every 66 bp of repeat-masked genome sequence. We defined probe sets for both orientations, encompassing the DMD exons, promoters, introns, predicted MiRNA (identified by PromiRII) and conserved non-coding sequences (CNSs) identified within dystrophin introns using the VISTA programme (http://genome.lbl.gov/vista/index.shtml). Two specific sets of probes were designed to cover, in both directions, the cDNA sequences of a group of control genes identified in the Gene Expression Omnibus (GEO) database http://www.ncbi.nlm.nih.gov/geo/) and expressed equally in both normal and dystrophic muscles. Each probe set was opportunely distributed and replicated several times in order to obtain two 4x44k microarrays, referred to as DMD GEx Sense and DMD GEx Antisense, respectively, able to detect transcripts in the same and opposite directions as that of DMD gene transcription. Three commercial poly A+ RNAs from normal human brain, heart and skeletal muscle tissues were utilised (Ambion). Skin poly A+ RNA was isolated from total Skin RNA (Stratagene) using the Qiagen Oligotex kit. All RNA samples were checked for purity using a ND-1000 spectrophotometer (NanoDrop Technologies), and for integrity by electrophoresis on a 2100 BioAnalyzer (Agilent Technologies). Sample labelling and hybridisation were performed according to the protocols provided by Agilent (One-Color Microarray-Based Gene Expression Analysis version 5.0.1). The array was analysed using the Agilent scanner and Feature Extraction software (v9.1).

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

Dataset Information

Dystrophin isoform Dp71eΔ71 is involved in the neurite outgrowth and neuronal differentiation of PC12 cells

Dataset's files

Publications

The dystrophin isoform Dp71e<sub>Δ71</sub> is involved in neurite outgrowth and neuronal differentiation of PC12 cells.

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