Divergent Modulation of Neuronal Differentiation by Caspase-2 and -9
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{"title"=>"Divergent modulation of neuronal differentiation by Caspase-2 and -9", "type"=>"journal", "authors"=>[{"first_name"=>"Giuseppa", "last_name"=>"Pistritto", "scopus_author_id"=>"57192989834"}, {"first_name"=>"Veruska", "last_name"=>"Papaleo", "scopus_author_id"=>"16203323700"}, {"first_name"=>"Pilar", "last_name"=>"Sanchez", "scopus_author_id"=>"34467842500"}, {"first_name"=>"Claudia", "last_name"=>"Ceci", "scopus_author_id"=>"55247679100"}, {"first_name"=>"Maria Luisa", "last_name"=>"Barbaccia", "scopus_author_id"=>"7003459192"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84862105779", "sgr"=>"84862105779", "issn"=>"19326203", "isbn"=>"1932-6203", "pmid"=>"22629307", "doi"=>"10.1371/journal.pone.0036002", "pui"=>"364850100"}, "id"=>"17c6d6e9-56e7-34e2-981d-2bd7c6c9230b", "abstract"=>"Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.", "link"=>"http://www.mendeley.com/research/divergent-modulation-neuronal-differentiation-caspase2-9", "reader_count"=>14, "reader_count_by_academic_status"=>{"Researcher"=>1, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>4}, "reader_count_by_user_role"=>{"Researcher"=>1, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>4}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Medicine and Dentistry"=>2, "Agricultural and Biological Sciences"=>8, "Neuroscience"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>8}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/635417"], "description"=>"<p>Notwithstanding the increased caspase activity, no noticeable cleavage of poly-(ADP-ribose) polymerase (PARP) to its 89 kDa fragment was detected at any time point during NT2 cell differentiation (A). Specific si-RNA treatment ( see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036002#s2\" target=\"_blank\">Material and Methods</a> section) achieved an almost complete depletion of caspase-9 (B, si-Casp9) and caspase-2 (C, si-Casp2), that in both cases persisted throughout the RA-induced differentiation period of NT2 cells.</p>", "links"=>[], "tags"=>["apoptotic", "differentiating", "nt2", "cells", "caspase-9", "caspase-2", "si-rna", "transfection"], "article_id"=>305895, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Giuseppa Pistritto", "Veruska Papaleo", "Pilar Sánchez", "Claudia Ceci", "Maria Luisa Barbaccia"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036002.g002", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lack_of_apoptotic_activity_in_differentiating_NT2_cells_A_and_expression_of_caspase_9_B_and_caspase_2_C_after_si_RNA_transfection_of_NT2_cells_/305895", "title"=>"Lack of apoptotic activity in differentiating NT2 cells (A) and expression of caspase-9 (B) and caspase-2 (C) after si-RNA transfection of NT2 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 04:55:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/635618"], "description"=>"<p>NT2 cells stably transfected with scrambled si-RNA or with caspase-2 si-RNA (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036002#s2\" target=\"_blank\">Material and Methods</a> section) were harvested at the indicated time points during RA treatment. A, C and D: relative expression of NCAM, MAP2 and TH mRNA, respectively, analyzed by real-time PCR. Results were normalized to GAPDH mRNA levels. For each neuronal marker, the expression in control undifferentiated NT2 cells (day 0) was set at a value of 1. Each point represents the mean ± SD of three experiments. B: representative western blot of NCAM protein expression. Each blot was repeated three times with similar results. Densitometric analysis revealed, after normalization to actin, a ∼3, 32, 2.4 and 1.9 fold increase in the intensity of the NCAM-180 protein band in si-casp-2 cells, with respect to control cells at day 10, 14, 21 and 28, respectively. E: representative western blot of TH protein expression in control (si-RNA) and si-Casp-2 cells. The TH protein band appeared much earlier (7 versus 14 days) in si-Casp2 cells and densitometric analysis (n = 3) revealed higher intensity than in control cells. *P<0.05, versus the respective value control NT2 cells.</p>", "links"=>[], "tags"=>["caspase-2", "increases", "neuronal", "differentiation", "markers", "differentiating", "nt2"], "article_id"=>306100, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Giuseppa Pistritto", "Veruska Papaleo", "Pilar Sánchez", "Claudia Ceci", "Maria Luisa Barbaccia"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036002.g004", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_si_RNA_mediated_caspase_2_silencing_increases_the_expression_of_neuronal_differentiation_markers_in_differentiating_NT2_cells_/306100", "title"=>"si-RNA-mediated caspase-2 silencing increases the expression of neuronal differentiation markers in differentiating NT2 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 04:56:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/635713"], "description"=>"<p>NT2 cells stably transfected with scrambled si-RNA, caspase-9 or caspase-2 si-RNA (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036002#s2\" target=\"_blank\">Material and Methods</a> section) were harvested at the indicated time points during RA treatment. A: representative western blot of Sirt1 showing the ∼100 kDa fragment recognized by the anti-Sirt1 antibody (directed against an epitope in the C-terminus of the protein) in control and si-Casp9 cells during RA-induced differentiation. In si-Casp9 cells the intensity of the band corresponding to the 100 kDa Sirt1 fragment is clearly reduced between day 3 and 14 days. B: representative Sirt1 western blot in control and si-Casp2 cells, showing that the abundance of the 100 kDa Sirt1 fragment in si-Casp2 cells is comparable to that in control cells. C: results of densitometric analysis for the 100 kDa Sirt1 fragment (each bar represents the mean ± SD of 3 experiments). D: results of densitometric analysis for the full length Sirt1 (120 kDa, each bar represents the mean ± SD of 3 experiments), showing its constant expression in control, si-Casp9 and si-Casp2 cells.</p>", "links"=>[], "tags"=>["sirt1"], "article_id"=>306210, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Giuseppa Pistritto", "Veruska Papaleo", "Pilar Sánchez", "Claudia Ceci", "Maria Luisa Barbaccia"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036002.g005", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_si_RNA_mediated_silencing_of_caspase_9_but_not_of_caspase_2_reduces_Sirt1_cleavage_/306210", "title"=>"si-RNA-mediated silencing of caspase-9, but not of caspase-2, reduces Sirt1 cleavage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 04:56:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/635327"], "description"=>"<p>Total population (RA, filled circles) and non-apoptotic (NA, filled triangles) were harvested at the indicated time points during RA treatment and their lysates were incubated for the assay of caspase-2 (A), -3 (B) and -9 (C) activity with the respective fluorogenic substrate (VDVAD-AFC, for caspase-2, DEVD-AFC, for caspase-3/7 and LEHD-AFC for caspase-9). Control samples, in the presence of the pan-caspase inhibitor Z-VAD-fmk, were run in parallel (Z-VAD-fmk, filled squares). Each point represents the mean ± SEM of 3 experiments, each run in duplicate. *P<0.05, compared to the respective value in undifferentiated NT2 cells (day 0 ). The activity of caspase-1, -5, -6 and -8, expressed as percentage of the respective basal activity in undifferentiated cells, failed to show significant changes throughout the RA-induced differentiation (D).</p>", "links"=>[], "tags"=>["-3", "-9", "selectively", "activated", "ra-induced", "nt2", "neuronal"], "article_id"=>305807, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Giuseppa Pistritto", "Veruska Papaleo", "Pilar Sánchez", "Claudia Ceci", "Maria Luisa Barbaccia"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036002.g001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Caspase_2_3_and_9_are_selectively_activated_during_RA_induced_NT2_cell_neuronal_differentiation_/305807", "title"=>"Caspase-2, -3 and -9 are selectively activated during RA-induced NT2 cell neuronal differentiation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 04:54:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/635804"], "description"=>"<p>. A: caspase-3/7 activity assay showing that caspase-3/7 activation is similar in si-Casp9 and in control cells, while is markedly reduced in si-Casp2 cells. *P<0.05, versus the value in the respective undifferentiated cells (day 0); <sup>a</sup>P<0.05, versus the respective value in control cells. B: relative expression of MASH1 analyzed by real-time PCR in control and si-Casp9 cells. Results were normalized to GAPDH mRNA levels. The MASH1 expression in control undifferentiated NT2 cells (day 0) was set at a value of 1. Each point represents the mean ± SD of three experiments. The insert shows on a smaller scale the results at day 0 and 3 of RA-induction. C: relative expression of MASH1 analyzed by real-time PCR in control and si-Casp2 cells. *P<0.05, versus the respective value in control NT2 cells.</p>", "links"=>[], "tags"=>["si-rna-mediated", "caspase-9", "caspase-2", "mash1"], "article_id"=>306286, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Giuseppa Pistritto", "Veruska Papaleo", "Pilar Sánchez", "Claudia Ceci", "Maria Luisa Barbaccia"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036002.g006", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_si_RNA_mediated_silencing_of_caspase_9_and_caspase_2_on_Caspase_3_7_activity_and_MASH1_expression_/306286", "title"=>"Effects of si-RNA-mediated silencing of caspase-9 and caspase-2 on Caspase-3/7 activity and MASH1 expression", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 04:57:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/635517"], "description"=>"<p>NT2 cells stably transfected with scrambled si-RNA or with caspase-9 si-RNA (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036002#s2\" target=\"_blank\">Material and Methods</a> section) were harvested at the indicated time points during RA treatment. A, C and D: relative expression of NCAM, MAP2 and TH mRNA, respectively, analyzed by real-time PCR. Results were normalized to GAPDH mRNA levels. For each neuronal marker, the expression in control undifferentiated NT2 cells (day 0) was set at a value of 1. Each point represents the mean ± SD of three experiments. B: representative western blots of NCAM protein expression in not interfered (si-RNA) and si-Casp-9 NT2 cells. The experiment was repeated three times with similar results. Densitometric analysis revealed, after normalization to actin, a decrease of NCAM-180 protein band to ∼10 and 40% of the intensity in control cells at day 21 and 28. E: representative western blots of TH protein expression in control (si-RNA) and si-Casp-9 cells showing that TH protein expression was both delayed and reduced in si-Casp9 cells, with respect to control cells. The doublets in the TH Western blots are likely the result of the expression of alternatively spliced TH mRNA. Inserts show on a smaller scale the results at the earlier time points of differentiation for MAP2 (insert 2C) and TH (insert 2D) mRNA expression. *P<0.05, versus the respective value in control NT2 cells.</p>", "links"=>[], "tags"=>["decreases", "neuronal", "markers", "differentiating", "nt2"], "article_id"=>306009, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Giuseppa Pistritto", "Veruska Papaleo", "Pilar Sánchez", "Claudia Ceci", "Maria Luisa Barbaccia"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036002.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Caspase_9_silencing_decreases_the_expression_of_neuronal_markers_in_differentiating_NT2_cells_/306009", "title"=>"Caspase-9 silencing decreases the expression of neuronal markers in differentiating NT2 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 04:55:50"}

PMC Usage Stats | Further Information

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Relative Metric

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