Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups

Piersanti, Stefania; Burla, Romina; Licursi, Valerio; Brito, Catarina; Torre, Mattia La; Alves, Paula M.; Simão, Daniel; Mottini, Carla; Salinas, Sara; Negri, Rodolfo; Tagliafico, Enrico; Kremer, Eric J.; Saggio, Isabella

doi:10.1371/journal.pone.0133607

Cited by 11 publications

(14 citation statements)

References 46 publications

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“…Total cellular RNA was isolated from the cell populations using an RNeasy RNA isolation kit (Qiagen) as described in [71]. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit) were used to determine the concentration and purity/integrity of RNA samples using an Agilent 2100 bioanalyzer.…”

Section: Qpcr Gene Expression Profiling and Data Analysismentioning

confidence: 99%

“…Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit) were used to determine the concentration and purity/integrity of RNA samples using an Agilent 2100 bioanalyzer. cDNA synthesis, biotin-labeled target synthesis, HG-U133 plus 2.0 Gen-eChip (Affymetrix, Santa Clara, CA) array hybridization, staining and scanning were performed as described in [71,72]. The amount of a transcript mRNA (signal) was determined by the Affymetrix GeneChip Operative Software (GCOS) 1.2 absolute analysis algorithm [73].…”

Section: Qpcr Gene Expression Profiling and Data Analysismentioning

confidence: 99%

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Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone

et al. 2020

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Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (Gsα R201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the Gsα R201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for

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Section: Qpcr Gene Expression Profiling and Data Analysismentioning

confidence: 99%

Section: Qpcr Gene Expression Profiling and Data Analysismentioning

confidence: 99%

Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone

et al. 2020

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“…After DNase treatment (Invitrogen), RNA was reverse transcribed into cDNA as already described (30). q-PCR reactions were carried out as previously described (29), using the following primers: AKTIP Forward 5’-TCCACGCTTGGTGTTCGAT-3’; AKTIP Reverse 5’-TCACCTGAGGTGGGATCAACT-3’; GAPDH Forward 5’-TGGGCTACACTGAGCACCAG-3’; GAPDH Reverse 5’-GGGTGTCGCTGTTGAAGTCA-3’ and analyzed with the 2 −ΔΔCq method as previously described (31). For Western blotting, 72hrs post-transfection with siRNAs, protein extracts were obtained as previously described (12) and quantified by Bradford assay.…”

Section: Methodsmentioning

confidence: 99%

Human AKTIP interacts with ESCRT proteins and functions at the midbody in cytokinesis

Merigliano

Burla

Torre

et al. 2020

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28To complete mitosis, the intercellular bridge that links daughter cells needs to be cleaved. 29This abscission step is carried out by the sequential recruitment of ESCRT proteins at the 30 midbody. We report here that a new factor, named AKTIP, works in association with 31ESCRTs. We find that AKTIP binds to the ESCRT I subunit VPS28, and show by high 32 resolution microscopy that AKTIP forms a ring in the dark zone of the intercellular bridge. 33This ring is positioned in between the circular structures formed by ESCRTs type III. 34Functionally, we observe that the reduction of AKTIP impinges on the recruitment of the 35 ESCRT III member IST1 at the midbody and causes abscission defects. Taken together, 36these data indicate that AKTIP is a new factor that contributes to the formation of the 37 ESCRT complex at the midbody and is implicated in the performance of the ESCRT 38 machinery during cytokinetic abscission. 39 40 41

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“…After DNaseI treatment (Invitrogen) RNA from cells and brains was reverse transcribed into cDNA with oligo d(T) primer and OMNISCRIPT RT KIT (Qiagen). QPCRs were performed as described (Piersanti et al, 2015 ) using the following primers:…”

Section: Methodsmentioning

confidence: 99%

p53-Sensitive Epileptic Behavior and Inflammation in Ft1 Hypomorphic Mice

et al. 2018

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Epilepsy is a complex clinical condition characterized by repeated spontaneous seizures. Seizures have been linked to multiple drivers including DNA damage accumulation. Investigation of epilepsy physiopathology in humans imposes ethical and practical limitations, for this reason model systems are mostly preferred. Among animal models, mouse mutants are particularly valuable since they allow conjoint behavioral, organismal, and genetic analyses. Along with this, since aging has been associated with higher frequency of seizures, prematurely aging mice, simulating human progeroid diseases, offer a further useful modeling element as they recapitulate aging over a short time-window. Here we report on a mouse mutant with progeroid traits that displays repeated spontaneous seizures. Mutant mice were produced by reducing the expression of the gene Ft1 (AKTIP in humans). In vitro, AKTIP/Ft1 depletion causes telomere aberrations, DNA damage, and cell senescence. AKTIP/Ft1 interacts with lamins, which control nuclear architecture and DNA function. Premature aging defects of Ft1 mutant mice include skeletal alterations and lipodystrophy. The epileptic behavior of Ft1 mutant animals was age and sex linked. Seizures were observed in 18 mutant mice (23.6% of aged ≥ 21 weeks), at an average frequency of 2.33 events/mouse. Time distribution of seizures indicated non-random enrichment of seizures over the follow-up period, with 75% of seizures happening in consecutive weeks. The analysis of epileptic brains did not reveal overt brain morphological alterations or severe neurodegeneration, however, Ft1 reduction induced expression of the inflammatory markers IL-6 and TGF-β. Importantly, Ft1 mutant mice with concomitant genetic reduction of the guardian of the genome, p53, showed no seizures or inflammatory marker activation, implicating the DNA damage response into these phenotypes. This work adds insights into the connection among DNA damage, brain function, and aging. In addition, it further underscores the importance of model organisms for studying specific phenotypes, along with permitting the analysis of genetic interactions at the organismal level.

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Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups

Cited by 11 publications

References 46 publications

Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone

Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone

Human AKTIP interacts with ESCRT proteins and functions at the midbody in cytokinesis

p53-Sensitive Epileptic Behavior and Inflammation in Ft1 Hypomorphic Mice

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