The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

La Displasia Fibrosa in modelli in vitro e in vivo

La displasia fibrosa (DF) è una malattia genetica dell’osso e del midollo osseo causata da mutazioni missenso attivanti nel gene codificante per la subunità α della proteina G stimolatoria, Gs (Gsα). Fratture patologiche, deformità e dolori ossei rappresentano comunemente l’espressione clinica della malattia, correlata a sostituzione di osso normale e midollo osseo con tessuto abnorme, a carente mineralizzazione ed instabilità dell’osso, a midollo osseo fibrotico e non ematopoietico. Tali anomalie dipendono dalla disfunzione delle cellule che formano l’osso (osteoblasti), causata dalla presenza della mutazione nelle cellule stesse e nei loro progenitori, le cellule stromali del midollo osseo (BMSC). Ad oggi, non sono note le modificazioni molecolari generate dalla mutazione, né quale sia il contributo dei diversi tipi cellulari al fenotipo malattia, né è disponibile una cura efficace per la DF. Per definire esaustivamente a livello molecolare, cellulare ed organismico gli eventi fisiopatologici della DF, e per identificare nuove strategie terapeutiche, abbiamo generato e studiato modelli di DF in vitro e in vivo. Per analizzare la modulazione trascrizionale indotta dalla mutazione attivante di Gsα (R201C), abbiamo esaminato con i microarray il profilo di espressione di BMSC umane, ingegnerizzate per esprimere stabilmente la mutazione GsαR201C. L’analisi interpretativa dei dati di microarray ha evidenziato la modulazione di geni che sottendono i fondamentali cambiamenti tissutali osservati nella DF, tra cui MGP (artefice della sotto-mineralizzazione dell’osso) e RANKL (responsabile dell’eccessivo riassorbimento osseo), entrambi possibili bersagli terapeutici. D’altra parte, per valutare il contributo di specifiche popolazioni cellulari al fenotipo malattia abbiamo generato modelli murini che consentono l’espressione tessuto-specifica di GsαR201C. In particolare, abbiamo prodotto topi con l’espressione della GsαR201C confinata alle cellule murali, ossia i progenitori scheletrici intesi come cellule microvascolari. L’analisi ai raggi X di questi animali ha messo in luce un’alterazione radiografica delle ossa femorali dei topi, suggerendo che l’espressione della Gsα mutata nelle cellule murali causi anormalità del tessuto scheletrico. Inoltre, per ottenere nuovi modelli murini tessuto-specifici, abbiamo prodotto topi condizionali per l’espressione tessuto-specifica della GsαR201C (Lox-Stop-Lox-GsαR201C). Lo studio radiografico di questi animali ha confermato l’assenza di anomalie ossee. Questi animali potranno essere incrociati con topi che esprimano la ricombinasi Cre nei diversi tessuti di interesse, per ottenere un’ ampia gamma di topi GsαR201C tessuto-specifici. Nel suo insieme, questo lavoro è stato importante per l’identificazione di possibili bersagli terapeutici, ha contribuito a definire l’istopatogenesi molecolare della DF, e, in particolare, dall’uso/analisi di topi GsαR201C tessuto-specifici, potrà derivare un’ulteriore caratterizzazione della patologia

La Displasia Fibrosa in modelli in vitro e in vivo

La displasia fibrosa (DF) è una malattia genetica dell’osso e del midollo osseo causata da mutazioni missenso attivanti nel gene codificante per la subunità α della proteina G stimolatoria, Gs (Gsα). Fratture patologiche, deformità e dolori ossei rappresentano comunemente l’espressione clinica della malattia, correlata a sostituzione di osso normale e midollo osseo con tessuto abnorme, a carente mineralizzazione ed instabilità dell’osso, a midollo osseo fibrotico e non ematopoietico. Tali anomalie dipendono dalla disfunzione delle cellule che formano l’osso (osteoblasti), causata dalla presenza della mutazione nelle cellule stesse e nei loro progenitori, le cellule stromali del midollo osseo (BMSC). Ad oggi, non sono note le modificazioni molecolari generate dalla mutazione, né quale sia il contributo dei diversi tipi cellulari al fenotipo malattia, né è disponibile una cura efficace per la DF. Per definire esaustivamente a livello molecolare, cellulare ed organismico gli eventi fisiopatologici della DF, e per identificare nuove strategie terapeutiche, abbiamo generato e studiato modelli di DF in vitro e in vivo. Per analizzare la modulazione trascrizionale indotta dalla mutazione attivante di Gsα (R201C), abbiamo esaminato con i microarray il profilo di espressione di BMSC umane, ingegnerizzate per esprimere stabilmente la mutazione GsαR201C. L’analisi interpretativa dei dati di microarray ha evidenziato la modulazione di geni che sottendono i fondamentali cambiamenti tissutali osservati nella DF, tra cui MGP (artefice della sotto-mineralizzazione dell’osso) e RANKL (responsabile dell’eccessivo riassorbimento osseo), entrambi possibili bersagli terapeutici. D’altra parte, per valutare il contributo di specifiche popolazioni cellulari al fenotipo malattia abbiamo generato modelli murini che consentono l’espressione tessuto-specifica di GsαR201C. In particolare, abbiamo prodotto topi con l’espressione della GsαR201C confinata alle cellule murali, ossia i progenitori scheletrici intesi come cellule microvascolari. L’analisi ai raggi X di questi animali ha messo in luce un’alterazione radiografica delle ossa femorali dei topi, suggerendo che l’espressione della Gsα mutata nelle cellule murali causi anormalità del tessuto scheletrico. Inoltre, per ottenere nuovi modelli murini tessuto-specifici, abbiamo prodotto topi condizionali per l’espressione tessuto-specifica della GsαR201C (Lox-Stop-Lox-GsαR201C). Lo studio radiografico di questi animali ha confermato l’assenza di anomalie ossee. Questi animali potranno essere incrociati con topi che esprimano la ricombinasi Cre nei diversi tessuti di interesse, per ottenere un’ ampia gamma di topi GsαR201C tessuto-specifici. Nel suo insieme, questo lavoro è stato importante per l’identificazione di possibili bersagli terapeutici, ha contribuito a definire l’istopatogenesi molecolare della DF, e, in particolare, dall’uso/analisi di topi GsαR201C tessuto-specifici, potrà derivare un’ulteriore caratterizzazione della patologia

Soma-to-Germline Transmission of RNA in Mice Xenografted with Human Tumour Cells: Possible Transport by Exosomes

Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information

High-throughput transcriptional analysis of gene therapy viral vectors effects on brain cells

The gene therapy approach using viral vectors currently represents one of the best hopes for treating numerous genetic and acquired brain disorders. Different viral vector platforms have been extensively studied and utilised in clinical trials on the Central Nervous System (CNS), by taking advantage of the specific viral features. However, the improvement of viral systems to mediate safe and long-lasting expression of therapeutic transgenes in brain is particularly challenging due to the post-mitotic nature of nervous cells, the high level of compartmentalisation of the CNS, the potential toxicity and the alteration of the neuronal physiology triggered by the virus. Although many studies have proved the efficacy of the viral sources in transducing the brain in vivo, little is known on neuronal cells perturbations following the vector interaction. To address this issue, we have analysed the global transcriptome of differentiated midbrain-derived human neuronal progenitor cells transduced in vitro with HIV-1-, AAV9-, Helper Dependent human adenoviral (HD hAd)- and Helper Dependent canine adenoviral (HD CAV-2)- vectors, at early and late time points. In particular, canine adenovectors have proved to be an interesting alternative to the human Ad, because of their efficiency in transducing human cells and the absence of CAV-specific neutralizing antibodies in human serum, that inhibit the vector effect. This study intends to provide insights in vector development for CNS, consisting in the ability to predict the neuronal functions altered by the vectors and the possibility to act on these with tools aimed at improving the efficacy and reducing the toxicity

Canine adenovirus (CAV-2) vectors induce an innate immune response and the modulation of cell cycle genes in dopaminergic differentiated human midbrain neuronal precursors

CAV-2 vectors circumvent the ubiquitous human anti-human Adenovirus (hAd) memory immune response, are capable of long term neuron-specific expression (>1year in rat brains), do not induce the maturation of dendritic cells and have been proposed for the treatment of neurodegenerative diseases. In the prospect of clinical applications to brain diseases in humans, we investigated the toxicogenomic profile of helper-dependent (HD) CAV-2 vectors in human midbrain precursors differentiated into dopaminergic neurons. We transduced the cultures with HD CAV-2 and, for comparison, with third generation SIN lentiviral vectors (LV) and HD hAd. We evaluated gene modulation by Affymetrix gene chip at 2h and 5 days post transduction. Our analyses of the chip-contained 47,000 transcripts showed that, at the moi of 1000 genomes per cell, HD CAV-2 exhibited a specific modulation profile. It induced genes belonging to the cell cycle, DNA recombination and repair pathways, including p53, RAD51, BIRC5, FANCD2 and MAD2L1. It also up regulated genes involved in the immune response and in inflammation, including TL3 and 4, HAS3 and CD44, and genes involved in neuron projection morphogenesis. HD hAd was less efficient in transduction than HD CAV-2 and its effect on the trascriptome was milder. However, at 2h, it did have an impact on neuron remodeling and trafficking genes. LV transduced very efficiently, had a strong effect on the trascriptome, which overlapped with that of HD CAV-2 for TLR activation, and diverged in specific aspects of the immune and DNA repair gene groups. Single gene and pathway modulation data emerged from our analysis constitute useful information for toxicity prediction, vector comparison and evolution and virus-host interaction studies

EGFP RNA is present in spermatozoa of mice inoculated with EGFP-infected A-375-cells.

<p><b>A:</b> Murine protamine 2 gene (<i>Prm2</i>) amplification products used to select DNA-free RNA samples. Exemplifying gel of <i>Prm2-</i>specific PCR amplification products of intron-containing DNA from the mouse sperm genome (lane 1) and RT-PCR products from RNA extracted from spermatozoa of non-inoculated (lane 2) and EGFP-expressing A-375⁺ inoculated (lane 3) mice, both showing the spliced <i>Prm2</i> form. <b>B:</b> Southern blot hybridization of RT-PCR amplified RNA from<b>:</b> spermatozoa of non-inoculated control (lane 2) and A-375⁺ inoculated (lane 3) mice, and from non-infected (lane 4) and EGFP-infected (lane 5) A-375 whole cells. Hybridization was carried out with an EGFP-specific internal probe. Lane 1 is a no-RNA control. The bottom panel shows RT-PCR amplification products from the same samples using GAPDH-specific primers as a loading control. <b>C:</b> Southern blot hybridization of RT-PCR amplified RNA from spermatozoa from a control mouse (lane 2) and from a single EGFP-expressing A375⁺ inoculated mouse (lane 3); lane 1 shows a no RNA reaction. As in B, the bottom panel shows GAPDH amplification from the same samples. <b>D:</b> RT-PCR amplification with (+RT) or without (-RT) reverse transcription step of RNA extracted from sperm-depleted epididymis from two inoculated EGFP mice. No EGFP-specific amplification products were visible by ethidium bromide staining (EtBr) nor by Southern blot hybridization (Hyb) using an EGFP radioactive probe. The bottom panel shows GAPDH amplification from the same samples.</p

EGFP-specific RNA in circulating blood from A-375/EGFP-inoculated mice.

<p><b>A:</b> Ethidium bromide staining of specific RT-PCR products from RNA extracted from EGFP-infected A-375 cells (lane 1) and blood-purified extracellular exosomal fraction from inoculated (lane 4) and non-inoculated (lane 5) mice. No RNA and no primers were added to the amplification mix in lanes 2 and 3, respectively. <b>B:</b> EGFP hybridization pattern. The gel in A was blotted on filter, hybridized with ³²P-end labelled EGFP-specific probe, washed and autoradiographed. <b>C</b>: GAPDH-specific amplification products from the same samples.</p

Outline of the general procedure used for the stepwise detection of EGFP expression from tumor to sperm cells.

<p>An A-375 melanoma derivative cell line stably expressing the EGFP reporter gene was obtained by infecting with an engineered letiviral vector. EGFP RNA, DNA and proteins were detected both in whole A-375 cells and in A-375-released exosomes. Cells were then xenografted in nude mice, 45 days after inoculation the animals were sacrificed and both blood-released exosomes and epidydimal spermatozoa were analyzed for EGFP-containing RNA.</p