Identification of putative interactors of Fanconi anaemia proteins by yeast to hybrid system: characterization of two novel genes highly expressed during spermatogenesis

2007/2008Fanconi Anaemia (FA) is a rare human genetic disease characterized by bone marrow failure, malformations, chromosomal instability and cancer susceptibility. Thirteen genes belonging to a common pathway have been identified, but their function is still unclear even if evidence indicates a role in DNA-repair. In the attempt to gain new insights on FA-BRCA pathway, this work aimed at finding and characterizing novel putative interactors of the FA proteins. Using the yeast two-hybrid system, we screened a cDNA library of human testis and rescued two clones. Clone 54, which encoded for a putative ubiquitin-conjugating enzyme E2 (UBE2U), was first found to interact with FANCD2 and then with FANCL (E3 ubiquitin-ligase of the FA pathway), FANCC, FANCE and FANCF by direct interaction mating in yeast. Different assays indicated that the expression of this gene is limited to mouse and human testis (specifically in spermatocytes and spermatides). Interestingly, even mouse Fancd2 showed a high expression level in these two cell types, supporting the hypothesis of an interaction between the two proteins and a role of the FA-BRCA pathway during spermatogenesis. In order to confirm the binding between UBE2U and FANCD2, we transiently transfected cell lines with a tagged UBE2U. However, since we failed to detect the protein at any level, we tried to validate the interaction using mouse testis extract. Using the specific antibody we generated, we were however not able to confirm the binding, but, before excluding definitively the interaction, we should further investigate using more suitable antibodies. Clone 4, encoding for a novel putative exonuclease (ISG20L2), was instead found to interact with the C-terminus of FANCG. Though it was ubiquitously present at low levels in all the cells tested, it showed a stronger expression in mouse testis. In transiently transfected cells, ISG20L2 was detected primarily in nucleoli by immunofluorescence, but it was revealed also in the cytoplasmic fraction by western blot. Both nuclear and cytoplasmic distributions of the protein were confirmed at endogenous levels, after production of a specific antibody. Coimmunoprecipitation studies between ISG20L2 and FANCG did not confirm their interaction, but this might be in agreement with a recent report for ISG20L2 as a nucleolar exoribonuclease, not directly involved with DNArepair.197

Genomic stability of self-inactivating rabies.

Peer reviewed: TrueAcknowledgements: We thank Elena Williams for comments on the manuscript. We thank Jerome Boulanger for writing the script for the 3d-alignment of 2-photon recordings, Nicolas Alexandre for the help with the bioinformatic analysis of the NGS datasets, the Laboratory of Molecular Biology (LMB) workshops for the help with software and hardware development, and members of the Biological Service Group for their support with the in vivo work. This study was supported by the Medical Research Council (MC_UP_1201/2), the European Research Council (STG 677029 to MT), the European Union’s Horizon 2020 research and innovation program with the Marie Sklodowska-Curie fellowship to DdM (894697), the Cambridge Philosophical Society and St. Edmund’s College (University of Cambridge) with the Henslow Research Fellowship to AGR, the Rosetrees Trust with an MBPhD fellowship to HL (M598). For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising. All data are stored on the LMB server. All materials described in this paper can be obtained upon reasonable request and for non-commercial purposes after signing a material transfer agreement (MTA) with the MRC.Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that "revertant" mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments

Genomic stability of self-inactivating rabies

Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that “revertant” mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments

Combining long-term circuit mapping and network transcriptomics with SiR-N2c

An exciting frontier in circuit neuroscience lies at the intersection between neural network mapping and single-cell genomics. Monosynaptic rabies viruses provide a promising platform for the merger of circuit mapping methods with -omics approaches. However, three key limitations have hindered the extraction of physiologically meaningful gene expression profiles from rabies-mapped circuits: inherent viral cytotoxicity, high viral immunogenicity and virus-induced alteration of cellular transcriptional regulation. These factors alter the transcriptional and translational profiles of infected neurons and their neighboring cells. To overcome these limitations we applied a self-inactivating genomic modification to the less immunogenic rabies strain, CVS-N2c, to generate a self-inactivating CVS-N2c rabies virus (SiR-N2c). SiR-N2c not only eliminates undesired cytotoxic effects but also substantially reduces gene expression alterations in infected neurons and dampens the recruitment of innate and acquired immune responses, thus enabling open-ended interventions on neural networks and their genetic characterization using single-cell genomic approaches

Neurobiological links between depression and AD: The role of TGF-β1 signaling as a new pharmacological target

In the last several years a large number of studies have demonstrated the neurobiological and clinical continuum between depression and Alzheimer's disease (AD). Depression is a risk factor for the development of AD, and the presence of depressive symptoms significantly increases the conversion of Mild Cognitive Impairment (MCI) into AD. Common pathophysiological events have been identified in depression and AD, including neuroinflammation with an aberrant Tumor Necrosis Factor-α (TNF-α) signaling, and an impairment of Brain-Derived Neurotrophic Factor (BDNF) and Transforming-Growth-Factor-β1 (TGF-β1) signaling.TGF-β1 is an anti-inflammatory cytokine that exerts neuroprotective effects against amyloid-β (Aβ)-induced neurodegeneration, and it has a key role in memory formation and synaptic plasticity. TGF-β1 plasma levels are reduced in major depressed patients (MDD), correlate with depression severity, and significantly contribute to treatment resistance in MDD. The deficit of Smad-dependent TGF-β1 signaling is also an early event in AD pathogenesis, which contributes to inflammaging and cognitive decline in AD. A long-term treatment with antidepressants such as selective-serotonin-reuptake inhibitors (SSRIs) is known to reduce the risk of AD in patients with depression and, SSRIs, such as fluoxetine, increase the release of TGF-β1 from astrocytes and exert relevant neuroprotective effects in experimental models of AD.We propose the TGF-β1 signaling pathway as a common pharmacological target in depression and AD, and discuss the potential rescue of TGF-β1 signaling by antidepressants as a way to prevent the transition from depression to AD

Reducing mortality in acute kidney injury patients: systematic review and international web-based survey.

To identify all interventions that increase or reduce mortality in patients with acute kidney injury (AKI) and to establish the agreement between stated beliefs and actual practice in this setting

The quality of life of children and adolescents with X-linked agammaglobulinemia

INTRODUCTION: The health-related quality of life in X-linked agammaglobulinemia was investigated in 25 children and adolescents patients through the Italian version of Pediatric Quality of Life Inventory 4.0 Generic Core Scale for patients aged less then 18 years, comparing child perception to that of the parents and the physician's evaluation. The data were compared with the ones of 80 healthy controls and the literature data of a group of patients with rheumatic diseases. DISCUSSION: The agammaglobulinemia subjects perceived a lower global quality of life than the healthy subjects, but significantly higher than the rheumatic diseases controls. The clinical relevance of health-related quality of life assessment in X-linked agammaglobulinemia pediatric patients is discussed

The quality of life of children and adolescents with X-linked agammaglobulinemia

INTRODUCTION: The health-related quality of life in X-linked agammaglobulinemia was investigated in 25 children and adolescents patients through the Italian version of Pediatric Quality of Life Inventory 4.0 Generic Core Scale for patients aged less then 18 years, comparing child perception to that of the parents and the physician's evaluation. The data were compared with the ones of 80 healthy controls and the literature data of a group of patients with rheumatic diseases. DISCUSSION: The agammaglobulinemia subjects perceived a lower global quality of life than the healthy subjects, but significantly higher than the rheumatic diseases controls. The clinical relevance of health-related quality of life assessment in X-linked agammaglobulinemia pediatric patients is discussed