Characterization of Neuronal-Specific Tra2b Knock-Out Mice and Identification of Tra2b Splicing Targets

TRA2B is a serine-arginine-rich splicing factor that contributes to the alternative splicing of exons and depletion of Tra2b in the mouse causes early embryonic lethality. It modulates splice site selection in a concentration dependent fashion and associates to target exons either directly via GAA binding motifs or indirectly via interactions with other splice factors. TRA2B is highest expressed in neuronal tissue and testis and its expression and activation are controlled via an autoregulatory self-splicing mechanism and by phosphorylation. Depending on interactors and on the position of binding, TRA2B can promote either inclusion or skipping of exons. TRA2B has been associated with splicing processes involved in development, vascularization, spermatogenesis and neuronal function and deregulation of splicing processes has been linked to conditions like Alzheimer’s disease, dementia and Parkinson’s disease. Importantly, TRA2B is involved in the splicing of the SMN transcript and has been demonstrated to promote inclusion of exon 7. The functional loss of the SMN1 gene causes spinal muscular atrophy, whereas the copy number of SMN2, which is alternatively spliced to mainly block exon 7 inclusion, is the major determinant of the disease severity. Correction of the SMN2 splicing pattern to increase the number of functional full-length transcripts is a promising avenue for SMA therapy. The use of HDAC inhibitors like VPA has been demonstrated to correct the SMN2 splicing pattern to a great extent by transcriptional upregulation of TRA2B. Despite promising trial of VPA in SMA therapy and successful application in other conditions like epilepsy and bipolar disorder, it can be assumed that the splicing of other transcripts targeted by TRA2B besides SMN2 will be affected by its upregulation. In the present work a neuronal-specific Tra2b knock-out mouse was generated to identify transcripts targeted by Tra2b in the central nervous system. Mice homozygously depleted of Tra2b in the central nervous system died shortly after birth and presented severe abnormalities in cortical development. These included a loss of cortical patterning, a reduction of the total cortical width and ventriculomegaly. By immunohistochemical analyses of brain sections at developmental stages reaching from 14.5 dpc to birth, massive apoptosis was detected in the ventricular and subventricular layers of the lateral ventricles and in the thalamic region. Apoptosis at 14.5 dpc was followed by a loss of the proliferative potential in a timely fashion, which caused a progressive loss of cortical material and ventricular dilation that reached its end stage at 16-17 dpc. Based on a mosaic knock-out of Tra2b in the murine brain, mRNA and protein levels of Tra2b were drastically reduced, but not fully absent as demonstrated by quantitative PCR and semi-quantitative Western Blot. Heterozygous knock-out animals were fully viable, showed no apoptosis, normal brain development and Tra2b protein levels comparable to control mice. Analysis of Tra2b isoforms revealed that the autoregulatory splicing feedback-loop of Tra2b is functional in vivo in the mouse brain and compensates for the loss of a single gene copy by upregulation of functional Tra2b transcripts due to skipping of exon 2. By the use of quantitative PCR, previously and in minigene approaches identified Tra2b-dependent splicing processes of Mapt, Cltb, Tra2a and Nasp were indentified in vivo providing proof of concept for the identification of splicing processes in the Tra2b-deficient murine brain. For the in vivo identification of novel Tra2b target exons, whole transcriptome sequencing and mouse exon array analysis were performed using whole brain RNA from neuronal-specific knock-out mice. Based on dramatic differences between control and knock-out brains and drastically altered cellular compositions, the reliable identification of targeted exons proved to be difficult. Exons of the Shugoshin-like 2 and Tubulin delta chain were identified via exon array analysis to be promoted by Tra2b in vivo. These splicing processes were confirmed by quantitative PCR and both identified exons were shown to be responsive to increased Tra2b concentrations in a minigene splicing assay. Some of the in vivo identified Tra2b-dependent splicing processes have possible implications in neuronal development and function, and might therefore contribute to the observed aberrations in brain development. Moreover, the cell cycle regulating gene Cdkn1a (encoding p21) was found upregulated in brains of neuronal-specific knock-out mice using exon arrays, and p21 upregulation was validated by quantitative PCR. P21 is a well-known inhibitor of cell cycle progression that causes cells to enter cell cycle arrest at the G1-S-phase transition. RNAi-mediated depletion of Tra2b in NSC34 neuronal precursor cells caused a drastic increase of p21 expression on RNA and on protein level. Strikingly, NSC34 cells died shortly after p21 expression had increased, indicating an apoptotic effect. This suggests that an increased p21 expression is likely the reason for apoptosis and the loss of the proliferative potential in neurogenic areas of the developing Tra2b-depleted brains. Previous studies have linked deficiency of the histone chaperone Nasp to upregulation of p21 and to apoptosis. Further investigations need to clarify, whether Tra2b-related missplicing of Nasp (tNasp depletion) is the underlying reason for p21 upregulation and apoptosis

Multi-staged deposition of trench-gate oxides for power MOSFETs

Here, silicon oxide was formed in a U-shaped trench of a power metal-oxide semiconductor field-effect transistor device by various processes. One SiO₂ formation process was performed in multiple steps to create a low-defect Si-SiO₂ interface, where first a thin initial oxide was grown by thermal oxidation followed by the deposition of a much thicker oxide layer by chemical vapor deposition (CVD). In a second novel approach, silicon nitride CVD was combined with radical oxidation to form silicon oxide in a stepwise sequence. The resulting stack of silicon oxide films was then annealed at temperatures between 1000 and 1100 °C. All processes were executed in an industrial environment using 200 mm-diameter (100)-oriented silicon wafers. The goal was to optimize the trade-off between wafer uniformity and conformality of the trenches. The thickness of the resulting silicon oxide films was determined by ellipsometry of the wafer surface and by scanning electron microscopy of the trench cross sections. The insulation properties such as gate leakage and electrical breakdown were characterized by current–voltage profiling. The electrical breakdown was found to be highest for films treated with rapid thermal processing. The films fabricated via the introduced sequential process exhibited a breakdown behavior comparable to films deposited by the common low-pressure CVD technique, while the leakage current at electric fields higher than 5 MV/cm was significantly lower

Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier

Pride in technology-based health interventions: A double-edged sword

his is the peer reviewed version of the following article: Manika, D., Gregory‐Smith, D. and Antonetti, P. (2017), Pride in Technology‐Based Health Interventions: A Double‐Edged Sword. Psychol. Mark., 34: 410-427., which has been published in final form at https://doi.org/10.1002/mar.20997. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

Longitudinal Multi-Parametric Liquid Biopsy Approach Identifies Unique Features of Circulating Tumor Cell, Extracellular Vesicle, and Cell-Free DNA Characterization for Disease Monitoring in Metastatic Breast Cancer Patients

Dynamics of mRNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs), and cell-free DNA (cfDNA) were assessed to examine the relevance of a longitudinal multi-parametric liquid biopsy strategy. Eighteen milliliters of blood was drawn from 27 hormone receptor-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) patients at disease progression and at two subsequent radiologic staging time points. CTC mRNA and EV mRNA were analyzed using multi-marker qPCR, and cfDNA was analyzed using targeted next-generation sequencing (NGS). The presence of ERBB2 or ERBB3 overexpression signals in CTCs significantly correlated with disease progression (87% specificity, 36% sensitivity, p-value = 0.023), and the presence of either ERBB3 signals in CTCs or EVs or cfDNA variants in ERBB3 also showed a significant association with progressive MBC. Fluctuations during treatment were detected in the EV fraction with the appearance of hitherto undetected ERCC1 signals correlating with progressive disease (97% specificity, 18% sensitivity, p-value = 0.030). Allele frequency development of ESR1 and PIK3CA variants detected at subsequent staging time points could be used as a predictor for therapy success and, importantly, might help guide therapy decisions. The three analytes, each with their own unique features for disease monitoring, were shown to be complementary, underlining the usefulness of the longitudinal multi-parametric liquid biopsy approach

Cell-Free DNA Variant Sequencing Using CTC-Depleted Blood for Comprehensive Liquid Biopsy Testing in Metastatic Breast Cancer

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube„ format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube„ format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies

Integrative statistical analyses of multiple liquid biopsy analytes in metastatic breast cancer

Background!#!Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA).!##!Methods!#!Blood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes.!##!Results!#!A combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited-only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster.!##!Conclusions!#!The additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice

PRUNE1: a disease-causing gene for secondary microcephaly

WOS: 000414358500001PubMed ID: 28969376European Community's Seventh Framework Programme (FP7)European Union (EU) [2012-305121]The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 2012-305121 'Integrated European -omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)'

Neuronal-specific deficiency of the splicing factor Tra2b causes apoptosis in neurogenic areas of the developing mouse brain.

Alternative splicing (AS) increases the informational content of the genome and is more prevalent in the brain than in any other tissue. The splicing factor Tra2b (Sfrs10) can modulate splicing inclusion of exons by specifically detecting GAA-rich binding motifs and its absence causes early embryonic lethality in mice. TRA2B has been shown to be involved in splicing processes of Nasp (nuclear autoantigenic sperm protein), MAPT (microtubule associated protein tau) and SMN (survival motor neuron), and is therefore implicated in spermatogenesis and neurological diseases like Alzheimer's disease, dementia, Parkinson's disease and spinal muscular atrophy. Here we generated a neuronal-specific Tra2b knock-out mouse that lacks Tra2b expression in neuronal and glial precursor cells by using the Nestin-Cre. Neuronal-specific Tra2b knock-out mice die immediately after birth and show severe abnormalities in cortical development, which are caused by massive apoptotic events in the ventricular layers of the cortex, demonstrating a pivotal role of Tra2b for the developing central nervous system. Using whole brain RNA on exon arrays we identified differentially expressed alternative exons of Tubulinδ1 and Shugoshin-like2 as in vivo targets of Tra2b. Most interestingly, we found increased expression of the cyclin dependent kinase inhibitor 1a (p21) which we could functionally link to neuronal precursor cells in the affected brain regions. We provide further evidence that the absence of Tra2b causes p21 upregulation and ultimately cell death in NSC34 neuronal-like cells. These findings demonstrate that Tra2b regulates splicing events essential for maintaining neuronal viability during development. Apoptotic events triggered via p21 might not be restricted to the developing brain but could possibly be generalized to the whole organism and explain early embryonic lethality in Tra2b-depleted mice