Functional analysis of the mospd gene family

Mospd3, a gene located on mouse chromosome 5, was identified in a gene trap screen in ES cells. The gene trap vector integration in multiple copies into the putative promoter of the gene, resulted in a loss of expression of Mospd3 at the trapped allele. In mice generated from ES cells carrying the vector integration it was found that the lack of Mospd3 expression resulted in the death of a proportion of the homozygote mutants within the first day after birth. Homozygote neonates exhibited a thinning of the right ventricular free heart wall which resembles other mouse mutant phenotypes as well as human congenital heart defects caused by a loss of desmosome and adherens junction mediated cell adhesion between cardiomyocytes. The protein encoded by Mospd3, contains an N-terminal Major Sperm Protein (MSP) domain implicated as a mediator of protein- protein interactions, as well as a two C-terminal transmembrane domains. Both, protein structure and phenotypic similarities with defects in desmosomal and adherens junction proteins suggests that Mospd proteins might play a role in cell adhesion and maintaining the structural integrity of the heart. The phenotype of Mospd3 mutants was highly dependent on genetic background, which led us to speculate that there might be genetic redundancy between Mospd3 and its closest family member the X-linked Mospd1. The aims of this thesis were to generate tools to better understand the function of the Mospd gene family in cardiac development as well as assessing genetic redundancy between Mospd1 and Mospd3. A conditional gene targeting strategy was designed for both Mospd genes. Large genomic regions of the Mospd1 and Mospd3 loci were subcloned from bacterial artificial chromosomes (BACs) and using a recombineering approach, loxP sites and a drug selection cassette (neomycin) were placed in precise locations surrounding the MSP domain of both genes. The conditional targeting vectors were electroporated into both CGR8 and E14 ES cells and homologous recombinant clones were identified at a frequency of 2% and 1.3% for Mospd1 and Mospd3 respectively. Five euploid targeted clones for both Mospd1 and Mospd3 have been generated. Transient expression of Cre recombinase in ES cells carrying the conditional Mospd1 allele was used to delete the one copy of this X-linked gene. Phenotypic characterisation of this null ES cell line revealed that Mospd1 is neither essential for ES cell viability and self-renewal, nor for the early differentiation of these cells towards a cardiac fate. In order to investigate the mechanism of action of Mospd proteins, specific polyclonal antibodies were generated to detect either Mospd1 or Mospd3. These antibodies were purified and tested by western blotting using COS7 cells overexpressing either Mospd protein as well as mouse tissue lysates. Whilst the antibodies were found to detect the proteins and differentiate between Mospd1 and Mospd3, they showed insufficient purification to be used in co-localisation and co-immunoprecipitation experiments to identify interacting proteins and determine whether Mospd proteins are involved in cell adhesion complexes. Monoclonal antibodies were subsequently generated and initial western blotting experiments showed promising results, indicating that these antibodies may be better suited for immunohistochemical analysis of Mospd proteins

A role for mospd1 in mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1‐null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self‐renewal capacity of MOSPD1‐null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)‐like cells expressing CD73, CD90, and CD105 were generated from MOSPD1‐null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1‐null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA‐sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre‐MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation. Stem Cells 2015;33:3077–308

The comorbidity and co-medication profile of patients with progressive supranuclear palsy

BackgroundProgressive supranuclear palsy (PSP) is usually diagnosed in elderly. Currently, little is known about comorbidities and the co-medication in these patients.ObjectivesTo explore the pattern of comorbidities and co-medication in PSP patients according to the known different phenotypes and in comparison with patients without neurodegenerative disease.MethodsCross-sectional data of PSP and patients without neurodegenerative diseases (non-ND) were collected from three German multicenter observational studies (DescribePSP, ProPSP and DANCER). The prevalence of comorbidities according to WHO ICD-10 classification and the prevalence of drugs administered according to WHO ATC system were analyzed. Potential drug-drug interactions were evaluated using AiDKlinik (R).ResultsIn total, 335 PSP and 275 non-ND patients were included in this analysis. The prevalence of diseases of the circulatory and the nervous system was higher in PSP at first level of ICD-10. Dorsopathies, diabetes mellitus, other nutritional deficiencies and polyneuropathies were more frequent in PSP at second level of ICD-10. In particular, the summed prevalence of cardiovascular and cerebrovascular diseases was higher in PSP patients. More drugs were administered in the PSP group leading to a greater percentage of patients with polypharmacy. Accordingly, the prevalence of potential drug-drug interactions was higher in PSP patients, especially severe and moderate interactions.ConclusionsPSP patients possess a characteristic profile of comorbidities, particularly diabetes and cardiovascular diseases. The eminent burden of comorbidities and resulting polypharmacy should be carefully considered when treating PSP patients

Functional analysis of the Mospd gene family

Mospd3, a gene located on mouse chromosome 5, was identified in a gene trap screen in ES cells. The gene trap vector integration in multiple copies into the putative promoter of the gene, resulted in a loss of expression of Mospd3 at the trapped allele. In mice generated from ES cells carrying the vector integration it was found that the lack of Mospd3 expression resulted in the death of a proportion of the homozygote mutants within the first day after birth. Homozygote neonates exhibited a thinning of the right ventricular free heart wall which resembles other mouse mutant phenotypes as well as human congenital heart defects caused by a loss of desmosome and adherens junction mediated cell adhesion between cardiomyocytes. The protein encoded by Mospd3, contains an N-terminal Major Sperm Protein (MSP) domain implicated as a mediator of protein- protein interactions, as well as a two C-terminal transmembrane domains. Both, protein structure and phenotypic similarities with defects in desmosomal and adherens junction proteins suggests that Mospd proteins might play a role in cell adhesion and maintaining the structural integrity of the heart. The phenotype of Mospd3 mutants was highly dependent on genetic background, which led us to speculate that there might be genetic redundancy between Mospd3 and its closest family member the X-linked Mospd1. The aims of this thesis were to generate tools to better understand the function of the Mospd gene family in cardiac development as well as assessing genetic redundancy between Mospd1 and Mospd3. A conditional gene targeting strategy was designed for both Mospd genes. Large genomic regions of the Mospd1 and Mospd3 loci were subcloned from bacterial artificial chromosomes (BACs) and using a recombineering approach, loxP sites and a drug selection cassette (neomycin) were placed in precise locations surrounding the MSP domain of both genes. The conditional targeting vectors were electroporated into both CGR8 and E14 ES cells and homologous recombinant clones were identified at a frequency of 2% and 1.3% for Mospd1 and Mospd3 respectively. Five euploid targeted clones for both Mospd1 and Mospd3 have been generated. Transient expression of Cre recombinase in ES cells carrying the conditional Mospd1 allele was used to delete the one copy of this X-linked gene. Phenotypic characterisation of this null ES cell line revealed that Mospd1 is neither essential for ES cell viability and self-renewal, nor for the early differentiation of these cells towards a cardiac fate. In order to investigate the mechanism of action of Mospd proteins, specific polyclonal antibodies were generated to detect either Mospd1 or Mospd3. These antibodies were purified and tested by western blotting using COS7 cells overexpressing either Mospd protein as well as mouse tissue lysates. Whilst the antibodies were found to detect the proteins and differentiate between Mospd1 and Mospd3, they showed insufficient purification to be used in co-localisation and co-immunoprecipitation experiments to identify interacting proteins and determine whether Mospd proteins are involved in cell adhesion complexes. Monoclonal antibodies were subsequently generated and initial western blotting experiments showed promising results, indicating that these antibodies may be better suited for immunohistochemical analysis of Mospd proteins.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Telephone-assisted self-help for parents of children with attention-deficit/hyperactivity disorder who have residual functional impairment despite methylphenidate treatment: a randomized controlled trial

BackgroundSelf-help parenting interventions have been shown to be effective in the management of children with attention-deficit/hyperactivity disorder (ADHD) and may be useful when there are barriers to face-to-face therapist-led parent trainings. Previous studies indicate that behavioral interventions might be a useful adjunct to medication in children with residual ADHD symptoms, and regarding comorbid oppositional symptoms and multiple domains of functional impairment. In the present study, we examined whether a telephone-assisted self-help (TASH) parenting behavioral intervention (written materials plus telephone counseling) enhanced the effects of methylphenidate treatment in children with ADHD. MethodsIn this randomized controlled trial, parents of 103 school-aged children with ADHD and residual functional impairment despite methylphenidate treatment were randomly assigned to either the enhancement group, which received the TASH intervention as adjunct to routine clinical care (including continued medication), or to the active control group, which received routine clinical care only (including continued medication). Parent-completed outcome measures at baseline and at 12months (postassessment) included functional impairment, ADHD symptoms, oppositional defiant disorder (ODD) symptoms, parenting behavior, and parental satisfaction with the intervention (ClinicalTrials.gov: NCT01660425; URL: ). ResultsIntention-to-treat analyses of covariance (ANCOVAs), which controlled for baseline data, revealed significant and moderate intervention effects for ODD symptoms and negative parenting behavior at the postassessment, whereas per-protocol analyses additionally showed significant and moderate effects on functional impairment (primary outcome). Parents expressed high satisfaction with the program. ConclusionsThe TASH program enhances effects of methylphenidate treatment in families who complete the intervention. Thediscontinuation rate of about 30% and comparison between completing and discontinuing families suggest that the program may be more suitable for families with a higher educational level and fewer additional stresses

Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4(+) T cells display impaired Th1 polarization, as reflected by reduced interferon-gamma production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c(+) dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-gamma concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway. Previous studies have shown that the CD40L-CD40 signaling axis plays a role in atherosclerosis. Here the authors investigate the cell-specific functions of the most relevant CD40L-expressing cell types in atherosclerosis. Deficiency of T cell-derived CD40L reduces and stabilizes plaques through impaired Th1 polarization while platelet-derived CD40L ameliorates atherothrombosis