Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients

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Characterization of PTZ-Induced Seizure Susceptibility in a Down Syndrome Mouse Model That Overexpresses CSTB

Down syndrome (DS) is a complex genetic syndrome characterized by intellectual disability, dysmorphism and variable additional physiological traits. Current research progress has begun to decipher the neural mechanisms underlying cognitive impairment, leading to new therapeutic perspectives. Pentylenetetrazol (PTZ) has recently been found to have positive effects on learning and memory capacities of a DS mouse model and is foreseen to treat DS patients. But PTZ is also known to be a convulsant drug at higher dose and DS persons are more prone to epileptic seizures than the general population. This raises concerns over what long-term effects of treatment might be in the DS population. The cause of increased propensity for epilepsy in the DS population and which Hsa21 gene(s) are implicated remain unknown. Among Hsa21 candidate genes in epilepsy, CSTB, coding for the cystein protease inhibitor cystatin B, is involved in progressive myoclonus epilepsy and ataxia in both mice and human. Thus we aim to evaluate the effect of an increase in Cstb gene dosage on spontaneous epileptic activity and susceptibility to PTZ-induced seizure. To this end we generated a new mouse model trisomic for Cstb by homologous recombination. We verified that increasing copy number of Cstb from Trisomy (Ts) to Tetrasomy (Tt) was driving overexpression of the gene in the brain, we checked transgenic animals for presence of locomotor activity and electroencephalogram (EEG) abnormalities characteristic of myoclonic epilepsy and we tested if those animals were prone to PTZ-induced seizure. Overall, the results of the analysis shows that an increase in Cstb does not induce any spontaneous epileptic activity and neither increase or decrease the propensity of Ts and Tt mice to myoclonic seizures suggesting that Ctsb dosage should not interfere with PTZ-treatment

Brain inflammation is accompanied by peripheral inflammation in Cstb(-/-) mice, a model for progressive myoclonus epilepsy

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene (CSTB) underlie EPM1. The CSTB-deficient (Cstb(-/-)) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb(-/-) mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb(-/-) mice and higher CXCL13 expression in activated microglia in Cstb(-/-) compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb(-/-) mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb(-/-)mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.Peer reviewe

Gene-Expression Profiling Suggests Impaired Signaling via the Interferon Pathway in Cstb(-/-) Microglia

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive neurodegenerative disorder characterized by stimulus-sensitive and action-activated myoclonus, tonic-clonic epileptic seizures, and ataxia. Loss-of-function mutations in the gene encoding the cysteine protease inhibitor cystatin B (CSTB) underlie EPM1. The deficiency of CSTB in mice (Cstb(-/-) mice) generates a phenotype resembling the symptoms of EPM1 patients and is accompanied by microglial activation at two weeks of age and an upregulation of immune system-associated genes in the cerebellum at one month of age. To shed light on molecular pathways and processes linked to CSTB deficiency in microglia we characterized the transcriptome of cultured Cstb(-/-) mouse microglia using microarray hybridization and RNA sequencing (RNA-seq). The gene expression profiles obtained with these two techniques were in good accordance and not polarized to either pro- or anti-inflammatory status. In Cstb(-/-) microglia, altogether 184 genes were differentially expressed. Of these, 33 genes were identified by both methods. Several interferon-regulated genes were weaker expressed in Cstb(-/-) microglia compared to control. This was confirmed by quantitative real-time PCR of the transcripts Irf7 and Stat1. Subsequently, we explored the biological context of CSTB deficiency in microglia more deeply by functional enrichment and canonical pathway analysis. This uncovered a potential role for CSTB in chemotaxis, antigen-presentation, and in immune-and defense response-associated processes by altering JAK-STAT pathway signaling. These data support and expand the previously suggested involvement of inflammatory processes to the disease pathogenesis of EPM1 and connect CSTB deficiency in microglia to altered expression of interferon-regulated genes.Peer reviewe

The transcriptome of follicular cells: biological insights and clinical implications for the treatment of infertility.

BACKGROUND: Oocyte maturation is under strict regulatory control, not only from intrinsic cellular processes, but also extrinsic influences. While the oocyte is directly connected to the surrounding cumulus cells (CCs) via a network of gap junctions facilitating communication and exchange of molecules, it is also influenced by the greater follicular environment. In order to produce an oocyte capable of successfully transmitting the female genetic material and able to support the earliest stages of preimplantation development, cytoplasmic and nuclear maturation must be achieved. Granulosa and CCs play an essential role in the maturation and competence acquisition of the developing oocyte. The fact that these cells are closely associated with the oocyte, share the same microenvironment and can be easily collected during IVF procedures makes them attractive targets for basic research and the development of clinically relevant assays. Analysis of follicular cells is likely to reveal important information concerning the viability and genetic constitution of their associated oocyte, as well as increase our understanding of normal follicular processes and the impact of disorders or of medical interventions such as controlled ovarian stimulation (COS). This review summarizes results obtained during the investigation of granulosa and CCs, and considers the possibilities of using follicular cells as surrogate markers of stimulation response during IVF, oocyte/embryo competence and clinical outcome. METHODS: In order to summarize the current knowledge obtained from the analysis of follicular cells, a thorough literature search was carried out. Relevant research articles published in English up to March 2013 were reviewed. RESULTS: Multiple groups of genes expressed in follicular cells have been identified as possible indicators of ovulation, oocyte maturity, fertilization, chromosome status, ability to generate embryos capable of reaching the blastocyst stage of development, embryo morphology and the establishment of a pregnancy. However, there is a general lack of uniformity concerning groups of gene biomarkers among different studies. CONCLUSIONS: Extensive investigation of genes and proteins of granulosa and CCs has provided a detailed insight into the follicular microenvironment surrounding oocytes. It was evident from the data reviewed that the gene expression of follicular cells influences and is influenced by the oocyte, affecting factors such as maturity, chromosomal constitution, viability and competence. However, a general lack of overlap among genes identified as potentially useful biomarkers suggests that the transcriptome of follicular cells could be affected by multiple intrinsic factors, having to do with the patient and possibly the aetiology of infertility, as well as extrinsic factors, such as hormonal stimulation. Further work is required in order to establish a universally applicable, non-invasive test for the determination of oocyte competence based upon follicular cell assessment

The transcriptome of follicular cells: biological insights and clinical implications for the treatment of infertility.

BACKGROUND: Oocyte maturation is under strict regulatory control, not only from intrinsic cellular processes, but also extrinsic influences. While the oocyte is directly connected to the surrounding cumulus cells (CCs) via a network of gap junctions facilitating communication and exchange of molecules, it is also influenced by the greater follicular environment. In order to produce an oocyte capable of successfully transmitting the female genetic material and able to support the earliest stages of preimplantation development, cytoplasmic and nuclear maturation must be achieved. Granulosa and CCs play an essential role in the maturation and competence acquisition of the developing oocyte. The fact that these cells are closely associated with the oocyte, share the same microenvironment and can be easily collected during IVF procedures makes them attractive targets for basic research and the development of clinically relevant assays. Analysis of follicular cells is likely to reveal important information concerning the viability and genetic constitution of their associated oocyte, as well as increase our understanding of normal follicular processes and the impact of disorders or of medical interventions such as controlled ovarian stimulation (COS). This review summarizes results obtained during the investigation of granulosa and CCs, and considers the possibilities of using follicular cells as surrogate markers of stimulation response during IVF, oocyte/embryo competence and clinical outcome. METHODS: In order to summarize the current knowledge obtained from the analysis of follicular cells, a thorough literature search was carried out. Relevant research articles published in English up to March 2013 were reviewed. RESULTS: Multiple groups of genes expressed in follicular cells have been identified as possible indicators of ovulation, oocyte maturity, fertilization, chromosome status, ability to generate embryos capable of reaching the blastocyst stage of development, embryo morphology and the establishment of a pregnancy. However, there is a general lack of uniformity concerning groups of gene biomarkers among different studies. CONCLUSIONS: Extensive investigation of genes and proteins of granulosa and CCs has provided a detailed insight into the follicular microenvironment surrounding oocytes. It was evident from the data reviewed that the gene expression of follicular cells influences and is influenced by the oocyte, affecting factors such as maturity, chromosomal constitution, viability and competence. However, a general lack of overlap among genes identified as potentially useful biomarkers suggests that the transcriptome of follicular cells could be affected by multiple intrinsic factors, having to do with the patient and possibly the aetiology of infertility, as well as extrinsic factors, such as hormonal stimulation. Further work is required in order to establish a universally applicable, non-invasive test for the determination of oocyte competence based upon follicular cell assessment