RhoBTB3 interacts with the 5-HT7a receptor and inhibits its proteasomal degradation

The 5-hydroxytryptamine (5-HT)7 receptor is the most recently identified serotonin receptor and is involved in a wide variety of central nervous system (CNS) functions, namely circadian rhythm, REM sleep, depression, thermoregulation, obsessive-compulsive disorder (OCD), anxiety, schizophrenia, epilepsy, nociception, migraine, sensation-seeking behavior, impulsivity, learning and memory. These numerous (patho)physiological processes of the CNS, in which the 5-HT7 receptor is involved, most likely reflect a diverse set of signaling pathways arising from this receptor. In order to reveal new interaction partners and possibly new signaling and/or trafficking pathways, we performed a yeast two-hybrid screening, using the C-terminal tail of the 5-HT7a receptor as bait and an adult-human brain cDNA library as prey. In this way we identified RhoBTB3 as a new interaction partner of the 5-HT7a receptor. By means of co-immunoprecipitation we were able to confirm the interaction between full length 5-HT7a receptor and RhoBTB3 in HEK293T cells. Subsequent domain mapping of this interaction revealed that not only the C-terminal tail, but also the third intracellular loop of the 5-HT7a receptor is involved. In addition, immunofluorescence microscopy showed clear co-localization between the 5-HT7a receptor and RhoBTB3 at the plasma membrane and in the endoplasmic reticulum. Despite the fact that RhoBTB3 has been shown to interact with Cul3, which in turn interacts with the E3 ubiquitin ligase, Roc1, we show here that RhoBTB3 neither recruits Cul3/Roc1 to the 5-HT7a receptor nor does it mediate ubiquitination of this receptor. Instead, we demonstrate that RhoBTB3 strongly inhibits proteasomal degradation of the 5-HT7a receptor.     

Metabolomic Analysis of the Effect of Postnatal Hypoxia on the Retina in a Newly Born Piglet Model

The availability of reliable biomarkers of brain injury secondary to birth asphyxia could substantially improve clinical grading, therapeutic intervention strategies, and prognosis. In this study, changes in the metabolome of retinal tissue caused by profound hypoxia in an established neonatal piglet model were investigated using an ultra performance liquid chromatography – quadrupole time of flight mass spectrometry (UPLC-QTOFMS) untargeted metabolomic approach, which included Partial Least Squares – Discriminant Analysis (PLSDA) multivariate data analysis. The initial identification of a set of discriminant metabolites from UPLC-QTOFMS data was confirmed by target UPLC-MS/MS and allowed the selection of endogenous CDP-choline as a promising candidate biomarker for hypoxia-derived brain damage assessing intensity of retinal hypoxia. Results from this study will foster further research on CDP-choline changes occurring during resuscitation.     

Induction of Circadian Rhythm in Cultured Human Mesenchymal Stem Cells by Serum Shock and cAMP Analogs in Vitro

Circadian clocks have been shown to operate developmentally in mouse and human hematopoietic stem and progenitor cells in vivo, but little is known about their possible oscillations in vitro. Here, we show that repeated circadian oscillations could be induced in both cultured bone marrow‐derived mesenchymal‐ and adipose‐derived stem cells (MSCs and ASCs, respectively) by serum shock. In particular, the novel finding of rhythmic clock gene expression induced by cAMP analogs showed similarities as well as differences to serum‐induced oscillations. Rhythmic PER1 expression was found in serum‐shocked MSCs, suggesting the phosphorylation status of PER1 is important for its activity in circadian rhythms. Furthermore, immunofluoresent staining showed that the localization of PER1 was dependent on the level of PER1 expression. These inducible self‐sustained circadian clocks in primary cultures of human MSCs in vitro with rhythmic changes in expression levels, phosphorylation, and localization of clock protein, PER1, may be of importance for maintaining the induced oscillations in stem cells. Therefore, the established cell models described here appear to be valuable for studying the molecular mechanism driving and coordinating the circadian network between stem and stromal cells.     

Zebrafish kidney marrow contains ABCG2-dependent side population cells exhibiting hematopoietic stem cell properties

Zebrafish (Danio rerio) has emerged as a powerful genetic model for the study of vertebrate hematopoiesis. However, methods for detection and isolation of hematopoietic stem cells (HSCs) have not yet been reported. In mammals, the combination of Hoechst 33342 staining with flow cytometry can be used for separation of a bone marrow side population (SP), which is highly enriched for HSCs. We applied a similar procedure to hematopoietic kidney marrow cells from adult zebrafish, and identified a segregated cohort of SP cells, which demonstrate a set of features typical of stem cells. SP cells show extremely low scatter characteristics, and are small in size with a minimum of cytoplasm. Treatment of zebrafish kidney marrow cells with reserpine or fumitremorgin C, which inhibit the ABCG2 transporter responsible for Hoechst 33342 efflux, caused a clear reduction in the number of SP cells. Consistent with the quiescent state of HSCs, the SP cells are strongly resistant to the myelosuppressive agent 5-fluorouracil. In addition, SP cells specifically demonstrate higher expression of genes known to be markers of HSCs of mammals. Hence, our results show that the SP phenotype is conserved between mammals and teleosts, and the properties of the zebrafish SP cells indicate a significant enrichment for HSCs. These rapid flow cytometric methods for purification of HSCs from zebrafish may greatly facilitate genetic analysis of stem cells using the advantages of this vertebrate model.     

The majority of the genetic risk for Paget’s disease of bone is explained by genetic variants close to the CSF1, OPTN, TM7SF4, and TNFRSF11A genes

Paget’s disease of bone (PDB) is one of the most frequent metabolic bone disorders (1–5%), next to osteoporosis, affecting individuals above age 55. Sequestosome1 mutations explain a part of the PDB patients, but still the disease pathogenesis in the remaining PDB patients is largely unknown. Therefore, association studies investigating the relationship between genetic polymorphisms and sporadic PDB have been performed to find the genetic risk variants. Previously such studies indicated a role of the OPG and RANK gene. The latter was recently confirmed in a genome-wide association study (GWAS) which also indicated the involvement of chromosomal regions harbouring the CSF1 and OPTN gene. In this study, we sought to replicate these findings in a Belgian and a Dutch population. Similar significant results were obtained for the single nucleotide polymorphisms and the haplotypes. The most significant results are found in the CSF1 gene region, followed by the OPTN and TNFRSF11A gene region (p values ranging from 1.3 × 10^?4 to 3.8 × 10^?8, OR = 1.523–1.858). We next obtained significant association with a polymorphism from the chromosomal region around the TM7SF4 gene (p = 2.7 × 10^?3, OR = 1.427), encoding DC-STAMP which did not reach genome-wide significance in the GWAS, but based on its function in osteoclasts it can be considered a strong candidate gene. After meta-analysis with the GWAS data, p values ranged between 2.6 × 10^?4 and 8.8 × 10^?32. The calculated cumulative population attributable risk of these four loci turned out to be about 67% in our two populations, indicating that most of the genetic risk for PDB is coming from genetic variants close to these four genes.     

Van Buchem disease (hyperostosis corticalis generalisata) maps to chromosome 17q12-q21.

Van Buchem disease (hyperostosis corticalis generalisata; OMIM 239100 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?239100]) is an autosomal recessive disorder characterized by hyperostosis of the skull, mandible, clavicles, ribs, and diaphyseal cortices of the long bones. The most striking clinical features are the enlargement of the jaw and the thickness of the skull, which may lead to facial nerve palsy, hearing loss, and optic atrophy. Increased formation, by osteoblasts, of qualitatively normal bone has been proposed as the underlying pathological mechanism, but the molecular defect is unknown. We studied 11 van Buchem patients and their highly inbred family, who live in The Netherlands in a small ethnic isolate, that had a common ancestor approximately 9 generations ago. A genomewide search with highly polymorphic microsatellite markers showed linkage to marker D17S1299 on chromosome 17q12-21 (maximum LOD score of 8.82 at a recombination fraction [straight theta] of .01). Analysis of additional markers from that region delineated a candidate region of <1 cM, between markers D17S1787 and D17S934. Interestingly, the only marker not showing recombination with the disease locus was an intragenic marker of the thyroid-hormone receptor alpha1 (THRA1) gene, which generated a LOD score of 12.84 at straight theta=.00. Since thyroid hormones are known to stimulate bone resorption, the THRA1 gene might be involved in the etiology and pathogenesis of van Buchem disease. Unraveling the underlying mechanism for this disorder could contribute to the understanding of the regulatory processes conditioning bone density and the underlying pathological processes.     

Reaggregation of fetal rat brain cells in a stationary culture system I: Methodology and cell identification

Summary A stationary tissue culture system for reaggregation cultures of rat brain cells is described. Aggregates were formed by placing cells at high concentrations in liquid overlay cultures on a nonadherent nutrient agar surface. No physical stress in the form of rotation or shaking was applied to the aggregating cell population. Transmission electron microscopy and immunohistochemistry showed that the cells developed from homogeneously dispersed, immature cells in Day 4 aggregates, to mature astrocytes, oligodendrocytes, and neurons in Day 20 aggregates. Twenty days and older aggregates had a tightly packed neuropil which was most prominent in a cell-sparse outer layer of the aggregates. When the aggregates were allowed to adhere to a substrate, both glial fibrillary acidic protein (GFAP) positive and negative cells were observed migrating out from the aggregates. Cells giving a positive reaction for neuron specific enolase (NSE) were also present. This reaggregation procedure, with transfer of selected brain cell aggregates into agar-coated multiwells is an alternative three-dimensional culture system which can be potentially useful in the study of morphogenesis and cell interactions in the nervous system. This project was supported by the Norwegian Cancer Society.     

Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors (exostoses). Besides suffering complications caused by the pressure of these exostoses on the surrounding tissues, EXT patients are at an increased risk for malignant chondrosarcoma, which may develop from an exostosis. EXT is genetically heterogeneous, and three loci have been identified so far: EXT1, on chromosome 8q23-q24; EXT2, on 11p11-p12; and EXT3, on the short arm of chromosome 19. The EXT1 and EXT2 genes were cloned recently, and they were shown to be homologous. We have now analyzed the EXT1 and EXT2 genes, in 26 EXT families originating from nine countries, to identify the underlying disease-causing mutation. Of the 26 families, 10 families had an EXT1 mutation, and 10 had an EXT2 mutation. Twelve of these mutations have never been described before. In addition, we have reviewed all EXT1 and EXT2 mutations reported so far, to determine the nature, frequency, and distribution of mutations that cause EXT. From this analysis, we conclude that mutations in either the EXT1 or the EXT2 gene are responsible for the majority of EXT cases. Most of the mutations in EXT1 and EXT2 cause premature termination of the EXT proteins, whereas missense mutations are rare. The development is thus mainly due to loss of function of the EXT genes, consistent with the hypothesis that the EXT genes have a tumor- suppressor function.     

Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.     

Molecular integrity and usefulness of episomal expression vectors derived from BK and Epstein-Barr virus

High-level and stable production of a protein of interest is one of the most important parameters when considering the development of an efficient vector system for heterologous gene expression. In order to achieve this goal, we have used episomal vector elements derived from Epstein-Barr virus (EBV) or BK virus (BKV) in combination with the strictly regulated interferon-inducible Mx promoter.Here we demonstrate that EBV-derived vectors replicate efficiently in all cell lines tested (i.e. HEK293, HeLaH21 and Vero), yielding stable transfectants with a high, inducible expression level and almost no background. In contrast, BKV-derived vectors are much more restricted to particular cell types and hampered by DNA rearrangements, which is a serious drawback for use over a longer timespan.