MMP13 mutations are the cause of recessive metaphyseal dysplasia, Spahr type.

Metaphyseal dysplasia, Spahr type (MDST; OMIM 250400) was described in 1961 based on the observation of four children in one family who had rickets-like metaphyseal changes but normal blood chemistry and moderate short stature. Its molecular basis and nosologic status remained unknown. We followed up on those individuals and diagnosed the disorder in an additional member of the family. We used exome sequencing to ascertain the underlying mutation and explored its consequences on three-dimensional models of the affected protein. The MDST phenotype is associated with moderate short stature and knee pain in adults, while extra-skeletal complications are not observed. The sequencing showed that MDST segregated with a c.619T>G single nucleotide transversion in MMP13. The predicted non-conservative amino acid substitution, p.Trp207Gly, disrupts a crucial hydrogen bond in the calcium-binding region of the catalytic domain of the matrix metalloproteinase, MMP13. The MDST phenotype is associated with recessive MMP13 mutations, confirming the importance of this metalloproteinase in the metaphyseal growth plate. Dominant MMP13 mutations have been associated with metaphyseal anadysplasia (OMIM 602111), while a single child homozygous for a MMP13 mutation had been previously diagnosed as "recessive metaphyseal anadysplasia," that we conclude is the same nosologic entity as MDST. Molecular confirmation of MDST allows distinction of it from dominant conditions (e.g., metaphyseal dysplasia, Schmid type; OMIM # 156500) and from more severe multi-system conditions (such as cartilage-hair hypoplasia; OMIM # 250250) and to give precise recurrence risks and prognosis. © 2014 Wiley Periodicals, Inc.

Inflammatory role of ASC in antigen-induced arthritis is independent of caspase-1, NALP-3, and IPAF.

Because IL-1beta plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA, decreased levels of synovial IL-1beta, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-gamma, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.

Peripheral projections of calretinin-immunoreactive primary sensory neurons in chick hindlimbs.

In chicken dorsal root ganglia, calretinin immunoreactivity is expressed by a subpopulation of large A-neurons, most of which co-express calbindin D-28k. The myelinated axons of these neurons selectively innervate all muscle spindles and most Herbst corpuscles associated to feathers in hindlimbs. It is suggested that the presence of calretinin in primary afferents may be correlated with the electrophysiological properties of rapidly adapting mechanoreceptors.

Expression of calbindin immunoreactivity by subpopulations of primary sensory neurons in chick embryo dorsal root ganglion cells grown in coculture or conditioned medium.

Primary sensory neurons which innervate neuromuscular spindles in the chicken are calbindin-immunoreactive. The influence exerted by developing skeletal muscle on the expression of calbindin immunoreactivity by subpopulations of dorsal root ganglion (DRG) cells in the chick embryo was tested in vitro in coculture with myoblasts, in conditioned medium (CM) prepared from myoblasts and in control cultures of DRG cells alone. Control cultures of DRG cells grown at the 6th embryonic day (E6) did not show any calbindin-immunostained ganglion cell. In coculture of myoblasts previously grown for 14 days, about 3% of calbindin-immunoreactive ganglion cells were detected while about 1% were observed in some cultures grown in CM. Fibroblasts from various sources were devoid of effect. Skin or kidney cells were more active than myoblasts to initiate calbindin expression by subpopulations of DRG cells in coculture or, to a lesser degree, in CM. The results suggest that cellular factors would rather induce calbindin expression in certain sensory neurons than ensure a selective neuronal survival.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.

Mouse fetal trisomy 13 and hypotrophy of the spinal cord: effect on calbindin-D28k and calretinin expressed by neurons of the spinal cord and dorsal root ganglia.

Trisomy 13 was detected in 10% of mouse embryos obtained from pregnant females which were doubly heterozygous for Robertsonian chromosomes involving chromosome 13. The developing dorsal root ganglia and spinal cords were examined in trisomy 13 and littermate control mice between days 12 and 18 of gestation (E12-18). The overall size of the dorsal root ganglia and number of ganglion cells within a given ganglion were not altered, but the number of neurons immunoreactive for calbindin and calretinin was reduced. The trisomic spinal cord was reduced in size with neurons lying in a tightly compact distribution in the gray matter. In trisomic fetuses, the extent of the neuropil of the spinal cord was reduced, and may represent a diminished field of interneuronal connectivity, due to reduced arborization of dendritic processes of the neurons present, particularly of calbindin-immunostained neurons. Furthermore, the subpopulation of calretinin-immunoreactive neurons and axons was also reduced in developing trisomic gray and white matter, respectively. Thus, overexpression of genes on mouse chromosome 13 exerts a deleterious effect on the development of neuropil, affecting both dendritic and axonal arborization in the trisomy 13 mouse. The defect of calbindin or calretinin expression by subsets of dorsal root ganglion or spinal cord neurons may result from deficient cell-to-cell interactions with targets which are hypoplastic.

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8⁺ T cells.

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

Genotyping microarray for CSNB-associated genes.

PURPOSE: Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches. METHODS: To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, GRM6, NYX, PDE6B, and SAG and spotted on the chip. RESULTS: Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations. CONCLUSIONS: This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects.

Whole-mount confocal immunofluorescence of mammalian CNS.

Bright-field wholemount labeling techniques applied to the mammalian central nervous system (CNS) offer advantages over conventional methods based on sections since an immediate and three-dimensional view of the stained components is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The ability of confocal laser scanning microscopy to visualize in one focal plane the fluorescence associated with multiple markers could be most valuable by the availability of reliable wholemount fluorescent techniques. Accordingly, based in our previously published bright-field wholemount protocols [Brain Res. Prot. 2 (1998) 165-173], we have devised an effective immmunofluorescence wholemount procedure. We show that reliable wholemount fluorescent staining can be obtained using isolated complete CNS aged up to rat embryonic day 17, with antibodies penetration in the millimeter range. Examples are shown of preparations in which colocalization can be observed in nerve cells of cytoskeletal and calcium-binding proteins.

New pool of cortical interneuron precursors in the early postnatal dorsal white matter.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Dopamine affects parvalbumin expression during cortical development in vitro.

This study was undertaken to determine how dopamine influences cortical development. It focused on morphogenesis of GABAergic neurons that contained the calcium-binding protein parvalbumin (PV). Organotypic slices of frontoparietal cortex were taken from neonatal rats, cultured with or without dopamine, harvested daily (4-30 d), and immunostained for parvalbumin. Expression of parvalbumin occurred in the same regional and laminar sequence as in vivo. Expression in cingulate and entorhinal preceded that in lateral frontoparietal cortices. Laminar expression progressed from layer V to VI and finally II-IV. Somal labeling preceded fiber labeling by 2 d. Dopamine accelerated PV expression. In treated slices, a dense band of PV-immunoreactive neurons appeared in layer V at 7 d in vitro (DIV), and in all layers of frontoparietal cortex at 14 DIV, whereas in control slices such labeling did not appear until 14 and 21 DIV, respectively. The laminar distribution and dendritic branching of PV-immunoreactive neurons were quantified. More labeled neurons were in the superficial layers, and their dendritic arborizations were significantly increased by dopamine. Treatment with a D1 receptor agonist had little effect, whereas a D2 agonist mimicked dopamine's effects. Likewise, the D2 but not the D1 antagonist blocked dopamine-induced changes, indicating that they were mediated primarily by D2 receptors. Parvalbumin expression was accelerated by dopaminergic reinnervation of cortical slices that were cocultured with mesencephalic slices. Coapplication of the glutamate NMDA receptor antagonist MK801 or AP5 blocked dopamine-induced increases in dendritic branching, suggesting that changes were mediated partly by interaction with glutamate to alter cortical excitability.

Pre-existing astrocytes form functional perisynaptic processes on neurons generated in the adult hippocampus.

The adult dentate gyrus produces new neurons that morphologically and functionally integrate into the hippocampal network. In the adult brain, most excitatory synapses are ensheathed by astrocytic perisynaptic processes that regulate synaptic structure and function. However, these processes are formed during embryonic or early postnatal development and it is unknown whether astrocytes can also ensheathe synapses of neurons born during adulthood and, if so, whether they play a role in their synaptic transmission. Here, we used a combination of serial-section immuno-electron microscopy, confocal microscopy, and electrophysiology to examine the formation of perisynaptic processes on adult-born neurons. We found that the afferent and efferent synapses of newborn neurons are ensheathed by astrocytic processes, irrespective of the age of the neurons or the size of their synapses. The quantification of gliogenesis and the distribution of astrocytic processes on synapses formed by adult-born neurons suggest that the majority of these processes are recruited from pre-existing astrocytes. Furthermore, the inhibition of astrocytic glutamate re-uptake significantly reduced postsynaptic currents and increased paired-pulse facilitation in adult-born neurons, suggesting that perisynaptic processes modulate synaptic transmission on these cells. Finally, some processes were found intercalated between newly formed dendritic spines and potential presynaptic partners, suggesting that they may also play a structural role in the connectivity of new spines. Together, these results indicate that pre-existing astrocytes remodel their processes to ensheathe synapses of adult-born neurons and participate to the functional and structural integration of these cells into the hippocampal network.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.