Skeletal deterioration in COL2A1-related spondyloepiphyseal dysplasia occurs prior to osteoarthritis.

OBJECTIVE: Spondyloepiphyseal dysplasia, a combination of progressive arthropathy with variable signs of skeletal dysplasia, can be a result of mutations in the collagen, type II, alpha 1 (COL2A1) gene. However, the bone involvement (e.g., density, microstructure) in this disorder has hitherto not been studied. DESIGN: A 50-year-old female patient and her 8-year-old son with flattening of vertebral bodies and early-onset osteoarthritis were genetically tested using a custom designed gene bone panel including 386 genes. Bone microstructure and turnover were assessed using high-resolution peripheral quantitative computed tomography (HR-pQCT) and serum bone turnover markers, respectively. Furthermore, the bone and cartilage phenotype of male mice heterozygous for the loss-of-function mutation of Col2a1 (Col2a1+/d) was analyzed compared to wildtype littermates using µ-CT and histomorphometry. RESULTS: We identified a dominant COL2A1 mutation (c.620G > A p.(Gly207Glu)) indicating spondyloepiphyseal dysplasia in the female patient and her son, both being severely affected by skeletal deterioration. Although there was no osteoarthritis detectable at first visit, the son was affected by trabecular osteopenia, which progressed over time. In an iliac crest biopsy obtained from the mother, osteoclast indices were remarkably increased. Col2a1+/d mice developed a moderate skeletal phenotype expressed by reduced cortical and trabecular parameters at 4 weeks. Importantly, no articular defects could be observed in the knee joints at 4 weeks, while osteoarthritis was only detectable in 12-week-old mice. CONCLUSIONS: Our results indicate that collagen type II deficiency in spondyloepiphyseal dysplasia leads to skeletal deterioration with early-onset in humans and mice that occurs prior to the development of osteoarthritis.

Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice.

Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, ß-hexosaminidase, α-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease.

SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Neuronal acetylcholine receptors in Drosophila: mature and immature transcripts of the ard gene in the developing central nervous system.

The ARD protein is a Drosophila homolog of vertebrate nicotinic acetylcholine receptor (AChR) polypeptides. Here, an analysis of transcripts of the corresponding ard gene is presented. In situ hybridization experiments revealed ard gene expression in nervous tissue only. During development, ard transcripts are prevalent in late embryos, pupae, and newly eclosed flies. Both the spatial and the temporal pattern of ard gene expression is consistent with the ARD protein being part of a neuronal AChR that is produced in large amounts during major periods of neuronal differentiation. In situ hybridization with an intron-specific probe indicated codistribution of immature and mature ard RNAs in pupae and adult flies. In addition to the mature 3.2 kb RNA species, two large immature transcripts are found in newly eclosed flies but not in embryos, suggesting a developmentally regulated processing of ard RNA.

Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition.

Synaptic inhibition by GABA(A) and glycine receptors, which are ligand-gated anion channels, depends on the electrochemical potential for chloride. Several potassium-chloride cotransporters can lower the intracellular chloride concentration [Cl(-)](i), including the neuronal isoform KCC2. We show that KCC2 knockout mice died immediately after birth due to severe motor deficits that also abolished respiration. Sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered spinal cord responses to peripheral electrical stimuli. In the spinal cord of wild-type animals, the KCC2 protein was found at inhibitory synapses. Patch-clamp measurements of embryonic day 18.5 spinal cord motoneurons demonstrated an excitatory GABA and glycine action in the absence, but not in the presence, of KCC2, revealing a crucial role of KCC2 for synaptic inhibition.

Cloning of a putative glutamate receptor: a low affinity kainate-binding subunit.

Kainate, a glutamate receptor agonist, is a potent neuroexcitatory agent that produces epileptiform activity and selective neuronal degeneration. Binding studies using neuronal membrane homogenates or brain sections have identified sites having either high or low affinity for [3H]kainate. Here we report the cloning of a gene, GluR7, with approximately 75% sequence identity with the previously cloned GluR5 and GluR6 subunit genes. Transcripts of the GluR7 gene are evident in brain areas that bind [3H]kainate and are susceptible to kainate-induced neurotoxicity. We have performed ligand binding studies with membranes of transfected HeLa cells expressing GluR6 or GluR7 subunits. Our data show that the GluR6 and GluR7 subunits have a rank order of agonist affinity (domoate greater than kainate much greater than L-glutamate, quisqualate much greater than AMPA, NMDA) and a dissociation constant for kainate (95 and 77 nM, respectively) characteristic of the low affinity kainate-binding sites described in the brain.

Cloning of a novel glutamate receptor subunit, GluR5: expression in the nervous system during development.

We have isolated cDNAs encoding a glutamate receptor subunit, designated GluR5, displaying 40%-41% amino acid identity with the kainate/AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4. This level of sequence similarity is significantly below the approximately 70% intersubunit identity characteristic of kainate/AMPA receptors. The GluR5 protein forms homomeric ion channels in Xenopus oocytes that are weakly responsive to L-glutamate. The GluR5 gene is expressed in subsets of neurons throughout the developing and adult central and peripheral nervous systems. During embryogenesis, GluR5 transcripts are detected in areas of neuronal differentiation and synapse formation.

The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress.

In Alzheimer's disease (AD) brains, selected populations of neurons degenerate heavily, whereas others are frequently spared from degeneration. To address the cellular basis for this selective vulnerability of neurons in distinct brain regions, we compared gene expression between the severely affected inferior temporal lobes and the mostly unaffected fronto-parietal cortices by using an mRNA differential display. We identified seladin-1, a novel gene, which was downregulated in large pyramidal neurons in vulnerable regions in AD but not control brains. Seladin-1 is a human homolog of the DIMINUTO/DWARF1 gene described in plants and Caenorhabditis elegans. Its sequence shares similarities with flavin-adenin-dinucleotide (FAD)-dependent oxidoreductases. In human control brain, seladin-1 was highly expressed in almost all neurons. In PC12 cell clones that were selected for resistance against AD-associated amyloid-beta peptide (Abeta)-induced toxicity, both mRNA and protein levels of seladin-1 were approximately threefold higher as compared with the non-resistant wild-type cells. Functional expression of seladin-1 in human neuroglioma H4 cells resulted in the inhibition of caspase 3 activation after either Abeta-mediated toxicity or oxidative stress and protected the cells from apoptotic cell death. In apoptotic cells, however, endogenous seladin-1 was cleaved to a 40 kDa derivative in a caspase-dependent manner. These results establish that seladin-1 is an important factor for the protection of cells against Abeta toxicity and oxidative stress, and they suggest that seladin-1 may be involved in the regulation of cell survival and death. Decreased expression of seladin-1 in specific neurons may be a cause for selective vulnerability in AD.

Developmental expression of the estrogen receptor-related receptor gamma in the nervous system during mouse embryogenesis.

The ERR's (estrogen receptor-related receptors) are constitutive activators of the classical estrogen response element. In this report, we demonstrate that ERRgamma is highly expressed in the nervous system of the developing mouse embryo and that the adult pattern of expression of ERRgamma is, with few exceptions, established during embryogenesis. Transcripts are preferentially detected in already differentiating areas of the nervous system.

Expression of the Na-K-2Cl-cotransporter NKCC1 during mouse development.

Here we describe the expression pattern of the Na-K-2Cl-cotransporter NKKC1 during embryonal and early postnatal mouse development. During early stages hybridization signals were detected over single cells of the developing neuroepithelia, whereas the neuroepithelium of the basal telencephalon was labeled continuously. With ongoing differentiation a distinct pattern of hybridization became apparent, which switched from a neuronal to a more glial pattern in the adult. Outside the nervous system NKCC1 transcripts were present in many organs and were mostly confined to epithelia.

Identification of SorCS2, a novel member of the VPS10 domain containing receptor family, prominently expressed in the developing mouse brain.

We report the identification of a fourth member of the VPS10 domain containing receptor family, SorCS2, highly expressed in the developing and mature murine central nervous system. During early central nervous system development its main site of expression is the floor plate. In addition, high transcript levels were detected transiently in a variety of brain regions including the dopaminergic midbrain nuclei and the dorsal thalamus. Outside the nervous system expression is detected in lung and heart and transiently in a variety of mesodermally derived tissues.

Unique expression pattern of a novel mosaic receptor in the developing cerebral cortex.

Recently, a new type of transmembrane protein with a unique combination of protein domains was characterized from human, rabbit and chicken. This protein exhibits features of the low-density lipoprotein receptor family and shows homology to the receptor of the neuropeptide head activator isolated from hydra. To study the temporal and spatial pattern of expression of this unusual new receptor we have isolated a murine homolog and, in accordance with its human counterpart, named it mSorLA. Northern blot analysis revealed the highest abundance of mSorLA transcripts in the adult brain, lower levels in a variety of other organs and expression during embryogenesis. In situ hybridization showed predominant localization in neurons of the cortex, the hippocampus and the cerebellum. During embryonic development mSorLA displayed a unique pattern of expression in the cerebral cortex, where a subpopulation of neurons was labeled before final differentiation. Transcripts of mSorLA were also detected outside the central nervous system in regions active in morphogenesis.

Neuronal control of development in hydra.

Hydra is an excellent model system for developmental biology, because pattern formation processes can be easily studied in regeneration, transplantation, and reaggregation experiments. At the cellular level hydra has the advantage that it contains only a few basic cell types and that differentiation pathways are short. Two types of signals, produced and released by nerve cells, control the spatial and temporal patterns of differentiation. Positive signals induce specific local differentiation events, and negative signals inhibit the spread of such inductions to larger areas. Head-specific growth and differentiation are controlled by head activator and head inhibitor, food-specific processes are regulated by foot activator and foot inhibitor. The activators are peptides, the inhibitors are low-molecular-weight substances. The sequence of the head activator is known, and it is conserved throughout the animal kingdom. At the cellular level head activator exerts three types of effects in hydra. It stimulates cells to divide, and it is responsible for the determination and the final differentiation of nerve cells and head-specific epithelial cells. For nerve-cell differentiation the cAMP pathway is used as second messenger system. Components of this pathway were identified in hydra. In mammals head activator is produced by nerve and neuro-endocrine cells, and it acts as mitogen on cells of neural origin. It is present early in neural development and in abnormal neural development, such as brain and neuroendocrine tumours.

Characterization of an invertebrate nicotinic acetylcholine receptor gene: the ard gene of Drosophila melanogaster.

The ard gene encodes a neuronal nicotinic acetylcholine receptor (AChR) protein from Drosophila (ARD protein). Cytogenetically this gene maps at position 64B/C on the left arm of the 3rd chromosome. Five introns interrupt the protein coding region of the gene, and one is found upstream of the translation start site. The ard gene thus contains less introns than vertebrate muscle AChR genes, but, with one exception, the positions of the resident introns are precisely conserved. Implications for the evolution of AChR genes are discussed.

Ionotropic glutamate-receptor gene expression in hypothalamus: localization of AMPA, kainate, and NMDA receptor RNA with in situ hybridization.

In situ hybridization and Northern blots were used to study the ionotropic subtypes of the glutamate receptor in the rat hypothalamus. Widespread expression of AMPA, kainate, and NMDA receptor RNA was found in the hypothalamus with the transcripts the same size and number as found in other regions of the brain. Most of the glutamate-receptor subunits studied were expressed in greater amounts in hippocampus than in hypothalamus; GluR5, on the other hand, showed a greater expression in hypothalamus than in hippocampus. On the basis of Northern blot analysis, all regions of the brain examined, including hypothalamus, cerebral cortex, cerebellum, olfactory bulb, and hippocampus, expressed all eight of the subunits studied. Each subunit showed different relative expressions in the different regions. In the hypothalamus, GluR1 and GluR2 were among the most widely expressed of the non-NMDA ionotropic receptors. Other AMPA-preferring receptors, GluR3 and -R4, were also found, but to a lesser extent. Scattered cells expressed the kainate-preferring receptors GluR5, -R6, and -R7. The NMDA receptor NMDAR1 was detected throughout the hypothalamus. In many regions of the hypothalamus, only scattered cells showed detectable expression of the glutamate-receptor mRNA as detected by autoradiographic silver grains over neurons; unlabeled cells were mixed among labeled cells. Every region of the hypothalamus had several different glutamate receptors. The expression of many different types of ionotropic glutamate receptors throughout the hypothalamus suggests that multiple modes of ion channel regulation by glutamate probably operate here and provides further support for the importance of the excitatory transmitter glutamate in hypothalamic regulation.

Expression of the sodium-driven chloride bicarbonate exchanger NCBE during prenatal mouse development.

In the immature central nervous system (CNS) GABA-mediated excitation is thought to be an important developmental signal. It depends on a high intracellular chloride concentration ([Cl(-)](i)) of the particular neuron. [Cl(-)](i) is a consequence of chloride transport processes across the plasma membrane. The ongoing expression of the KCl-co-transporter KCC2 eventually lowers [Cl(-)](i) in most CNS neurons and thus renders GABA hyperpolarizing. As NCBE, a sodium-dependent chloride-bicarbonate exchanger, also lowers [Cl(-)](i) and may thus modulate the GABA-response, we analyzed its expression during prenatal mouse development before establishment of the mature KCC2 expression. Indeed, NCBE is expressed very early in CNS neurons and precedes the expression of KCC2. Unlike KCC2, NCBE is expressed in the peripheral nervous system and in non-neuronal tissues as the choroid plexus, the dura, and some epithelia including the acid secreting epithelium of the stomach and the duodenal epithelium.

Chronic stress induces opposite changes in the mRNA expression of the cell adhesion molecules NCAM and L1.

The effects of 21-day exposure to restraint stress on mRNA levels of the cell adhesion molecules NCAM and L1 were evaluated in different hippocampal regions (CA1, CA3, and dentate gyrus) and other structures (thalamus, prefrontal and frontal cortices, and striatum) of the rat brain. A general decrease in gene expression of the neural cell adhesion molecule (NCAM) was found throughout the brain, particularly in all hippocampal subregions. On the contrary, transcripts for the adhesion molecule L1 were specifically increased at the level of the hippocampus, especially in the dorsal dentate gyrus and area CA3. mRNA for the NCAM180 isoform was detected unchanged in all brain areas examined after chronic stress. A second experiment explored whether there would be cognitive alterations associated with this stress procedure and molecular regulation. Thus, after exposure to the same restraint regimen, performance in the water maze was evaluated. Although stressed rats displayed the ability to learn the task throughout the training session, they showed a transient deficit in the initial phase of the acquisition. In conclusion, our findings indicate that chronic stress interferes with the mechanisms involved in the synthesis of cell adhesion molecules of the immunoglobulin superfamily. Furthermore, they suggest that these effects might be involved in the mechanisms by which stress induces structural and functional alterations in the central nervous system and, particularly, in the hippocampus.

Central nicotinic acetylcholine receptors in the chicken and Drosophila CNS: biochemical and molecular biology approaches.

Putative neuronal nicotinic acetylcholine receptors (nAChRs) were investigated using biochemical and molecular biology approaches. In the chick visual system, a nicotinic cholinergic binding site was localized on a polypeptide of Mr 57,000 using the potent antagonist, alpha-bungarotoxin (alpha-Btx). Ion flux experiments indicate that this membrane protein is different from the toxin-insensitive nAChR present in vertebrate neurons. Crosshybridization with a Torpedo nAChR cDNA probe allowed isolation of a cholinergic receptor cDNA (ARD) from the Drosophila CNS. Analysis of the corresponding gene, its transcripts and the ARD protein are presented here.

Differential expression of the estrogen receptor-related receptor gamma in the mouse brain.

To elucidate estrogen functions, the expression of the estrogen receptor-related receptor ERRgamma, a novel orphan nuclear receptor regulating transcription via estrogen responsive elements, has been localized by in situ hybridization in adult murine brain. ERRgamma transcripts were abundantly present in the isocortex, the olfactory system, cranial nerve nuclei and major parts of the coordination centers, e.g. reticular formation and major parts of the extrapyramidal motor systems. In addition, ERRgamma expression was detected in trigeminal ganglion neurons. ERRgamma distribution was clearly distinguished from that described for ERRalpha, for ERRbeta, and for estrogen receptors (ER) pointing at functional differences between ERRgamma and these receptors.

Expression of the 100-kDa neurotensin receptor sortilin during mouse embryonal development.

Recently, sortilin a non G-protein-coupled receptor has been identified as the 100-kDa neurotensin receptor. In this paper we describe the expression of its gene during mouse embryonal development. We show that the nervous system is the main location of sortilin gene expression and that with ongoing development the forebrain exhibits the highest accumulation of transcripts.