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.

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.

SorCS1, a member of the novel sorting receptor family, is localized in somata and dendrites of neurons throughout the murine brain.

The expression of the sorCS1 protein in the central nervous system of adult mice was studied by immunohistochemistry. A detailed mapping revealed a distribution of sorCS1 immunoreactivity in a widespread population of neurons throughout the brain. Two different types of cellular localization were observed. Many neurons exhibited a punctate cytoplasmic staining which extended into the dendrites, in other neurons sorCS1 immunoreactivity was associated with the plasma membrane. This suggests variable functions for sorCS1 in the neurons of the brain.

The genes for the human VPS10 domain-containing receptors are large and contain many small exons.

The two human proteins with a VPS10 domain, SorLA and sortilin, both bind neuropeptides. Searching for other VPS10-domain proteins in the database revealed three new putative human neuropeptide receptors. The new receptors were designated SorCS1, SorCS2 and SorCS3, due to their identical domain composition, which, except for the N-terminal VPS10 domain, differs from that of SorLA and sortilin. Using the databases of the human genome project we elucidated the exon-intron structures of the human VPS10-receptor genes. They contain many short exons, separated by introns, several of which extend over more than 50 kb. The three SorCS genes encompass more than 500 kb of genomic DNA and therefore represent some of the largest known human genes. All these genes map to chromosomal localisations of known genetic diseases, many of them neurological disorders, corresponding to the strong expression of these receptors in the brain. CpG islands are located in the first exon of each of the VPS10-receptor genes and might be involved in developmental or tissue-specific regulation of gene expression.

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.

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.

Transient expression of SorCS in developing telencephalic and mesencephalic structures of the mouse.

Here we describe the expression of a third member of the VPS10 domain containing receptor family, SorCS, during mouse embryonal and early postnatal nervous system development. SorCS is expressed in a unique transient and dynamic pattern in regions where cells proliferate, as well as in areas where already differentiated cells reside, including the cerebral cortex, the ventral tegmental area, and the globus pallidus. Transcripts were absent from fiber tracts hinting at a neuronal expression. The only exception was hybridization signals on the developing optic nerve correlating with the appearance of astrocytes migrating into the retina.

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.

Gpr85, a novel member of the G-protein coupled receptor family, prominently expressed in the developing mouse cerebral cortex.

The G-protein coupled receptors (GPCRs) characterized by seven transmembrane domains represent the largest receptor superfamily to date and are implied in diverse cell signaling events, its members being present in a diversity of organs and tissues. Here we report the expression of Gpr85, a novel member of this gene family during mouse embryonal development and in the adult brain. Transcripts of Gpr85 were detected predominantly in tissues of neuroectodermal origin. In the central nervous system Gpr85 was expressed during phases of early neuronal differentiation. Highest transcript levels were observed in the developing cerebral cortex, pointing to a specific function of this gene for differentiation processes in the cerebral cortex. In addition, expression was also detected in derivatives of the neural crest and developing teeth.

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.

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.

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.

Alternative splicing and expression of the mouse estrogen receptor-related receptor gamma.

Estrogen receptor-related receptors (ERRs) are orphan members of the nuclear receptor superfamily, closely related to the estrogen receptor. With a PCR-based cloning strategy we have identified two cDNA isoforms from a neonatal mouse whole brain cDNA library encoding ERRgamma. We show that alternative splicing gives rise to different 5'-ends. The predicted peptide sequence of ERRgamma2 differs from ERRgamma1 by additional 23 N-terminal residues. The presence of identical isoforms in human suggests a pivotal function of ERRgamma in mammalia. Northern blot analysis reveals that ERRgamma is expressed as early as day 11 of embryonic development (E11). Whole mount in situ hybridization analysis shows major expression of ERRgamma in the central nervous system at E12.5. In the adult mouse a 5.7 kb transcript is detected in heart, brain, kidney, and skeletal muscle. Therefore, ERRgamma may be involved in the differentiation, as well as the maintenance of the differentiated properties in the brain.

Identification and characterization of SorCS, a third member of a novel receptor family.

A novel receptor, SorCS, was isolated from murine brain. It shows homology to the mosaic receptor SorLA and the neurotensin receptor sortilin based on a common VPS10 domain which is the hallmark of this new receptor family. In the N-terminus of SorCS two putative cleavage sites for the convertase furin mark the beginning of the VPS10 domain, followed by a module of imperfect leucine-rich repeats and a transmembrane domain. The short intracellular C-terminus contains consensus signals for rapid internalization. The identified putative binding motifs for SH2 and SH3 domains are unique in the family of VPS10 domain receptors. SorCS is predominantly expressed in brain, but also in heart, liver, and kidney. SorCS transcripts detected by in situ hybridization in the murine central nervous system point to a neuronal expression.

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.

Identification of a novel seven-transmembrane receptor with homology to glycoprotein receptors and its expression in the adult and developing mouse.

Using a PCR-based cloning strategy we have isolated a cDNA from mouse brain and named it fex, because it codes for a novel putative G protein-coupled receptor expressed in follicles. The deduced amino acid sequence shows a higher degree of homology to the family of glycoprotein receptors, namely those for FSH, LH, and TSH, than to other G protein-coupled receptors. With 18 leucine-rich repeats FEX exhibits features in its N-terminal portion characterizing it as unique within the glycoprotein receptor family. In the adult mouse fex expression was detected in the male and female gonads, the adrenal medulla, and the olfactory bulb of the brain. During embryonic development fex transcripts were detected transiently in various tissues, particularly in selected regions of the central nervous system, the developing face, the intervertebral discs anlagen, and the limb buds. Because fex was expressed during periods of active morphogenesis, it may be an important receptor for signals controlling growth and differentiation of specific embryonic tissues.

Identification of shyc, a novel gene expressed in the murine developing and adult nervous system.

The embryonal carcinoma cell line P19 responds to treatment with retinoid acid by differentiation into neuronal cell types [2]. Using radioactively labeled cDNA derived from differentiating P19 cells we screened an adult mouse brain cDNA library and isolated a gene named shyc for selective hybridizing clone. The encoded protein did not reveal homology to any known protein. We used in situ hybridization on mouse embryonic and adult brain sections to study shyc expression. The developing and embryonic nervous system showed the most prominent hybridization signals. In the adult brain the olfactory pathway was marked by shyc expression.