Low density lipoprotein receptor-related protein 5 (LRP5) mutations and osteoporosis, impaired glucose metabolism and hypercholesterolaemia.

OBJECTIVE: Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) underlie osteoporosis-pseudoglioma syndrome. Animal models implicate a role for LRP5 in lipid and glucose homeostasis. The objective was to evaluate metabolic consequences of LRP5 mutations in humans. DESIGN AND PATIENTS: Thirteen Finnish individuals with homozygous or heterozygous LRP5 mutations were assessed for bone health, glucose and lipid metabolism, and for serum serotonin concentration. Results were compared with findings in family members without mutations. MEASUREMENTS: Bone mineral density (BMD), vertebral morphology, oral and intravenous glucose tolerance tests, lipid profile and serum serotonin concentrations. RESULTS: Two individuals were homozygous for R570W, one compound heterozygous for R570W and R1036Q, and 10 were heterozygous (six for R570W, three for R1036Q and one for R925C). Subjects with two LRP5 mutations had multiple spinal fractures and low BMD. Subjects with one mutation had significantly lower median lumbar spine (P = 0.004) and femoral neck (P = 0.005) BMD Z-scores, and more often vertebral fractures than the 18 individuals without mutations. Of the 12 subjects with LRP5 mutation six had diabetes and one had impaired glucose tolerance. Intravenous glucose tolerance tests suggested impaired beta-cell function; no insulin resistance was observed. Prevalence of hypercholesterolaemia was similar in mutation positive and negative subjects. Serum serotonin concentrations showed a trend towards higher concentrations in subjects with LRP5 mutation. CONCLUSIONS: We found high prevalence of osteoporosis and abnormal glucose metabolism in subjects with LRP5 mutation(s). Further studies are needed to establish the role of LRP5 in glucose and lipid metabolism.

Physical performance of individuals with intellectual disability: a 30 year follow up.

Physical performance of Finnish adolescents (33 females, 44 males) with moderate intellectual disability (ID) was studied over a 30 year period. This study is an extension of Lahtinen's previous work on documenting the performance of individuals with intellectual disabilities over time. This study consisted of analyzing data from a total of four data collection periods (1973, 1979, 1996 and 2003 in which participants ranged in age from 11-16, 17-22, 34-39 and 41-46 years old, respectively. Improvement from early to late adolescence, and decline during adulthood in abdominal strength/endurance, static balance, and manual dexterity were identified. The male adults with ID were moderately overweight (BMI), but the females with ID were obese. The IQ effect was significant on balance and manual dexterity. The gender differences in adulthood were significant, but differences were not noted for DS when controlling for IQ.

The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter.

The late-infantile-onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile-onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide-polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted "MGC33302"), which encodes a novel polytopic 518-amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs.

Functional biology of the neuronal ceroid lipofuscinoses (NCL) proteins.

Neuronal ceroid lipofucinoses (NCLs) are a group of severe neurodegenerative disorders characterized by accumulation of autofluorescent ceroid lipopigment in patients' cells. The different forms of NCL share many similar pathological features but result from mutations in different genes. The genes affected in NCLs encode both soluble and transmembrane proteins and are localized to ER or to the endosomes/lysosomes. Due to selective vulnerability of the central nervous system in the NCL disorders, the corresponding proteins are proposed to have important, tissue specific roles in the brain. The pathological similarities of the different NCLs have led not only to the grouping of these disorders but also to suggestion that the NCL proteins function in the same biological pathway. Despite extensive research, including the development of several model organisms for NCLs and establishment of high-throughput techniques, the precise biological function of many of the NCL proteins has remained elusive. The aim of this review is to summarize the current knowledge of the functions, or proposed functions, of the different NCL proteins.

Oligomerization and interacellular localization of the glycoprotein receptor ERGIC-53 is independent of disulfide bonds.

ERGIC-53 is a type I transmembrane lectin facilitating the efficient export of a subset of secretory glycoproteins from the endoplasmic reticulum. Previous results have shown that ERGIC-53 is present as reduction-sensitive homo-oligomers, i.e. as a balanced mixture of disulfide-linked hexamers and dimers, with the two cysteine residues located close to the transmembrane domain playing a crucial role in oligomerization. Here, we demonstrate, using sucrose gradient sedimentation, cross-linking analyses, and non-denaturing gel electrophoresis, that ERGIC-53 is present exclusively as a hexameric complex in cells. However, the hexamers exist in two forms, one as a disulfide-linked, Triton X-100, perfluoro-octanic acid, and SDS-resistant complex, and the other as a non-covalent, Triton X-100, perfluoro-octanoic acid-resistant, but SDS-sensitive, complex made up of three disulfide-linked dimers that are likely to interact through the coiled-coil domains present in the luminal part of the protein. In contrast to what was previously believed, neither of the membrane-proximal cysteine residues plays an essential role in the formation, or maintenance, of the latter form of hexamers. Subcellular fractionation revealed that the double-cysteine mutant was present in the endoplasmic reticulum-Golgi-intermediate compartment, indicating that the two cysteine residues are not essential for the intracellular distribution of ERGIC-53. Based on these results, we present a model for the formation of the two hexameric forms.

Mass spectrometric analysis reveals changes in phospholipid, neutral sphingolipid and sulfatide molecular species in progressive epilepsy with mental retardation, EPMR, brain: a case study.

Progressive epilepsy with mental retardation, EPMR, belongs to a group of inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The CLN8 gene that underlies EPMR encodes a novel transmembrane protein that localizes to the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment. Recently, CLN8 was linked to a large eukaryotic protein family of TLC (TRAM, Lag1, CLN8) domain homologues with postulated functions in lipid synthesis, transport or sensing. By using liquid chromatography/mass spectrometry we analysed molecular species of major phosholipid and simple sphingolipid classes from cerebral samples of two EPMR patients representing a progressive and advanced state of the disease. The progressive state brain showed reduced levels of ceramide, galactosyl- and lactosylceramide and sulfatide as well as a decrease in long fatty acyl chain containing molecular species within these classes. Among glycerophospholipid classes, an increase in species containing polyunsaturated acyl chains was detected especially in phosphatidylserines and phosphatidylethanolamines. By contrast, saturated and monounsaturated species were overrepresented among phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol classes in the advanced state sample. The observed changes in brain sphingo- and phospholipid molecular profiles may result in altered membrane stability, lipid peroxidation, vesicular trafficking or neurotransmission and thus may contribute to the progression of the molecular pathogenesis of EPMR.

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase.

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations.

Loss-of-function mutations in the cystatin B (CSTB), a cysteine protease inhibitor, gene underlie progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1), characterized by myoclonic and tonic-clonic seizures, ataxia and a progressive course. A minisatellite repeat expansion in the promoter region of the CSTB gene is the most common mutation in EPM1 patients and leads to reduced mRNA levels. Seven other mutations altering the structure of CSTB, or predicting altered splicing, have been described. Using a novel monoclonal CSTB antibody and organelle-specific markers in human primary myoblasts, we show here that endogenous CSTB localizes not only to the nucleus and cytoplasm but also associates with lysosomes. Upon differentiation to myotubes, CSTB becomes excluded from the nucleus and lysosomes, suggesting that the subcellular distribution of CSTB is dependent on the differentiation status of the cell. Four patient mutations altering the CSTB polypeptide were transiently expressed in BHK-21 cells. The p.Lys73fsX2-truncated mutant protein shows diffuse cytoplasmic and nuclear distribution, whereas p.Arg68X is rapidly degraded. Two missense mutations, the previously described p.Gly4Arg affecting the highly conserved glycine, critical for cathepsin binding, and a novel mutation, p.Gln71Pro, fail to associate with lysosomes. These data imply an important lysosome-associated physiological function for CSTB and suggest that loss of this association contributes to the molecular pathogenesis of EPM1.

Cystatin-B is expressed by neural stem cells and by differentiated neurons and astrocytes.

Mutation in the gene encoding cystatin-B (CSTB) has been shown to cause progressive myoclonus epilepsy. Mice with a gene deletion of CSTB exhibit increased apoptosis of specific neurons but the physiological role of CSTB in brain cells is not fully understood. In the present study, we have examined the expression of CSTB in neural stem cells (NSC) and in differentiating mature brain cells. The results show that CSTB is present in embryonic and adult NSC and in the neuroepithelium. CSTB was expressed by both neurons and glial cells differentiated from NSC and in hippocampal cultures. CSTB localized mainly to the nucleus in NSC and in neurons, whilst in astrocytes CSTB was also in the cytoplasm. Double labeling showed that CSTB was present in the lysosomes in glial cells. The results demonstrate a nuclear expression of CSTB in NSC and in neurons, suggesting novel function for this molecule.

Involvement of caveolin-2 in caveolar biogenesis in MDCK cells.

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.

Expression, purification, refolding and crystallization of the carbohydrate-recognition domain of p58/ERGIC-53, an animal C-type lectin involved in export of glycoproteins from the endoplasmic reticulum.

p58/ERGIC-53 is a mammalian calcium-dependent lectin that serves as a glycoprotein-sorting receptor between the endoplasmic reticulum (ER) and the Golgi complex. It is a type I transmembrane protein with two lumenal domains, one of which is a carbohydrate-recognition domain (CRD) and homologous to leguminous lectins. The CRD of p58, the rat homologue of human ERGIC-53, was overexpressed in insect cells and Escherichia coli, purified and crystallized using Li(2)SO(4) as a precipitant. The crystals belong to space group I222, with unit-cell parameters a = 49.6, b = 86.1, c = 128.1 A, and contain one molecule per asymmetric unit, corresponding to a packing density of 2.4 A(3)Da(-1). Knowledge of the structure of p58/ERGIC-53 will provide a starting model for understanding receptor-mediated glycoprotein sorting between the ER and the Golgi.