ABSTRACT
Proximal spinal muscular atrophy (SMA) is a common motor neuron disorder caused by mutation of the telomeric survival of motor neuron gene SMN1. The centromeric survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. The SMN1 and SMN2 genes differ functionally by a single nucleotide change. This change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. Thus, SMN2 produces less full-length mRNA and protein than SMN1. We have screened a library of compounds in order to identify ones that can alter the splicing pattern of the SMN2 gene. Here, we report that the compound aclarubicin increases the retention of exon 7 into the SMN2 transcript. We show that aclarubicin effectively induces incorporation of exon 7 into SMN2 transcripts from the endogenous gene in type I SMA fibroblasts as well as into transcripts from a SMN2 minigene in the motor neuron cell line NSC34. In type I fibroblasts, treatment resulted in an increase in SMN protein and gems to normal levels. Our results suggest that alteration of splicing pattern represents a new approach to modification of gene expression in disease treatment and demonstrate the feasibility of high throughput screens to detect compounds that affect the splicing pattern of a gene.
Subject(s)
Aclarubicin/pharmacology , Nerve Tissue Proteins/physiology , Spinal Muscular Atrophies of Childhood/drug therapy , Alternative Splicing/drug effects , Animals , Blotting, Western , Cell Line , Cyclic AMP Response Element-Binding Protein , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Exons , Feasibility Studies , Fibroblasts , Humans , Immunohistochemistry , Mice , Motor Neurons/drug effects , Motor Neurons/metabolism , Nerve Tissue Proteins/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , SMN Complex Proteins , Spinal Muscular Atrophies of Childhood/genetics , Survival of Motor Neuron 1 Protein , Survival of Motor Neuron 2 Protein , Transcription, Genetic/drug effects , TransfectionABSTRACT
Anticardiolipin antibodies (aCL) of the immunoglobulin (Ig) G isotype have been significantly associated with neurological manifestations of antiphospholipid syndrome (APS). In a previous study we described the direct pathogenic effects of IgG aCL on living neurons in culture. Therefore, we studied the IgG aCL titre as a factor influencing the extent of this effect. Seventeen patients with a history of primary antiphospholipid syndrome were grouped according to their IgG aCL titre into low positive (GPL < or = 40), high positive (40< GPL <100) and very high positive (GPL >100). IgG from these patients were incubated with cerebellar neurons in primary culture for 24h and the effect was evaluated by using the tetrazolium salt (MTT) assay. We found that almost all patients' IgGs reduced cell viability in vitro, but the differences in the extent of the effect were statistically significant only for patients with >40 GPL. Our results reinforce the causal association between increasing level of IgG aCL and clinical features of aPS.
Subject(s)
Antibodies, Anticardiolipin/analysis , Antiphospholipid Syndrome/blood , Central Nervous System Diseases/immunology , Immunoglobulin G/blood , Adult , Analysis of Variance , Biomarkers/analysis , Case-Control Studies , Cell Survival/immunology , Cell Survival/physiology , Cells, Cultured , Central Nervous System Diseases/diagnosis , Female , Humans , Male , Middle Aged , Neurons , Probability , Reference Values , Sampling Studies , Sensitivity and Specificity , Severity of Illness IndexABSTRACT
Excitatory amino acids which promote the survival of cerebellar granule cells in culture, also promote the expression of the survival of motor neuron (SMN) protein. Immunolocalization studies using SMN monoclonal antibody showed that SMN is decreased in cultures grown in low K+ or chemically defined medium with respect to cultures grown in high K+ medium and that an increase of SMN can be induced by treatment of low K+ cultures with glutamate or N-methyl-D-aspartate.
Subject(s)
Cerebellum/metabolism , Excitatory Amino Acids/metabolism , Nerve Tissue Proteins/biosynthesis , Animals , Cell Survival , Cells, Cultured , Cerebellum/drug effects , Culture Media , Cyclic AMP Response Element-Binding Protein , Fluorescent Antibody Technique , Glutamic Acid/pharmacology , Humans , Microscopy, Fluorescence , N-Methylaspartate/pharmacology , Neurons/metabolism , Potassium/pharmacology , RNA-Binding Proteins , Rats , SMN Complex Proteins , Survival of Motor Neuron 1 ProteinABSTRACT
Proximal spinal muscular atrophy (SMA) is a common motor neuron disease in humans and in its most severe form causes death by the age of 2 years. It is caused by defects in the telomeric survival motor neuron gene ( SMN1 ), but patients retain at least one copy of a highly homologous gene, centromeric SMN ( SMN2 ). Mice possess only one survival motor neuron gene ( Smn ) whose loss is embryonic lethal. Therefore, to obtain a mouse model of SMA we created transgenic mice that express human SMN2 and mated these onto the null Smn (-/-)background. We show that Smn (-/-); SMN2 mice carrying one or two copies of the transgene have normal numbers of motor neurons at birth, but vastly reduced numbers by postnatal day 5, and subsequently die. This closely resembles a severe type I SMA phenotype in humans and is the first report of an animal model of the disease. Eight copies of the transgene rescues this phenotype in the mice indicating that phenotypic severity can be modulated by SMN2 copy number. These results show that SMA is caused by insufficient SMN production by the SMN2 gene and that increased expression of the SMN2 gene may provide a strategy for treating SMA patients.
Subject(s)
Muscular Atrophy, Spinal/genetics , Nerve Tissue Proteins/genetics , Animals , Animals, Newborn , Blotting, Northern , Blotting, Western , Brain Stem/metabolism , Brain Stem/pathology , Centromere , Cyclic AMP Response Element-Binding Protein , Disease Models, Animal , Exons , Gene Dosage , Genotype , Humans , In Vitro Techniques , Mice , Mice, Transgenic , Microinjections , Motor Neurons/pathology , Muscular Atrophy, Spinal/mortality , Muscular Atrophy, Spinal/pathology , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , SMN Complex Proteins , Spinal Cord/metabolism , Spinal Cord/pathology , Survival of Motor Neuron 1 Protein , Survival of Motor Neuron 2 ProteinABSTRACT
Tetragonal zirconia polycrystal (TZP) is a new interesting ceramic for the manufacture of medical devices. Its wide use in orthopedic and odontoiatric implants was limited till now by the high chemical and radiochemical impurities of the raw materials. Purification processes now available allow to obtain high purity ceramic grade powders suitable for TZP ceramics manufacture, even if their possible mutagenic and transforming effects are still unclear. The aim of this work is to study in vitro the mutagenic and oncogenic effects of a new zirconia ceramic stabilized by yttria (Y-TZP). This ceramic was sintered from high purity powders obtained by a process developed under a project carried out within the Brite EuRam programme. For comparison, ceramics made from unpurified zirconia powder were also tested. Fibroblasts irradiated by a linear accelerator were used as positive control. The results obtained show that Y-TZP ceramic does not elicit either mutagenic or transforming effect on C3H/10T(1/2) (10T(1/2)) cells and demonstrate that ceramic from high purity powders can be considered suitable for biomedical applications from the point of view of the effects of its radioactive impurity content.
Subject(s)
Biocompatible Materials/toxicity , Carcinogens/toxicity , Cell Transformation, Neoplastic/drug effects , Ceramics/toxicity , Mutagens/toxicity , Zirconium/toxicity , Animals , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Division/drug effects , Cell Division/physiology , Cell Line , Dental Materials/toxicity , Embryo, Mammalian/cytology , Mice , Mutagenicity TestsABSTRACT
IgGs from sera containing antiphospholipid antibodies (aPL), detected as antibodies to cardiolipin, or control sera were incubated with rat cerebellar granule cells in primary culture. Using a mitochondrial dehydrogenase activity assay (MTT test), aPL IgGs were shown to decrease MTT metabolism after 24 h incubation with the cells, and to cause non-toxic amounts of glutamate to become neurotoxic when added to the cells for 45 min. Acute and chronic aPL toxicity were prevented by MK-801. Sera containing aPL bound to intact cerebellar neurons, as revealed by an immunofluorescent technique. These results suggest that antiphospholipid antibodies interfere with excitatory pathways in glutamatergic cerebellar granule cells by a mechanism involving overactivation of the NMDA glutamate receptor.
