RESUMEN
Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7ß1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.
Asunto(s)
Proteínas de la Membrana , Investigación Biomédica Traslacional , Animales , Humanos , Ratones , Distrofina/metabolismo , Distrofina/inmunología , Distrofina/genética , Integrinas/metabolismo , Integrinas/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Duchenne/inmunología , Distrofia Muscular de Duchenne/metabolismoRESUMEN
BACKGROUND: The dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdxTG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression. METHODS: The tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdxTG mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the ingenuity pathway analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators. RESULTS: Through our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdxTG muscle, including those that were (1) unrestored (significantly different from wild type, but not from mdx), (2) restored (significantly different from mdx, but not from wild type), and (3) compensatory (significantly different from both wild type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM-optimized proteomics revealed an increased abundance of collagens II, V, and XI, along with ß-spectrin in mdxTG samples. Using ingenuity pathway analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Yap1, Sox9, Rho, RAC, and Wnt. CONCLUSIONS: Our findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Ratones , Animales , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mecanotransducción Celular , Multiómica , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Citoesqueleto/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismoRESUMEN
In Duchenne muscular dystrophy (DMD), mutations in dystrophin result in a loss of the dystrophin-glycoprotein complex (DGC) at the myofiber membrane, which functions to connect the extracellular matrix with the intracellular actin cytoskeleton. The dystroglycan subcomplex interacts with dystrophin and spans the sarcolemma where its extensive carbohydrates (matriglycan and CT2 glycan) directly interact with the extracellular matrix. In the current manuscript, we show that sarcospan overexpression enhances the laminin-binding capacity of dystroglycan in DMD muscle by increasing matriglycan glycosylation of α-dystroglycan. Furthermore, we find that this modification is not affected by loss of Galgt2, a glycotransferase, which catalyzes the CT2 glycan. Our findings reveal that the matriglycan carbohydrates, and not the CT2 glycan, are necessary for sarcospan-mediated amelioration of DMD. Overexpression of Galgt2 in the DMD mdx murine model prevents muscle pathology by increasing CT2 modified α-dystroglycan. Galgt2 also increases expression of utrophin, which compensates for the loss of dystrophin in DMD muscle. We found that combined loss of Galgt2 and dystrophin reduced utrophin expression; however, it did not interfere with sarcospan rescue of disease. These data reveal a partial dependence of sarcospan on Galgt2 for utrophin upregulation. In addition, sarcospan alters the cross-talk between the adhesion complexes by decreasing the association of integrin ß1D with dystroglycan complexes. In conclusion, sarcospan functions to re-wire the cell to matrix connections by strengthening the cellular adhesion and signaling, which, in turn, increases the resilience of the myofiber membrane.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Animales , Carbohidratos , Distroglicanos/genética , Distroglicanos/metabolismo , Distrofina/genética , Distrofina/metabolismo , Laminina/genética , Laminina/metabolismo , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Utrofina/genética , Utrofina/metabolismoRESUMEN
Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a rare fatal autosomal dominant vasculopathy associated with mutations in the TREX1 gene. Only one de novo case has been reported in the literature. We report the long-term clinical, radiological, and pathological presentation of a patient with a de novo and novel mutation in this gene. Description of the clinical, genetic, imaging and pathologic characteristics is important to better characterize RVCL-S and prevent unnecessary interventions. RVCL-S should be considered in patients with tumefactive brain lesions unresponsive to immunotherapy.
Asunto(s)
Enfermedades Desmielinizantes , Leucoencefalopatías , Enfermedades Vasculares , Exodesoxirribonucleasas , Humanos , Mutación , FosfoproteínasRESUMEN
Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.
Asunto(s)
Enfermedades Cerebelosas/genética , Predisposición Genética a la Enfermedad/genética , Proteínas Mitocondriales/genética , Mutación/genética , Proteínas de Transporte de Fosfato/genética , Polimorfismo de Nucleótido Simple/genética , Aminoácidos/genética , Animales , Animales Modificados Genéticamente , Encéfalo/anomalías , Línea Celular Transformada , Células Cultivadas , Enfermedades Cerebelosas/diagnóstico por imagen , Estudios de Cohortes , Embrión no Mamífero , Femenino , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Dinámicas Mitocondriales/genética , Modelos Moleculares , Pez CebraRESUMEN
RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.
Asunto(s)
Exosomas , Neuronas Motoras , Degeneración Nerviosa/genética , Atrofias Olivopontocerebelosas/genética , Puente/patología , Proteínas de Unión al ARN/genética , Nervios Espinales , Animales , Cerebelo/patología , Complejo Multienzimático de Ribonucleasas del Exosoma , Técnicas de Silenciamiento del Gen , Humanos , Degeneración Nerviosa/patología , Atrofias Olivopontocerebelosas/patología , ARN/análisis , Nervios Espinales/patología , Pez Cebra/embriologíaRESUMEN
Episodic ataxia (EA) syndromes are heritable diseases characterized by dramatic episodes of imbalance and incoordination. EA type 2 (EA2), the most common and the best characterized subtype, is caused by mostly nonsense, splice site, small indel, and sometimes missense mutations in CACNA1A. Direct sequencing of CACNA1A fails to identify mutations in some patients with EA2-like features, possibly due to incomplete interrogation of CACNA1A or defects in other EA genes not yet defined. Previous reports described genomic deletions between 4 and 40 kb in EA2. In 47 subjects with EA (26 with EA2-like features) who tested negative for mutations in the known EA genes, we used multiplex ligation-dependent probe amplification to analyze CACNA1A for exonic copy number variations. Breakpoints were further defined by long-range PCR. We identified distinct multi-exonic deletions in three probands with classic EA2-like features: episodes of prolonged vertigo and ataxia triggered by stress and fatigue, interictal nystagmus, with onset during infancy or early childhood. The breakpoints in all three probands are located in Alu sequences, indicating errors in homologous recombination of Alu sequences as the underlying mechanism. The smallest deletion spanned exons 39 and 40, while the largest deletion spanned 200 kb, missing all but the first three exons. One deletion involving exons 39 through 47 arose spontaneously. The search for mutations in CACNA1A appears most fruitful in EA patients with interictal nystagmus and onset early in life. The finding of large heterozygous deletions suggests haploinsufficiency as a possible pathomechanism of EA2.
RESUMEN
The Na(+)/K(+)-ATPases are ion pumps of fundamental importance in maintaining the electrochemical gradient essential for neuronal survival and function. Mutations in ATP1A3 encoding the alpha3 isoform cause rapid-onset dystonia-parkinsonism (RDP). We report a de novo ATP1A3 mutation in a patient with typical RDP, consisting of an in-frame insertion of a tyrosine residue at the very C terminus of the Na(+)/K(+)-ATPase alpha3-subunit-the first reported RDP mutation in the C terminus of the protein. Expression studies revealed that there is no defect in the biogenesis or plasma membrane targeting, although cells expressing the mutant protein showed decreased survival in response to ouabain challenge. Functional analysis demonstrated a drastic reduction in Na(+) affinity in the mutant, which can be understood by structural modelling of the E1 and E2 conformations of the wild-type and mutant enzymes on the basis of the strategic location of the C terminus in relation to the third Na(+) binding site. The dramatic clinical presentation, together with the biochemical findings, provides both in vivo and in vitro evidence for a crucial role of the C terminus of the alpha-subunit in the function of the Na(+)/K(+)-ATPase and a key impact of Na(+) affinity in the pathophysiology of RDP.
Asunto(s)
Distonía/fisiopatología , Mutagénesis Insercional , Trastornos Parkinsonianos/fisiopatología , ATPasa Intercambiadora de Sodio-Potasio/genética , Sodio/metabolismo , Adolescente , Edad de Inicio , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Supervivencia Celular , Distonía/genética , Distonía/metabolismo , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Linaje , Unión Proteica , Conformación Proteica , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
BACKGROUND: Episodic ataxia (EA) is variably associated with additional neurologic symptoms. At least 4 genes have been implicated. Recently, a mutation in the SLC1A3 gene encoding the glutamate transporter EAAT1 was identified in a patient with severe episodic and progressive ataxia, seizures, alternating hemiplegia, and migraine headache. The mutant EAAT1 showed severely reduced uptake of glutamate. The syndrome was designated EA6 and shares overlapping clinical features with EA2, which is caused by mutations in CACNA1A. OBJECTIVE: To test the role of the SLC1A3 gene in EA. DESIGN: Genetic and functional studies. We analyzed the coding region of the SLC1A3 gene by direct sequencing. SETTING: Academic research. PATIENTS: DNA samples from 20 patients with EA (with or without interictal nystagmus) negative for CACNA1A mutations were analyzed. MAIN OUTCOME MEASURES: We identified 1 novel EAAT1 mutation in a family with EA and studied the functional consequences of this mutation using glutamate uptake assay. RESULTS: We identified a missense C186S mutation that segregated with EA in 3 family members. The mutant EAAT1 showed a modest but significant reduction of glutamate uptake. CONCLUSIONS: We broadened the clinical spectrum associated with SLC1A3 mutations to include milder manifestations of EA without seizures or alternating hemiplegia. The severity of EA6 symptoms appears to be correlated with the extent of glutamate transporter dysfunction.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/genética , Predisposición Genética a la Enfermedad/genética , Ácido Glutámico/metabolismo , Mutación/genética , Degeneraciones Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/metabolismo , Adulto , Química Encefálica/genética , Cerebelo/metabolismo , Cerebelo/fisiopatología , Análisis Mutacional de ADN , Femenino , Marcadores Genéticos/genética , Pruebas Genéticas , Genotipo , Hemiplejía/genética , Hemiplejía/fisiopatología , Humanos , Patrón de Herencia/genética , Masculino , Persona de Mediana Edad , Trastornos Migrañosos/genética , Trastornos Migrañosos/fisiopatología , Linaje , Fenotipo , Convulsiones/genética , Convulsiones/fisiopatología , Degeneraciones Espinocerebelosas/fisiopatologíaRESUMEN
Episodic ataxia type 1 (EA1) is an autosomal-dominant neurological disease caused by point mutations in the potassium channel-encoding gene KCNA1. It is characterized by attacks of ataxia and continuous myokymia. Respiratory muscle involvement has not been previously reported in EA1. We clinically evaluated a family with features of EA1 and paroxysmal shortness of breath. Coding and flanking intronic regions of KCNA1 were sequenced. We identified a novel 3-nucleotide deletion mutation in KCNA1 in the affected individuals. Our findings of a deletion mutation with unusual respiratory muscle involvement expand the genetic and clinical spectrum of EA1.
Asunto(s)
Ataxia/genética , Disnea Paroxística/genética , Predisposición Genética a la Enfermedad , Canal de Potasio Kv.1.1/genética , Mutación/genética , Adolescente , Adulto , Ataxia/complicaciones , Disnea Paroxística/complicaciones , Salud de la Familia , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.
Asunto(s)
Encefalopatías/genética , Exodesoxirribonucleasas/genética , Mutación , Fosfoproteínas/genética , Enfermedades de la Retina/genética , Secuencia de Aminoácidos , Encefalopatías/enzimología , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Genes Dominantes , Predisposición Genética a la Enfermedad , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Enfermedades de la Retina/enzimología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Benign recurrent vertigo (BRV) is a common disorder affecting up to 2% of the adult population and may be etiologically related to migraine because of similarities in the clinical spectrum of the phenotypes and a high co-morbidity within families. Many families have multiple-affected genetically related individuals suggesting familial transmission of the disorder with moderate to high penetrance. While clinically similar to episodic ataxias, there are currently no genes identified that contribute to BRV and no systematic linkage studies performed. In an initial effort to genetically define BRV, we have selected from our Neurology Clinic population a subset of 20 multigenerational families with apparent autosomal dominant transmission, and performed genetic linkage mapping using both parametric and non-parametric linkage (NPL) approaches. The Affymetrix 10K SNP Mapping Assay was used for the genotyping. Heterogeneity LOD (HLOD) analysis reveals the evidence of genetic heterogeneity for BRV and evidence of linkage in a subset of the families to 22q12 (HLOD = 4.02). An additional region was identified by NPL analysis at 5p15 (LOD = 2.63). As migraine is observed substantially more commonly both within the BRV-affected individuals and the related family members, it is possible that a form of migraine is allelic to the BRV locus at 22q12. However, testing linkage or the chromosome 22q12 region to a broader migraine/vertigo phenotype by defining affectation status as either migrainous headaches or BRV greatly weakened the linkage signal, and no significant other peaks were detected. Thus, BRV and migraine does not appear to be allelic disorders within these families. We conclude that BRV is a heterogeneous genetic disorder, appears genetically distinct from migraine with aura and is linked to 22q12. Additional family and population-based linkage and association studies will be needed to determine the causative alleles.