RESUMEN
Nonsense mutations, which occur in â¼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4-C1, and TRPC5-C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8-18 µM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake.
Asunto(s)
Aminoglicósidos , Codón sin Sentido , Indazoles , Canales Catiónicos TRPC , Aminoglicósidos/farmacología , Codón sin Sentido/efectos de los fármacos , Humanos , Indazoles/farmacología , Inhibidores de la Síntesis de la Proteína , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
Dysfunction and degeneration of synapses is a common feature of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene is the main genetic cause of ALS/FTD (C9ALS/FTD). The repeat expansion leads to reduced expression of the C9orf72 protein. How C9orf72 haploinsufficiency contributes to disease has not been resolved. Here we identify the synapsin family of synaptic vesicle proteins, the most abundant group of synaptic phosphoproteins, as novel interactors of C9orf72 at synapses and show that C9orf72 plays a cell-autonomous role in the regulation of excitatory synapses. We mapped the interaction of C9orf72 and synapsin to the N-terminal longin domain of C9orf72 and the conserved C domain of synapsin, and show interaction of the endogenous proteins in synapses. Functionally, C9orf72 deficiency reduced the number of excitatory synapses and decreased synapsin levels at remaining synapses in vitro in hippocampal neuron cultures and in vivo in the hippocampal mossy fibre system of C9orf72 knockout mice. Consistent with synaptic dysfunction, electrophysiological recordings identified impaired excitatory neurotransmission and network function in hippocampal neuron cultures with reduced C9orf72 expression, which correlated with a severe depletion of synaptic vesicles from excitatory synapses in the hippocampus of C9orf72 knockout mice. Finally, neuropathological analysis of post-mortem sections of C9ALS/FTD patient hippocampus with C9orf72 haploinsufficiency revealed a marked reduction in synapsin, indicating that disruption of the interaction between C9orf72 and synapsin may contribute to ALS/FTD pathobiology. Thus, our data show that C9orf72 plays a cell-autonomous role in the regulation of neurotransmission at excitatory synapses by interaction with synapsin and modulation of synaptic vesicle pools, and identify a novel role for C9orf72 haploinsufficiency in synaptic dysfunction in C9ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína C9orf72/metabolismo , Demencia Frontotemporal , Sinapsinas/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Ratones , Ratones Noqueados , Sinapsis/patologíaRESUMEN
This study investigated the vasorelaxant effects of schwarzinicine A, an alkaloid recently reported from Ficus schwarzii Koord. Regulation of calcium homeostasis in vascular smooth muscle cells (VSMC) is viewed as one of the main mechanisms for controlling blood pressure. L-type voltage-gated calcium channel (VGCC) blockers are commonly used for controlling hypertension. Recently, the transient receptor potential canonical (TRPC) channels were found in blood vessels of different animal species with evidence of their roles in the regulation of vascular contractility. In this study, we studied the mechanism of actions of schwarzinicine A focusing on its regulation of L-type VGCC and TRPC channels. Schwarzinicine A exhibited the highest vasorelaxant effect (123.1%) compared to other calcium channel blockers. It also overtly attenuated calcium-induced contractions of the rat isolated aortae in a calcium-free environment showing its mechanism to inhibit calcium influx. Fluorometric intracellular calcium recordings confirmed its inhibition of hTRPC3-, hTRPC4-, hTRPC5- and hTRPC6-mediated calcium influx into HEK cells with IC50 values of 3, 17, 19 and 7 µM, respectively. The evidence gathered in this study suggests that schwarzinicine A blocks multiple TRPC channels and L-type VGCC to exert a significant vascular relaxation response.
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Canales de Potencial de Receptor Transitorio , Vasodilatación , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/farmacología , Ratas , Canales de Potencial de Receptor Transitorio/farmacología , Vasodilatadores/farmacologíaRESUMEN
A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc-51-like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a-dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62-positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient-derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD-associated p62 pathology.
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Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Fenómenos Fisiológicos Celulares , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Proteína C9orf72 , Células Cultivadas , Demencia Frontotemporal/patología , Humanos , Neuronas/química , Neuronas/metabolismoRESUMEN
RLIM, also known as RNF12, is an X-linked E3 ubiquitin ligase acting as a negative regulator of LIM-domain containing transcription factors and participates in X-chromosome inactivation (XCI) in mice. We report the genetic and clinical findings of 84 individuals from nine unrelated families, eight of whom who have pathogenic variants in RLIM (RING finger LIM domain-interacting protein). A total of 40 affected males have X-linked intellectual disability (XLID) and variable behavioral anomalies with or without congenital malformations. In contrast, 44 heterozygous female carriers have normal cognition and behavior, but eight showed mild physical features. All RLIM variants identified are missense changes co-segregating with the phenotype and predicted to affect protein function. Eight of the nine altered amino acids are conserved and lie either within a domain essential for binding interacting proteins or in the C-terminal RING finger catalytic domain. In vitro experiments revealed that these amino acid changes in the RLIM RING finger impaired RLIM ubiquitin ligase activity. In vivo experiments in rlim mutant zebrafish showed that wild type RLIM rescued the zebrafish rlim phenotype, whereas the patient-specific missense RLIM variants failed to rescue the phenotype and thus represent likely severe loss-of-function mutations. In summary, we identified a spectrum of RLIM missense variants causing syndromic XLID and affecting the ubiquitin ligase activity of RLIM, suggesting that enzymatic activity of RLIM is required for normal development, cognition and behavior.
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Discapacidad Intelectual Ligada al Cromosoma X/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Adolescente , Adulto , Animales , Niño , Preescolar , Trastorno de la Conducta/genética , Femenino , Genes Ligados a X , Células HEK293 , Humanos , Recién Nacido , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Masculino , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Ratones , Persona de Mediana Edad , Mutación , Linaje , Factores de Transcripción/genética , Ubiquitinación , Inactivación del Cromosoma X , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.
Asunto(s)
Adenosina Desaminasa/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras/metabolismo , Adenosina Desaminasa/fisiología , Adulto , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Astrocitos/metabolismo , Proteína C9orf72/metabolismo , Muerte Celular , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Progresión de la Enfermedad , Metabolismo Energético/fisiología , Femenino , Fibroblastos/metabolismo , Humanos , Inosina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Células Madre/metabolismoRESUMEN
The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/biosíntesis , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/química , Canales de Calcio Tipo T/genética , Femenino , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratas , Ratas WistarRESUMEN
Defective axonal transport is an early neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously shown that ALS-associated mutations in Cu/Zn superoxide dismutase 1 (SOD1) impair axonal transport of mitochondria in motor neurons isolated from SOD1 G93A transgenic mice and in ALS mutant SOD1 transfected cortical neurons, but the underlying mechanisms remained unresolved. The outer mitochondrial membrane protein mitochondrial Rho GTPase 1 (Miro1) is a master regulator of mitochondrial axonal transport in response to cytosolic calcium (Ca2+) levels ([Ca2+]c) and mitochondrial damage. Ca2+ binding to Miro1 halts mitochondrial transport by modifying its interaction with kinesin-1 whereas mitochondrial damage induces Phosphatase and Tensin Homolog (PTEN)-induced Putative Kinase 1 (PINK1) and Parkin-dependent degradation of Miro1 and consequently stops transport. To identify the mechanism underlying impaired axonal transport of mitochondria in mutant SOD1-related ALS we investigated [Ca2+]c and Miro1 levels in ALS mutant SOD1 expressing neurons. We found that expression of ALS mutant SOD1 reduced the level of endogenous Miro1 but did not affect [Ca2+]c. ALS mutant SOD1 induced reductions in Miro1 levels were Parkin dependent. Moreover, both overexpression of Miro1 and ablation of PINK1 rescued the mitochondrial axonal transport deficit in ALS mutant SOD1-expressing cortical and motor neurons. Together these results provide evidence that ALS mutant SOD1 inhibits axonal transport of mitochondria by inducing PINK1/Parkin-dependent Miro1 degradation.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Transporte Axonal/fisiología , Proteínas Mitocondriales/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Animales , Axones/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Células HeLa , Humanos , Ratones Transgénicos , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Mutación , Proteínas Quinasas/metabolismo , RatasRESUMEN
Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.
Asunto(s)
Prueba de Complementación Genética , Variación Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Alelos , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Colesterol/metabolismo , Mapeo Cromosómico , Cricetulus , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Fenotipo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise â¼ 5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such "bivalent" chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.
Asunto(s)
Tipificación del Cuerpo/genética , Drosophila melanogaster/embriología , Discos Imaginales/embriología , Factores de Transcripción/genética , Alas de Animales/embriología , Animales , Animales Modificados Genéticamente , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína/genética , Interferencia de ARN , ARN Polimerasa II/genética , ARN Interferente PequeñoRESUMEN
Imaginal discs, especially the wing imaginal disc, are powerful model systems to study organ development. The traditional methods to analyze wing imaginal discs depend on the laborious and time-consuming dissection of larvae. "Omics"-based approaches, such as RNA-seq, ChIP-seq, proteomics and lipidomics, offer new opportunities for the systems-level investigation of organ development. However, it is impractical to manually isolate the required starting material. This is even more problematic when experiments strive for enhanced temporal and spatial resolution. The mass isolation workflow discussed in this review, solves this problem. The semi-automated sorting of 1000 wing imaginal discs in less than 3h forms the basis of a workflow that can be connected to biochemical analyses of organ patterning and growth. In addition to the mass isolation workflow we briefly describe key "omics" technologies and their applications. The combination of mass isolation and "omics"-approaches ensures that the wing imaginal disc will continue to be a key model organ for studying developmental processes, both on the genetic, but increasingly also on the biochemical level.
Asunto(s)
Biología Evolutiva/métodos , Proteínas de Drosophila/biosíntesis , Discos Imaginales/metabolismo , Alas de Animales/metabolismo , Animales , Movimiento Celular/genética , Drosophila , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Larva , Proteómica/métodos , Alas de Animales/crecimiento & desarrolloRESUMEN
Spinocerebellar ataxia 17 (SCA17) is an autosomal-dominant, late-onset neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat in the TATA-box-binding protein (TBP). To further investigate this devastating disease, we sought to create a first transgenic rat model for SCA17 that carries a full human cDNA fragment of the TBP gene with 64 CAA/CAG repeats (TBPQ64). In line with previous observations in mouse models for SCA17, TBPQ64 rats show a severe neurological phenotype including ataxia, impairment of postural reflexes, and hyperactivity in early stages followed by reduced activity, loss of body weight, and early death. Neuropathologically, the severe phenotype of SCA17 rats was associated with neuronal loss, particularly in the cerebellum. Degeneration of Purkinje, basket, and stellate cells, changes in the morphology of the dendrites, nuclear TBP-positive immunoreactivity, and axonal torpedos were readily found by light and electron microscopy. While some of these changes are well recapitulated in existing mouse models for SCA17, we provide evidence that some crucial characteristics of SCA17 are better mirrored in TBPQ64 rats. Thus, this SCA17 model represents a valuable tool to pursue experimentation and therapeutic approaches that may be difficult or impossible to perform with SCA17 transgenic mice. We show for the first time positron emission tomography (PET) and diffusion tensor imaging (DTI) data of a SCA animal model that replicate recent PET studies in human SCA17 patients. Our results also confirm that DTI are potentially useful correlates of neuropathological changes in TBPQ64 rats and raise hope that DTI imaging could provide a biomarker for SCA17 patients.
Asunto(s)
Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Ataxias Espinocerebelosas , Proteína de Unión a TATA-Box/genética , Expansión de Repetición de Trinucleótido/genética , Animales , Ansiedad/etiología , Ansiedad/genética , Peso Corporal/genética , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Procesamiento Automatizado de Datos , Femenino , Genotipo , Humanos , Masculino , Aprendizaje por Laberinto , Actividad Motora , Examen Neurológico , Tomografía de Emisión de Positrones , Desempeño Psicomotor/fisiología , Racloprida/farmacocinética , Ratas , Ratas Transgénicas , Prueba de Desempeño de Rotación con Aceleración Constante , Índice de Severidad de la Enfermedad , Ataxias Espinocerebelosas/diagnóstico , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/fisiopatología , Tubulina (Proteína)/metabolismoRESUMEN
The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL). We find that naive α2δ-1(-/-) mice show a marked behavioral deficit in mechanical and cold sensitivity, but no change in thermal nociception threshold. The lower mechanical sensitivity is mirrored by a reduced in vivo electrophysiological response of dorsal horn wide dynamic range neurons. The CaV2.2 level is reduced in brain and spinal cord synaptosomes from α2δ-1(-/-) mice, and α2δ-1(-/-) DRG neurons exhibit lower calcium channel current density. Furthermore, a significantly smaller number of DRG neurons respond to the TRPM8 agonist menthol. After PSNL, α2δ-1(-/-) mice show delayed mechanical hypersensitivity, which only develops at 11 d after surgery, whereas in wild-type littermates it is maximal at the earliest time point measured (3 d). There is no compensatory upregulation of α2δ-2 or α2δ-3 after PSNL in α2δ-1(-/-) mice, and other transcripts, including neuropeptide Y and activating transcription factor-3, are upregulated normally. Furthermore, the ability of pregabalin to alleviate mechanical hypersensitivity is lost in PSNL α2δ-1(-/-) mice. Thus, α2δ-1 is essential for rapid development of mechanical hypersensitivity in a nerve injury model of neuropathic pain.
Asunto(s)
Canales de Calcio/genética , Hiperalgesia/genética , Neuralgia/genética , Traumatismos de los Nervios Periféricos/genética , Células Receptoras Sensoriales/fisiología , Animales , Canales de Calcio/metabolismo , Frío , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiopatología , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Ratones , Ratones Noqueados , Neuralgia/metabolismo , Neuralgia/fisiopatología , Umbral del Dolor/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/fisiopatología , Estimulación Física , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatologíaRESUMEN
Conversion of soluble α-synuclein into insoluble and fibrillar inclusions is a hallmark of Parkinson's disease and other synucleinopathies. Accumulating evidence points towards a relationship between its generation at nerve terminals and structural synaptic pathology. Little is known about the pathogenic impact of α-synuclein conversion and deposition at nigrostriatal dopaminergic synapses in transgenic mice, mainly owing to expression limitations of the α-synuclein construct. Here, we explore whether both the rat as a model and expression of the bacterial artificial chromosome construct consisting of human full-length wild-type α-synuclein could exert dopaminergic neuropathological effects. We found that the human promoter induced a pan-neuronal expression, matching the rodent α-synuclein expression pattern, however, with prominent C-terminally truncated fragments. Ageing promoted conversion of both full-length and C-terminally truncated α-synuclein species into insolube and proteinase K-resistant fibres, with strongest accumulation in the striatum, resembling biochemical changes seen in human Parkinson's disease. Transgenic rats develop early changes in novelty-seeking, avoidance and smell before the progressive motor deficit. Importantly, the observed pathological changes were associated with severe loss of the dopaminergic integrity, thus resembling more closely the human pathology.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fenotipo , alfa-Sinucleína/genética , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Neuronas Dopaminérgicas/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , alfa-Sinucleína/biosíntesis , alfa-Sinucleína/toxicidadRESUMEN
Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the alpha1 subunit is associated with two auxiliary subunits, the intracellular beta subunit and the alpha(2)delta subunit; both of these subunits influence the trafficking and properties of Ca(V)1 and Ca(V)2 channels. The alpha(2)delta subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the alpha(2)delta proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to delta rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the alpha(2)delta subunits studied, alpha(2)delta-1, alpha(2)delta-2, and alpha(2)delta-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the alpha(2)delta proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of alpha(2)delta subunits. We also show that GPI anchoring of alpha(2)delta subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when alpha(2)delta subunits are coexpressed. In conclusion, this study redefines our understanding of alpha(2)delta subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.
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Canales de Calcio/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Células COS , Canales de Calcio/química , Canales de Calcio/genética , Canales de Calcio Tipo L , Chlorocebus aethiops , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , RatasRESUMEN
Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Animales , Ratones , Dipéptidos , Proteína C9orf72 , Transporte Activo de Núcleo Celular , Células HEK293 , Péptidos , Neuronas Motoras , ARN , Factores de Empalme Serina-ArgininaRESUMEN
Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.
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Células Endoteliales , Canales Iónicos , Ratones , Animales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Células Endoteliales/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Mecanotransducción Celular , Uniones Intercelulares/metabolismo , Endotelio/metabolismoRESUMEN
BACKGROUND: In spring 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico. During the following weeks the virus spread worldwide, prompting the World Health Organization to declare the first influenza pandemic of the 21st century. Sustained human-to-human transmission and severe disease progression observed in some patients urged public health authorities to respond rapidly to the disease outbreak and vaccine manufacturers to develop pandemic influenza vaccines for mass distribution. With the onset of the pandemic we began to explore the potential of academic/industrial collaboration to accelerate the production of vaccines during an outbreak of an emerging virus by combining the use of an academic BSL-4 laboratory with the expertise of a commercial vaccine manufacturer. METHODS AND RESULTS: To obtain virus seed stocks used for the production of a vaccine to combat the pandemic H1N1 2009 influenza virus (H1N1pdm), we followed various strategies: (i) optimization of cell culture conditions for growth of wild-type H1N1pdm isolates; (ii) classical reassortment of H1N1pdm and standard influenza vaccine donor strain PR8; and (iii) generation of corresponding reassortant viruses using reverse genetics. To ensure a rapid transition to production, the entire potential seed stock development process was carried out in a certified canine kidney suspension cell line (MDCK 33016-PF) under Good Manufacturing Practice (GMP) conditions. CONCLUSIONS: The outcome of this study indicates that a combination of different experimental strategies is the best way to cope with the need to develop vaccines rapidly in the midst of an emerging pandemic.
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Brotes de Enfermedades/prevención & control , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/síntesis química , Vacunas contra la Influenza/provisión & distribución , Gripe Humana/prevención & control , Animales , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Perros , Industria Farmacéutica , Humanos , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Gripe Humana/virología , Relaciones Interinstitucionales , Células de Riñón Canino Madin Darby , Ratones , Células 3T3 NIH , Infecciones por Orthomyxoviridae/virología , Pandemias , Proyectos Piloto , Porcinos , Enfermedades de los Porcinos/virología , Células VeroRESUMEN
BACKGROUND: To guide time- and cost-efficient analyses of the increasing number of autosomal-dominant spinocerebellar ataxia genes (SCAs), more information about frequency distributions, phenotypic characteristics and optimal diagnostic strategies is warranted. OBJECTIVE: To assess the prevalence and phenotypic spectrum of SCA15 and to confirm multiplex ligation-dependent probe amplification (MLPA) as a robust and efficient strategy for routine molecular diagnosis. METHODS: Fifty-six German SCA families negative for common repeat expansions were screened for ITPR1 deletions by MLPA. Samples with conspicuous MLPA data were additionally assessed by high-density single nucleotide polymorphism (SNP) array to confirm MLPA results and further determine the size of deletions. The phenotype of patients harbouring ITPR1 deletions was characterised by standardised clinical, electrophysiological and imaging assessment. RESULTS: SCA15 accounted for 8.9% (5/56) of SCA families negative for common SCA repeat expansions. All deletions detected by MLPA were confirmed by SNP array. One of the ITPR1 deletions preserved exons 1 and 2 in the 5' prime UTR of the ITPR1 gene. All patients with SCA15 (n=10) presented with slowly progressive cerebellar ataxia and vermal cerebellar atrophy, while clinical and electrophysiological signs of extracerebellar affection were mild and more variable. CONCLUSIONS: SCA15 is the most common non-trinucleotide repeat SCA in Central Europe. Screening for ITPR1 deletions should be considered in patients with slowly progressive SCA, vermal cerebellar atrophy and prominent tremor after excluding common SCA repeat expansions. Promoter and exon 2 of ITPR1 may be preserved from the deletion in some cases of SCA15.
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Receptores de Inositol 1,4,5-Trifosfato , Ataxias Espinocerebelosas/diagnóstico , Ataxias Espinocerebelosas/genética , Adulto , Alelos , Exones , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Pruebas Genéticas , Genotipo , Alemania , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Técnicas de Amplificación de Ácido Nucleico , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Eliminación de Secuencia , Ataxias Espinocerebelosas/epidemiologíaRESUMEN
There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.