Asunto(s)
Neuropatías Amiloides Familiares , Amiloidosis , Cardiomiopatías , Humanos , Neuropatías Amiloides Familiares/complicaciones , Neuropatías Amiloides Familiares/tratamiento farmacológico , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/etiología , Cardiomiopatías/inmunología , PrealbúminaRESUMEN
Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.
Asunto(s)
Liasas de Carbono-Carbono/genética , Lipidómica/métodos , Peroxisomas/metabolismo , Fitol/administración & dosificación , Proteómica/métodos , Animales , Encéfalo/metabolismo , Familia 2 del Citocromo P450/metabolismo , Familia 4 del Citocromo P450/metabolismo , Ácidos Grasos/metabolismo , Femenino , Técnicas de Inactivación de Genes , Riñón/metabolismo , Hígado/metabolismo , Masculino , Ratones , Oxidación-Reducción , Ácido Fitánico/análogos & derivados , Ácido Fitánico/metabolismo , Fitol/farmacologíaRESUMEN
Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments.
Asunto(s)
Neuropatías Amiloides Familiares/metabolismo , Amiloide/metabolismo , Fibrinolisina/genética , Fibrinolisina/metabolismo , Placa Amiloide/metabolismo , Animales , Cardiomiopatías , Humanos , Ratones Transgénicos , Prealbúmina/metabolismo , Pliegue de Proteína , ProteolisisRESUMEN
Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3 . Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.
Asunto(s)
Cutis Laxo/genética , Antígenos de Histocompatibilidad Menor/genética , Mutación Missense , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Piel/patología , Secuencia de Aminoácidos , Animales , Niño , Cutis Laxo/patología , Femenino , Glicosilación , Homocigoto , Humanos , Ratones , Ratones Noqueados , Linaje , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficienciaRESUMEN
The development of new therapies to slow down or halt the progression of Parkinson's disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6 weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.
Asunto(s)
Encéfalo/metabolismo , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Exosomas/genética , Enfermedad de Parkinson/terapia , ARN Interferente Pequeño/genética , alfa-Sinucleína/administración & dosificación , Animales , Regulación de la Expresión Génica , Terapia Genética , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , alfa-Sinucleína/antagonistas & inhibidores , alfa-Sinucleína/genéticaRESUMEN
In Alzheimer's disease (AD), the canonical Wnt inhibitor Dickkopf-1 (Dkk1) is induced by ß-amyloid (Aß) and shifts the balance from canonical towards non-canonical Wnt signalling. Canonical (Wnt-ß-catenin) signalling promotes synapse stability, while non-canonical (Wnt-PCP) signalling favours synapse retraction; thus Aß-driven synapse loss is mediated by Dkk1. Here we show that the Amyloid Precursor Protein (APP) co-activates both arms of Wnt signalling through physical interactions with Wnt co-receptors LRP6 and Vangl2, to bi-directionally modulate synapse stability. Furthermore, activation of non-canonical Wnt signalling enhances Aß production, while activation of canonical signalling suppresses Aß production. Together, these findings identify a pathogenic-positive feedback loop in which Aß induces Dkk1 expression, thereby activating non-canonical Wnt signalling to promote synapse loss and drive further Aß production. The Swedish familial AD variant of APP (APPSwe) more readily co-activates non-canonical, at the expense of canonical Wnt activity, indicating that its pathogenicity likely involves direct effects on synapses, in addition to increased Aß production. Finally, we report that pharmacological inhibition of the Aß-Dkk1-Aß positive feedback loop with the drug fasudil can restore the balance between Wnt pathways, prevent dendritic spine withdrawal in vitro, and reduce Aß load in vivo in mice with advanced amyloid pathology. These results clarify a relationship between Aß accumulation and synapse loss and provide direction for the development of potential disease-modifying treatments.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/fisiología , Sinapsis/patología , Vía de Señalización Wnt , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismoRESUMEN
INTRODUCTION: Synapse loss is the structural correlate of the cognitive decline indicative of dementia. In the brains of Alzheimer's disease sufferers, amyloid ß (Aß) peptides aggregate to form senile plaques but as soluble peptides are toxic to synapses. We previously demonstrated that Aß induces Dickkopf-1 (Dkk1), which in turn activates the Wnt-planar cell polarity (Wnt-PCP) pathway to drive tau pathology and neuronal death. METHODS: We compared the effects of Aß and of Dkk1 on synapse morphology and memory impairment while inhibiting or silencing key elements of the Wnt-PCP pathway. RESULTS: We demonstrate that Aß synaptotoxicity is also Dkk1 and Wnt-PCP dependent, mediated by the arm of Wnt-PCP regulating actin cytoskeletal dynamics via Daam1, RhoA and ROCK, and can be blocked by the drug fasudil. DISCUSSION: Our data add to the importance of aberrant Wnt signaling in Alzheimer's disease neuropathology and indicate that fasudil could be repurposed as a treatment for the disease.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Péptidos beta-Amiloides/metabolismo , Fármacos Neuroprotectores/farmacología , Nootrópicos/farmacología , Sinapsis/metabolismo , Vía de Señalización Wnt , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Relación Dosis-Respuesta a Droga , Femenino , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Fármacos Neuroprotectores/farmacocinética , Nootrópicos/farmacocinética , Cultivo Primario de Células , ARN Mensajero/metabolismo , Ratas , Sinapsis/efectos de los fármacos , Sinapsis/patología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/fisiologíaRESUMEN
Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of ß2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type ß2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The ß2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N ß2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In ß2-microglobulin knock out mice, the D76N ß2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type ß2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.
Asunto(s)
Amiloidosis/inmunología , Anticuerpos de Dominio Único/inmunología , Microglobulina beta-2/inmunología , Amiloide/efectos de los fármacos , Amiloide/inmunología , Amiloide/metabolismo , Amiloidosis/metabolismo , Amiloidosis/prevención & control , Animales , Línea Celular Tumoral , Doxiciclina/farmacocinética , Doxiciclina/farmacología , Humanos , Ratones de la Cepa 129 , Ratones Noqueados , Mutación Missense , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/prevención & control , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Distribución Tisular/efectos de los fármacos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
During the course of normal aging, certain populations of nerve growth factor (NGF)-responsive neurons become selectively vulnerable to cell death. Studies using dissociated neurons isolated from neonates have shown that c-Jun N-terminal kinases (JNKs) are important in regulating the survival and neurite outgrowth of NGF-responsive sympathetic neurons. Unlike neonatal neurons, adult sympathetic neurons are not dependent on NGF for their survival. Moreover, the NGF precursor, proNGF, is neurotoxic for aging but not young adult NGF-responsive neurons. Because of these age-related differences, the effects of JNK inhibition on the survival and growth of sympathetic neurons isolated from aged mice were studied. Aged neurons, as well as glia, were found to be dependent on JNK for their growth but not their survival. Conversely, proNGF neurotoxicity was JNK-dependent and mediated by the p75-interacting protein NRAGE, whereas neurite outgrowth was independent of NRAGE. These results have implications for the potential use of JNK inhibitors as therapies for ameliorating age-related neurodegenerative disease.
Asunto(s)
Envejecimiento/genética , Envejecimiento/patología , Procesos de Crecimiento Celular/genética , Supervivencia Celular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Neuronas/citología , Neuronas/fisiología , Sistema Nervioso Simpático/citología , Animales , Muerte Celular/genética , Muerte Celular/fisiología , Células Cultivadas , Masculino , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Proteínas de Neoplasias/fisiología , Factor de Crecimiento Nervioso/fisiología , Factor de Crecimiento Nervioso/toxicidad , Neuritas/fisiología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/terapia , Precursores de Proteínas/toxicidad , Ratas Sprague-DawleyRESUMEN
Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIß in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.
Asunto(s)
Células Endoteliales/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Cuerpos de Weibel-Palade/metabolismo , Factor de von Willebrand/genética , Animales , Células Endoteliales/patología , Exocitosis , Regulación de la Expresión Génica , Histamina/administración & dosificación , Humanos , Inflamación/genética , Inflamación/patología , Lípidos/genética , Ratones , Neovascularización Patológica/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Interferencia de ARN , Trombosis/genética , Trombosis/patología , Cuerpos de Weibel-Palade/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismo , Factor de von Willebrand/biosíntesisRESUMEN
Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid ß (Aß) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming 'Depletion of serum amyloid P component in Alzheimer's disease (DESPIAD)' clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Placa Amiloide/metabolismo , Agregación Patológica de Proteínas/metabolismo , Componente Amiloide P Sérico/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Angiopatía Amiloide Cerebral/metabolismo , Angiopatía Amiloide Cerebral/patología , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Agregación Patológica de Proteínas/tratamiento farmacológico , Componente Amiloide P Sérico/genéticaRESUMEN
Systemic amyloidoses are a group of debilitating and often fatal diseases in which fibrillar protein aggregates are deposited in the extracellular spaces of a range of tissues. The molecular basis of amyloid formation and tissue localization is still unclear. Although it is likely that the extracellular matrix (ECM) plays an important role in amyloid deposition, this interaction is largely unexplored, mostly because current analytical approaches may alter the delicate and complicated three-dimensional architecture of both ECM and amyloid. We describe here a decellularization procedure for the amyloidotic mouse liver which allows high-resolution visualization of the interactions between amyloid and the constitutive fibers of the extracellular matrix. The primary structure of the fibrillar proteins remains intact and the amyloid fibrils retain their amyloid enhancing factor activity.
Asunto(s)
Amiloide/fisiología , Amiloidosis/patología , Hepatopatías/patología , Hígado/patología , Secuencia de Aminoácidos , Amiloide/química , Animales , Matriz Extracelular/fisiología , Femenino , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Hígado/química , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Proteína Amiloide A Sérica/químicaRESUMEN
Alpha-synuclein (α-Syn) aggregates are the main component of Lewy bodies, which are the characteristic pathological feature in Parkinson's disease (PD) brain. Evidence that α-Syn aggregation can be propagated between neurones has led to the suggestion that this mechanism is responsible for the stepwise progression of PD pathology. Decreasing α-Syn expression is predicted to attenuate this process and is thus an attractive approach to delay or halt PD progression. We have used α-Syn small interfering RNA (siRNA) to reduce total and aggregated α-Syn levels in mouse brains. To achieve widespread delivery of siRNAs to the brain we have peripherally injected modified exosomes expressing Ravies virus glycoprotein loaded with siRNA. Normal mice were analyzed 3 or 7 days after injection. To evaluate whether this approach can decrease α-Syn aggregates, we repeated the treatment using transgenic mice expressing the human phosphorylation-mimic S129D α-Syn, which exhibits aggregation. In normal mice we detected significantly reduced α-Syn messenger RNA (mRNA) and protein levels throughout the brain 3 and 7 days after treatment with RVG-exosomes loaded with siRNA to α-Syn. In S129D α-Syn transgenic mice we found a decreased α-Syn mRNA and protein levels throughout the brain 7 days after injection. This resulted in significant reductions in intraneuronal protein aggregates, including in dopaminergic neurones of the substantia nigra. This study highlights the therapeutic potential of RVG-exosome delivery of siRNA to delay and reverse brain α-Syn pathological conditions.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Mutación/genética , ARN Interferente Pequeño/administración & dosificación , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Animales , Línea Celular Tumoral , Células Dendríticas/metabolismo , Exosomas/fisiología , Regulación de la Expresión Génica/fisiología , Vectores Genéticos/genética , Glicoproteínas/administración & dosificación , Glicoproteínas/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroblastoma/patología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Transfección , Proteínas Virales/administración & dosificación , Proteínas Virales/genéticaRESUMEN
No deficiency of human C-reactive protein (CRP), or even structural polymorphism of the protein, has yet been reported so its physiological role is not known. Here we show for the first time that CRP-deficient mice are remarkably susceptible to Streptococcus pneumoniae infection and are protected by reconstitution with isolated pure human CRP, or by anti-pneumococcal antibodies. Autologous mouse CRP is evidently essential for innate resistance to pneumococcal infection before antibodies are produced. Our findings are consistent with the significant association between clinical pneumococcal infection and non-coding human CRP gene polymorphisms which affect CRP expression. Deficiency or loss of function variation in CRP may therefore be lethal at the first early-life encounter with this ubiquitous virulent pathogen, explaining the invariant presence and structure of CRP in human adults.
Asunto(s)
Proteína C-Reactiva/inmunología , Inmunidad Innata , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/inmunología , Animales , Proteína C-Reactiva/deficiencia , Proteína C-Reactiva/genética , Humanos , Ratones , Ratones Noqueados , FenotipoRESUMEN
To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N ß2-microglobulin (ß2m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N ß2m. Using biophysical and structural approaches, we show that the MHCI containing D76N ß2m (MHCI76) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type ß2m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N ß2m within the MHCI76 as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N ß2m degradation within the cell. Accordingly, D76N ß2m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.
Asunto(s)
Amiloide/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Mutación Missense , Microglobulina beta-2/metabolismo , Sustitución de Aminoácidos , Amiloide/genética , Animales , Cristalografía por Rayos X , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ratones , Ratones Transgénicos , Microglobulina beta-2/genéticaRESUMEN
Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.
Asunto(s)
Amiloide/metabolismo , Amiloidosis/fisiopatología , Inflamación/complicaciones , Proteína Amiloide A Sérica/metabolismo , Amiloidosis/etiología , Animales , Rojo Congo , Cartilla de ADN/genética , Doxiciclina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We report the in vivo evaluation, in a murine model, of MRI measurements of the extracellular volume fraction (ECV) for the detection and monitoring of systemic amyloidosis. A new inducible transgenic model was used, with increased production of mouse serum amyloid A protein controlled by oral administration of doxycycline, that causes both the usual hepatic and splenic amyloidosis and also cardiac deposits. ECV was measured in vivo by equilibrium contrast MRI in the heart and liver of 11 amyloidotic and 10 control mice. There was no difference in the cardiac function between groups, but ECV was significantly increased in the heart, mean (standard deviation) 0.20 (0.05) versus 0.14 (0.04), p < 0.005, and liver, 0.27 (0.04) versus 0.15 (0.04), p < 0.0005, of amyloidotic animals and was strongly correlated with the histological amyloid score, myocardium, ρ = 0.67, p < 0.01; liver, ρ = 0.87, p < 0.01. In a further four mice that received human serum amyloid P component (SAP) followed by anti-human SAP antibody, a treatment to eliminate visceral amyloid deposits, ECV in the liver and spleen returned to baseline after therapy (p < 0.01). MRI measurement of ECV is a sensitive marker of amyloid deposits with potential application for early detection and monitoring therapies promoting their clearance.
Asunto(s)
Amiloide/metabolismo , Amiloidosis/diagnóstico , Líquido Extracelular/metabolismo , Hígado/patología , Imagen por Resonancia Magnética , Miocardio/patología , Bazo/patología , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Femenino , Humanos , Hígado/diagnóstico por imagen , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Radiografía , Proteína Amiloide A Sérica/metabolismo , Componente Amiloide P Sérico/inmunología , Componente Amiloide P Sérico/metabolismo , Bazo/diagnóstico por imagen , Bazo/metabolismoRESUMEN
Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.
Asunto(s)
Apolipoproteínas B/genética , LDL-Colesterol/sangre , Exones , Oligonucleótidos/genética , Precursores del ARN/genética , Empalme del ARN , Animales , Apolipoproteínas B/metabolismo , Células CACO-2 , Terapia Genética/métodos , Células Hep G2 , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Lipoproteínas VLDL/antagonistas & inhibidores , Lipoproteínas VLDL/sangre , Hígado/metabolismo , Ratones , Ratones Transgénicos , Oligonucleótidos/metabolismo , Precursores del ARN/metabolismo , Conejos , Receptores de Lipoproteína/genética , Receptores de Lipoproteína/metabolismo , Análisis de Secuencia de ARNRESUMEN
Numerous genes causing autosomal recessive hereditary spastic paraplegia (AR HSP) have been described. Despite this, in many families the causative gene and mutation are unknown. In this study we sequenced the Pi4k2a gene, whose knockout has been shown to cause a typical HSP model in mice, in 24 index cases of autosomal recessive HSP not known to be linked to any other HSP locus. No pathogenic changes were identified in exons or splice sites, suggesting the Pi4k2a gene may not be a cause of AR HSP in humans.
Asunto(s)
Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Paraplejía Espástica Hereditaria/enzimología , Paraplejía Espástica Hereditaria/genética , Adolescente , Adulto , Edad de Inicio , Animales , Niño , Preescolar , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Paraplejía Espástica Hereditaria/diagnóstico , Adulto JovenRESUMEN
The 5-HT(2C) receptor has been implicated in mood and eating disorders. In general, it is accepted that 5-HT(2C) receptor agonists increase anxiety behaviours and induce hypophagia. However, pharmacological analysis of the roles of these receptors is hampered by the lack of selective ligands and the complex regulation of receptor isoforms and expression levels. Therefore, the exact role of 5-HT(2C) receptors in mood disorders remain controversial, some suggesting agonists and others suggesting antagonists may be efficacious antidepressants, while there is general agreement that antagonists are beneficial anxiolytics. In order to test the hypothesis that increased 5-HT(2C) receptor expression, and thus increased 5-HT(2C) receptor signalling, is causative in mood disorders, we have undertaken a transgenic approach, directly altering the 5-HT(2C) receptor number in the forebrain and evaluating the consequences on behaviour. Transgenic mice overexpressing 5-HT(2C) receptors under the control of the CaMKIIalpha promoter (C2CR mice) have elevated 5-HT(2C) receptor mRNA levels in cerebral cortex and limbic areas (including the hippocampus and amygdala), but normal levels in the hypothalamus, resulting in > 100% increase in the number of 5-HT(2C) ligand binding sites in the forebrain. The C2CR mice show increased anxiety-like behaviour in the elevated plus-maze, decreased wheel-running behaviour and reduced activity in a novel environment. These behaviours were observed in the C2CR mice without stimulation by exogenous ligands. Our findings support a role for 5-HT(2C) receptor signalling in anxiety disorders. The C2CR mouse model offers a novel and effective approach for studying disorders associated with 5-HT(2C) receptors.