RESUMEN
Angioblasts of the developing vascular system require many signaling inputs to initiate their migration, proliferation and differentiation into endothelial cells. What is less studied is which intrinsic cell factors interpret these extrinsic signals. Here, we show the Lim homeodomain transcription factor islet2a (isl2a) is expressed in the lateral posterior mesoderm prior to angioblast migration. isl2a deficient angioblasts show disorganized migration to the midline to form axial vessels and fail to spread around the tailbud of the embryo. Isl2a morphants have fewer vein cells and decreased vein marker expression. We demonstrate that isl2a is required cell autonomously in angioblasts to promote their incorporation into the vein, and is permissive for vein identity. Knockout of isl2a results in decreased migration and proliferation of angioblasts during intersegmental artery growth. Since Notch signaling controls both artery-vein identity and tip-stalk cell formation, we explored the interaction of isl2a and Notch. We find that isl2a expression is negatively regulated by Notch activity, and that isl2a positively regulates flt4, a VEGF-C receptor repressed by Notch during angiogenesis. Thus Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas con Homeodominio LIM/fisiología , Neovascularización Fisiológica/fisiología , Factores de Transcripción/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Arterias/embriología , Movimiento Celular , Técnicas de Inactivación de Genes , Proteínas con Homeodominio LIM/deficiencia , Proteínas con Homeodominio LIM/genética , Mesodermo , Morfolinos/genética , Morfolinos/toxicidad , Neovascularización Patológica/genética , Neovascularización Patológica/patología , ARN Mensajero/genética , Receptores Notch/fisiología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/fisiología , Venas/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Tau is a microtubule-associated protein exerting several physiological functions in neurons. In Alzheimer's disease (AD) misfolded tau accumulates intraneuronally and leads to axonal degeneration. However, tau has also been found in the extracellular medium. Recent studies indicated that extracellular tau uploaded from neurons causes synaptic dysfunction and contributes to tau pathology propagation. Here we report novel evidence that extracellular tau oligomers are abundantly and rapidly accumulated in astrocytes where they disrupt intracellular Ca2+ signaling and Ca2+ -dependent release of gliotransmitters, especially ATP. Consequently, synaptic vesicle release, the expression of pre- and postsynaptic proteins, and mEPSC frequency and amplitude were reduced in neighboring neurons. Notably, we found that tau uploading from astrocytes required the amyloid precursor protein, APP. Collectively, our findings suggests that astrocytes play a critical role in the synaptotoxic effects of tau via reduced gliotransmitter availability, and that astrocytes are major determinants of tau pathology in AD.
Asunto(s)
Astrocitos/metabolismo , Hipocampo/citología , Neuronas/fisiología , Neurotransmisores/metabolismo , Sinapsis/efectos de los fármacos , Proteínas tau/toxicidad , Adenosina Trifosfato/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Embrión de Mamíferos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinos/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Cultivo de Órganos , Receptores AMPA/metabolismo , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) designed to induce skipping of dystrophin exon 51 and restore its expression in patients with Duchenne muscular dystrophy (DMD). Preclinically, restoration of dystrophin in the dystrophic mdx mouse model requires skipping of exon 23, achieved with the mouse-specific PMO, AVI-4225. Herein, we report the potential toxicological consequences of exon skipping and dystrophin restoration in mdx mice using AVI-4225. We also evaluated the toxicological effects of AVI-4658 in both mdx and wild-type mice. In both studies, animals were dosed once weekly for 12 weeks up to the maximum feasible dose of 960 mg/kg per injection. Both AVI-4658 and AVI-4225 were well-tolerated at all doses. Findings in AVI-4225-treated animals were generally limited to mild renal tubular basophilia/vacuolation, without any significant changes in renal function and with evidence of reversing. No toxicity associated with the mechanism of action of AVI-4225 in a dystrophic animal was observed.
Asunto(s)
Exones , Morfolinos/toxicidad , Oligonucleótidos/toxicidad , Animales , Ratones , Ratones Endogámicos mdx , Tamaño de los Órganos/efectos de los fármacosRESUMEN
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in childhood. Mutations of the DMD gene destabilize the dystrophin associated glycoprotein complex in the sarcolemma. Ongoing mechanical stress leads to unregulated influx of calcium ions into the sarcoplasm, with activation of proteases, release of proinflammatory cytokines, and mitochondrial dysfunction. Cumulative damage and reparative failure leads to progressive muscle necrosis, fibrosis, and fatty replacement. Although there is presently no cure for DMD, scientific advances have led to many potential disease-modifying treatments, including dystrophin replacement therapies, upregulation of compensatory proteins, anti-inflammatory agents, and other cellular targets. Recently approved therapies include ataluren for stop codon read-through and eteplirsen for exon 51 skipping of eligible individuals. The purpose of this chapter is to summarize the clinical features of DMD, to describe current outcome measures used in clinical studies, and to highlight new emerging therapies for affected individuals.
Asunto(s)
Distrofina/genética , Morfolinos/uso terapéutico , Distrofia Muscular de Duchenne/terapia , Oxadiazoles/uso terapéutico , Codón de Terminación/genética , Bases de Datos Genéticas , Distrofina/uso terapéutico , Exones/genética , Humanos , Morfolinos/toxicidad , Células Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , MutaciónRESUMEN
Purpose: Retinal degenerative diseases can progress to severe reductions of vision. In general, the changes are permanent in higher vertebrates, including humans; however, retinal regeneration can occur in lower vertebrates, such as amphibians and teleost fish. Progranulin is a secreted growth factor that is involved in normal development and wound-healing processes. We have shown that progranulin promotes the proliferation of retinal precursor cells in mouse retinas. The purpose of this study was to investigate the role played by granulin 1 (grn1) in the retinal regeneration in zebrafish. Methods: We injured the retina of zebrafish with needle puncturing, and the retinas were examined at different times after the injury. We also checked the proliferation and the expression of retinal regeneration-related genes after knockdown of grn1 by electroporation with morpholino oligonucleotides (MO) and intravitreal injection of recombinant grn1. Results: Our results showed that the level of grn1 was highly increased after retinal injury, and it was expressed in various types of retinal cells. A knockdown of grn1 reduced the proliferation of Müller glial cells in zebrafish eyes undergoing retinal regeneration. The knockdown of grn1 also reduced the expression of achaete-scute homolog 1a (ascl1a), an important factor in retinal regeneration. An intravitreal injection of recombinant grn1 led to a proliferation of Müller glial cells and an increase in the expression of retinal regeneration-related genes, such as ascl1a and lin28. Conclusions: These findings suggested that grn1 should be considered as a target for stimulating the dedifferentiation of Müller glial cells and retinal regeneration.
Asunto(s)
Granulinas/fisiología , Regeneración/fisiología , Retina/fisiología , Degeneración Retiniana/metabolismo , Proteínas de Pez Cebra/fisiología , Animales , Bromodesoxiuridina/metabolismo , Recuento de Células , Electroporación , Silenciador del Gen/fisiología , Granulinas/farmacología , Inmunohistoquímica , Morfolinos/toxicidad , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/farmacología , Retina/efectos de los fármacos , Degeneración Retiniana/etiología , Degeneración Retiniana/fisiopatología , Factores de Transcripción/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/farmacologíaRESUMEN
In the study, we examined whether the silent information regulator 1 (SIRT1) can attenuate oxidative stress in the brains of mice carrying the APP/PS1 double mutation and/or in primary neonatal rat neurons exposed to oligomers of amyloid-ß peptide (AßOs). Starting at 4 or 8 months of age, the transgenic mice were treated with resveratrol (RSV, a stimulator of SIRT1) or suramin (an inhibitor) (each 20âmg/kg BW/day) for two months. The primary neurons were exposed to AßOs (0.5 µM) for 48âh and thereafter RSV (20 µM) or suramin (300âmg/ml) for 24âh. Cell viability was assessed by the CCK-8 assay; SIRT1 protein and mRNA determined by western blotting and real-time PCR, respectively; senile plaques examined immunohistochemically; ROS monitored by flow cytometry; and the contents of OH-, H2O2, O2·-, and MDA, and the activities of SOD and GSH-Px measured by standard biochemical procedures. In comparison to wild-type mice or untreated primary neurons, the expression of SIRT1 was significantly lower in the brains of APP/PS1 mice or neurons exposed to AßOs. In these same systems, increased numbers of senile plaques and a high level of oxidative stress were apparent. Interestingly, these two latter changes were attenuated by treatment with RSV, but enhanced by suramin. These findings indicate that SIRT1 may be neuroprotective.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Encéfalo/fisiopatología , Estrés Oxidativo/fisiología , Presenilina-1/genética , Sirtuina 1/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Glutatión Peroxidasa/metabolismo , Hipocampo/citología , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinos/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/genética , Presenilina-1/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sirtuina 1/genética , Superóxido Dismutasa/metabolismoRESUMEN
Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum.
Asunto(s)
Cryptosporidium parvum/enzimología , Cryptosporidium parvum/genética , Técnicas de Silenciamiento del Gen , L-Lactato Deshidrogenasa/fisiología , Morfolinos/farmacología , Animales , Western Blotting , Línea Celular , Clonación Molecular , Cryptosporidium parvum/crecimiento & desarrollo , ADN Complementario/biosíntesis , ADN Protozoario/biosíntesis , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Sueros Inmunes/inmunología , Concentración 50 Inhibidora , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/inmunología , Morfolinos/metabolismo , Morfolinos/toxicidad , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/fisiología , Ratas , Esporozoítos/aislamiento & purificaciónRESUMEN
Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.
Asunto(s)
Corazón/embriología , Retinal-Deshidrogenasa/metabolismo , Transducción de Señal/fisiología , Tretinoina/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Morfolinos/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retinal-Deshidrogenasa/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Phosphorodiamidate morpholino oligomers (PMOs) are a class of exon skipping drugs including eteplirsen, which has shown considerable promise for treatment of the degenerative neuromuscular disease, Duchenne musculardystrophy (DMD). OBJECTIVE: Toxicity studies in non-human primates (NHPs) of 12 weeks duration with two new PMOs for DMD, SRP-4045 and SRP-4053, along with results from a chronic study in NHPs of 39 weeks duration for eteplirsen, are described here. METHODS: PMOs were administered once-weekly by bolus intravenous (IV) injections to male NHPs. Endpoints evaluated included plasma exposures, clinical observations, body weight/food consumption, eye exams, electrocardiograms, male reproductive hormones/endpoints, complement alternative pathway, clinical pathology, urinalysis, and macroscopic/light microscopic pathology. RESULTS: Findings in these studies were limited to the kidneys, with a common presentation of tubular basophilia, vacuolation, and/or minimal degeneration that was considered non-adverse. No necrosis, glomerular lesions, or effects on renal function tests such as serum creatinine or urea nitrogen were observed, suggesting that PMO-related kidney findings are not likely to develop into frank nephrotoxicity. There were no adverse effects on other potential target organs after repeated IV injections at the highest dose levels tested, 320âmg/kg. CONCLUSIONS: Nonclinical results in NHPs for these three PMOs, together with the excellent clinical safety established for eteplirsen to date, suggest that once-weekly IV administration of PMOs for lifetime durations at therapeutic doses will be well tolerated by patients with DMD.
Asunto(s)
Riñón/efectos de los fármacos , Morfolinos/toxicidad , Distrofia Muscular de Duchenne/tratamiento farmacológico , Animales , Basófilos/efectos de los fármacos , Basófilos/patología , Peso Corporal/efectos de los fármacos , Electrocardiografía , Exones , Corazón/efectos de los fármacos , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Macaca fascicularis , Masculino , Vacuolas/efectos de los fármacos , Vacuolas/patologíaRESUMEN
The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80-85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery.
Asunto(s)
Epilepsia/complicaciones , Degeneración Nerviosa/complicaciones , Lipofuscinosis Ceroideas Neuronales/complicaciones , Pez Cebra/fisiología , Animales , Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Axones/patología , Conducta Animal/efectos de los fármacos , Encéfalo/anomalías , Encéfalo/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia/patología , Técnicas de Silenciamiento del Gen , Gliosis/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Morfolinos/farmacología , Morfolinos/toxicidad , Actividad Motora/efectos de los fármacos , Miocardio/patología , Degeneración Nerviosa/patología , Lipofuscinosis Ceroideas Neuronales/patología , Subunidades de Proteína/metabolismo , ARN sin Sentido/metabolismo , Retina/efectos de los fármacos , Retina/patología , Análisis de Supervivencia , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Dendrimer-based gene delivery has been constrained by intrinsic toxicity and suboptimal nanostructure. Conjugation of neutral morpholino oligonucleotides (ONs) with PAMAM dendrimers resulted in neutral, uniform, and ultra-small (â¼10 nm) nanoconjugates. The nanoconjugates dramatically enhanced cellular delivery of the ONs in cancer cells. After release from the dendrimer in the cytosol, the ONs produced potent functional activity without causing significant cytotoxicity. When carrying an apoptosis-promoting ON, the nanoconjugates produced cancer cell killing directly. Thus, the dendritic nanoconjugates may provide an effective tool for delivering ONs to tumors and other diseased tissues.
Asunto(s)
Dendrímeros/química , Morfolinos/metabolismo , Nanoconjugados/química , Transfección/métodos , Apoptosis/efectos de los fármacos , Secuencia de Bases , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Humanos , Microscopía Confocal , Morfolinos/química , Morfolinos/toxicidad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismoRESUMEN
Vivo-morpholinos are a promising tool for gene silencing. These oligonucleotide analogs transiently silence genes by blocking either translation or pre-mRNA splicing. Little to no toxicity has been reported for vivo-morpholino treatment. However, in a recent study conducted in our lab, treatment of mice with vivo-morpholinos resulted in high mortality rates. We hypothesized that the deaths were the result of oligonucleotide hybridization, causing an increased cationic charge associated with the dendrimer delivery moiety of the vivo-morpholino. The cationic charge increased blood clot formation in whole blood treated with vivo-morpholinos, suggesting that clotting could have caused cardiac arrest in the deceased mice. Therefore, we investigate the mechanism by which some vivo-morpholinos increase mortality rates and propose techniques to alleviate vivo-morpholino toxicity.
Asunto(s)
Silenciador del Gen , Morfolinos/toxicidad , Oligonucleótidos Antisentido/toxicidad , Animales , Anexina A6/genética , Coagulación Sanguínea/efectos de los fármacos , Proteínas de Unión al Calcio/genética , Calsecuestrina , Relación Dosis-Respuesta a Droga , Inyecciones , Masculino , Ratones Endogámicos C57BL , Morfolinos/administración & dosificación , Mortalidad , Oligonucleótidos Antisentido/administración & dosificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Fatty acid binding protein 3 (FABP3, also termed heart-type fatty acid binding protein) is a member of the intracellular lipid-binding protein family that may be essential in fatty acid transport, cell growth, cellular signaling and gene transcription. Previously, we demonstrated that FABP3 was involved in apoptosis-associated congenital cardiac malformations; however, its mechanism of regulation remains unclear. Apoptosis has increasingly been considered to be important in cardiac development. In the present study, a zebrafish model was used to investigate the involvement of FABP3morpholino (MO)-induced apoptosis and mitochondrial dysfunction in cardiac development. During the early stages of cardiac development, injection of FABP3MO into zebrafish resulted in significant impairment in cardiac development and promoted the rate of apoptosis which was correlated with signiï¬cant dysfunction of the mitochondria. For example, the ATP content was markedly decreased at 24 and 48 h post-fertilization (pf), reactive oxygen species production was significantly enhanced at 24 and 48 h pf and the mitochondrial DNA copy number was reduced at 24, 48 and 72 h pf. Additionally, Nkx2.5 expression was upregulated in FABP3-MO zebrafish, and Wnt signaling molecules (Wnt1, Wnt5 and Wnt11) were also highly expressed in FABP3-MO zebrafish at 24, 48 and 72 h pf. In conclusion, the results indicated that FABP3 knockdown exhibited significant toxic effects on cardiac development and mitochondrial function, which may be responsible for the knockdown of FABP3-induced apoptosis. Apoptosis was one of the mechanisms underlying this effect, and was correlated with the activation of Wnt signaling. These studies identified FABP3 as a candidate gene underlying the etiology of congenital heart defects.