RESUMEN
Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.
Asunto(s)
Segmento Inicial del Axón , Canales de Sodio , Potenciales de Acción , Análisis por Conglomerados , EndocitosisRESUMEN
Parvalbumin-expressing (PV+) basket cells are fast-spiking inhibitory interneurons that exert critical control over local circuit activity and oscillations. PV+ axons are often myelinated, but the electrical and metabolic roles of interneuron myelination remain poorly understood. Here, we developed viral constructs allowing cell type-specific investigation of mitochondria with genetically encoded fluorescent probes. Single-cell reconstructions revealed that mitochondria selectively cluster to myelinated segments of PV+ basket cells, confirmed by analyses of a high-resolution electron microscopy dataset. In contrast to the increased mitochondrial densities in excitatory axons cuprizone-induced demyelination abolished mitochondrial clustering in PV+ axons. Furthermore, with genetic deletion of myelin basic protein the mitochondrial clustering was still observed at internodes wrapped by noncompacted myelin, indicating that compaction is dispensable. Finally, two-photon imaging of action potential-evoked calcium (Ca2+) responses showed that interneuron myelination attenuates both the cytosolic and mitochondrial Ca2+ transients. These findings suggest that oligodendrocyte ensheathment of PV+ axons assembles mitochondria to branch selectively fine-tune metabolic demands.
Asunto(s)
Enfermedades Desmielinizantes , Parvalbúminas , Humanos , Parvalbúminas/metabolismo , Axones/metabolismo , Interneuronas/fisiología , Potenciales de Acción/fisiología , Vaina de Mielina/metabolismo , Enfermedades Desmielinizantes/metabolismoRESUMEN
The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by intrinsic changes and a reduced spike output. Finally, consistent with excitability deficits, we show that cuprizone-treated mice exhibit impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS.
Asunto(s)
Región CA2 Hipocampal/metabolismo , Región CA2 Hipocampal/patología , Complemento C1q/metabolismo , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Sinapsis/fisiología , Anciano , Animales , Estudios de Casos y Controles , Cuprizona , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple/etiologíaRESUMEN
OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5' splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.
Asunto(s)
Dependovirus/genética , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/genética , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/terapia , Ornitina Carbamoiltransferasa/genética , Empalme del ARN , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Exones , Terapia Genética/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Intrones , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Mutación , Ornitina Carbamoiltransferasa/metabolismo , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/enzimología , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/patología , Sitios de Empalme de ARN , ARN Nuclear Pequeño/metabolismoRESUMEN
Personalized cancer treatments using combinations of drugs with a synergistic effect is attractive but proves to be highly challenging. Here we present an approach to uncover the efficacy of drug combinations based on the analysis of mono-drug effects. For this we used dose-response data from pharmacogenomic encyclopedias and represent these as a drug atlas. The drug atlas represents the relations between drug effects and allows to identify independent processes for which the tumor might be particularly vulnerable when attacked by two drugs. Our approach enables the prediction of combination-therapy which can be linked to tumor-driving mutations. By using this strategy, we can uncover potential effective drug combinations on a pan-cancer scale. Predicted synergies are provided and have been validated in glioblastoma, breast cancer, melanoma and leukemia mouse-models, resulting in therapeutic synergy in 75% of the tested models. This indicates that we can accurately predict effective drug combinations with translational value.
Asunto(s)
Sinergismo Farmacológico , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Biología Computacional , Combinación de Medicamentos , Glioblastoma/metabolismo , Humanos , Modelos Logísticos , Melanoma/metabolismoRESUMEN
The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5' splice site (5'ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects. Here, we challenged the U1snRNA in the FAH5961SB mouse model of hepatic fumarylacetoacetate hydrolase (FAH) deficiency (Hereditary Tyrosinemia type I, HT1) due to the FAH c.706G>A splicing mutation. Through minigene expression studies we selected a compensatory U1snRNA (U1F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1F (AAV8-U1F), but not of U1wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1F treated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1F on the splicing profile and overall gene expression, thus pointing toward gene specificity. These data provide early in vivo proof-of-principle of the correction potential of compensatory U1snRNAs in HTI and encourage further optimization on a therapeutic perspective, and translation to other splicing-defective forms of metabolic diseases.
Asunto(s)
Hidrolasas/genética , Empalme del ARN/genética , ARN Nuclear Pequeño/genética , Tirosinemias/enzimología , Tirosinemias/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
The role of the VEGF inhibitor bevacizumab in the treatment of diffuse intrinsic pontine glioma (DIPG) is unclear. We aim to study the biodistribution and uptake of zirconium-89 ((89)Zr)-labeled bevacizumab in DIPG mouse models. Human E98-FM, U251-FM glioma cells, and HSJD-DIPG-007-FLUC primary DIPG cells were injected into the subcutis, pons, or striatum of nude mice. Tumor growth was monitored by bioluminescence imaging (BLI) and visualized by MRI. Seventy-two to 96 hours after (89)Zr-bevacizumab injections, mice were imaged by positron emission tomography (PET), and biodistribution was analyzed ex vivo High VEGF expression in human DIPG was confirmed in a publically available mRNA database, but no significant (89)Zr-bevacizumab uptake could be detected in xenografts located in the pons and striatum at an early or late stage of the disease. E98-FM, and to a lesser extent the U251-FM and HSJD-DIPG-007 subcutaneous tumors, showed high accumulation of (89)Zr-bevacizumab. VEGF expression could not be demonstrated in the intracranial tumors by in situ hybridization (ISH) but was clearly present in the perinecrotic regions of subcutaneous E98-FM tumors. The poor uptake of (89)Zr-bevacizumab in xenografts located in the brain suggests that VEGF targeting with bevacizumab has limited efficacy for diffuse infiltrative parts of glial brain tumors in mice. Translating these results to the clinic would imply that treatment with bevacizumab in patients with DIPG is only justified after targeting of VEGF has been demonstrated by (89)Zr-bevacizumab immuno-PET. We aim to confirm this observation in a clinical PET study with patients with DIPG. Mol Cancer Ther; 15(9); 2166-74. ©2016 AACR.