RESUMO
Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.
Assuntos
Doença de Huntington , Organoides , Fenótipo , Telencéfalo , Doença de Huntington/patologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Organoides/patologia , Organoides/metabolismo , Animais , Telencéfalo/patologia , Telencéfalo/citologia , Telencéfalo/metabolismo , Humanos , Camundongos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Análise de Célula Única , Comunicação Celular , Mosaicismo , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington's disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.
Assuntos
Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Hipocampo , Doença de Huntington , Potenciação de Longa Duração , Proteínas de Membrana , Receptor trkB , Transdução de Sinais , Animais , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Hipocampo/metabolismo , Hipocampo/patologia , Receptor trkB/metabolismo , Receptor trkB/antagonistas & inibidores , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Caderinas/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Neuroproteção , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Camundongos KnockoutRESUMO
Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.
Assuntos
Diferenciação Celular , Ventrículos Laterais , Fator Inibidor de Leucemia , Organoides , Células-Tronco Pluripotentes , Humanos , Organoides/metabolismo , Organoides/citologia , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Fator de Transcrição STAT3/metabolismo , Neuroglia/metabolismo , Neuroglia/citologia , Transdução de SinaisRESUMO
Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.
Assuntos
Neoplasias , Proteínas Nucleares , Humanos , Instabilidade Genômica , Mutação , Taxa de Mutação , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Microambiente Tumoral , Animais , CamundongosRESUMO
Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.
RESUMO
Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.
Assuntos
Transtorno Autístico , Transtorno Autístico/genética , Humanos , Proteínas do Tecido Nervoso/metabolismo , Organoides/metabolismo , Protocaderinas , TelencéfaloRESUMO
OBJECTIVES: Evaluate the ability of current ion-releasing materials to remineralise bacteria-driven artificial caries lesions. MATERIALS AND METHODS: Standardised class I cavities were obtained in 60 extracted human molars. Specimens underwent a microbiological cariogenic protocol (28 days) to generate artificial caries lesions and then were randomly divided into four restorative groups: adhesive + composite (negative control); glass ionomer cement (GIC); calcium silicate cement (MTA); and resin-modified calcium silicate cement (RMTA). Microhardness analysis (ΔKHN) was performed on 40 specimens (10/group, t = 30 days, 45 days, 60 days in artificial saliva, AS). Micro-CT scans were acquired (3/group, t = 0 days, 30 days, and 90 days in AS). Confocal microscopy was employed for interfacial ultra-morphology analysis (2/group, t = 0 days and 60 days in AS). Additional specimens were prepared and processed for scanning electron microscopy (SEM) and FTIR (n = 3/group + control) to analyse the ability of the tested materials to induce apatite formation on totally demineralised dentine discs (60 days in AS). Statistical analyses were performed with a significance level of 5%. RESULTS: Adhesive + composite specimens showed the lowest ΔKHN values and the presence of gaps at the interface when assessed through micro-CT even after storage in AS. Conversely, all the tested ion-releasing materials presented an increase in ΔKHN after storage (p < 0.05), while MTA best reduced the demineralised artificial carious lesions gap at the interface. MTA and RMTA also showed apatite deposition on totally demineralised dentine surfaces (SEM and FTIR). CONCLUSIONS: All tested ion-releasing materials expressed mineral precipitation in demineralised dentine. Additionally, calcium silicate-based materials induced apatite precipitation and hardness recovery of artificial carious dentine lesions over time. CLINICAL RELEVANCE: Current ion-releasing materials can induce remineralisation of carious dentine. MTA shows enhanced ability of nucleation/precipitation of hydroxyapatite compared to RMTA and GIC, which may be more appropriate to recover severe mineral-depleted dentine.
Assuntos
Cárie Dentária , Dentina , Humanos , Apatitas , Compostos de Cálcio , Cárie Dentária/patologia , Cárie Dentária/terapia , Dentina/química , Cimentos de Ionômeros de Vidro , Hidroxiapatitas , Teste de Materiais , Minerais/análise , Cimentos de Resina , Saliva Artificial , SilicatosRESUMO
BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.
Assuntos
Mucosa Bucal/metabolismo , Antissépticos Bucais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Reatores Biológicos/microbiologia , DNA/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Géis/farmacologia , Engenharia Genética/métodos , Humanos , Mucosa Bucal/efeitos dos fármacos , Antissépticos Bucais/metabolismo , RNA/farmacologiaRESUMO
Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.
Assuntos
Atlas como Assunto , Corpo Estriado/citologia , Corpo Estriado/embriologia , Neurogênese/genética , RNA Longo não Codificante/genética , Análise de Célula Única , Fatores de Transcrição/genética , Feto , Neurônios GABAérgicos/metabolismo , Humanos , RNA-Seq , Transcrição GênicaRESUMO
Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.
Assuntos
Proteína ADAM10/metabolismo , Proteínas do Citoesqueleto/metabolismo , Doença de Huntington/metabolismo , Neuropeptídeos/metabolismo , Vesículas Sinápticas/metabolismo , Proteína ADAM10/genética , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Humanos , Doença de Huntington/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Vesículas Sinápticas/ultraestrutura , Sinaptossomos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Human mutations and haploinsufficiency of the SHANK family genes are associated with autism spectrum disorders (ASD) and intellectual disability (ID). Complex phenotypes have been also described in all mouse models of Shank mutations and deletions, consistent with the heterogeneity of the human phenotypes. However, the specific role of Shank proteins in synapse and neuronal functions remain to be elucidated. Here, we generated a new mouse model to investigate how simultaneously deletion of Shank1 and Shank3 affects brain development and behavior in mice. Shank1-Shank3 DKO mice showed a low survival rate, a developmental strong reduction in the activation of intracellular signaling pathways involving Akt, S6, ERK1/2, and eEF2 during development and a severe behavioral impairments. Our study suggests that Shank1 and Shank3 proteins are essential to developmentally regulate the activation of Akt and correlated intracellular pathways crucial for mammalian postnatal brain development and synaptic plasticity. Therefore, Akt function might represent a new therapeutic target for enhancing cognitive abilities of syndromic ASD patients.
Assuntos
Transtorno do Espectro Autista , Proteínas Proto-Oncogênicas c-akt , Animais , Transtorno do Espectro Autista/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso/genética , SinapsesRESUMO
Mechanotransduction is the ability of cells to translate mechanical stimuli into biochemical signals that can ultimately influence gene expression, cell morphology and cell fate. Tenocytes are responsible for tendon mechanical adaptation converting mechanical stimuli imposed during mechanical loading, thus affecting extracellular matrix homeostasis. Since we previously demonstrated that MD-Tissue, an injectable collagen-based medical compound containing swine-derived collagen as the main component, is able to affect tenocyte properties, the aim of this study was to analyze whether the effects triggered by MD-Tissue were based on mechanotransduction-related mechanisms. For this purpose, MD-Tissue was used to coat Petri dishes and cytochalasin B was used to deprive tenocytes of mechanical stimulation mediated by the actin cytoskeleton. Cell morphology, migration, collagen turnover pathways and the expression of key mechanosensors were analyzed by morphological and molecular methods. Our findings confirm that MD-Tissue affects collagen turnover pathways and favors cell migration and show that the MD-Tissue-induced effect represents a mechanical input involving the mechanotransduction machinery. Overall, MD-Tissue, acting as a mechanical scaffold, could represent an effective medical device for a novel therapeutic, regenerative and rehabilitative approach to favor tendon healing in tendinopathies.
Assuntos
Colágeno/química , Estresse Mecânico , Tenócitos/metabolismo , Citoesqueleto de Actina , Idoso , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Citocalasina B/farmacologia , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Suínos , Tenócitos/citologia , Tenócitos/efeitos dos fármacos , Tenócitos/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMO
Detecting the ultrastructure of brain tissue in human archaeological remains is a rare event that can offer unique insights into the structure of the ancient central nervous system (CNS). Yet ancient brains reported in the literature show only poor preservation of neuronal structures. Using scanning electron microscopy (SEM) and advanced image processing tools, we describe the direct visualization of neuronal tissue in vitrified brain and spinal cord remains which we discovered in a male victim of the AD 79 eruption in Herculaneum. We show exceptionally well preserved ancient neurons from different regions of the human CNS at unprecedented resolution. This tissue typically consists of organic matter, as detected using energy-dispersive X-ray spectroscopy. By means of a self-developed neural image processing network, we also show specific details of the neuronal nanomorphology, like the typical myelin periodicity evidenced in the brain axons. The perfect state of preservation of these structures is due to the unique process of vitrification which occurred at Herculaneum. The discovery of proteins whose genes are expressed in the different region of the human adult brain further agree with the neuronal origin of the unusual archaeological find. The conversion of human tissue into glass is the result of sudden exposure to scorching volcanic ash and the concomitant rapid drop in temperature. The eruptive-induced process of natural vitrification, locking the cellular structure of the CNS, allowed us to study possibly the best known example in archaeology of extraordinarily well-preserved human neuronal tissue from the brain and spinal cord.
Assuntos
Encéfalo/anatomia & histologia , Sistema Nervoso Central/anatomia & histologia , Erupções Vulcânicas , Arqueologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Sistema Nervoso Central/fisiologia , Bases de Dados Factuais , Humanos , Processamento de Imagem Assistida por Computador , Cinesinas/genética , Masculino , Microscopia Eletrônica de Varredura , Espectrometria por Raios X , Medula Espinal/anatomia & histologia , Medula Espinal/fisiologia , Preservação de Tecido , Adulto JovemRESUMO
PURPOSE: To investigate whether the addition of sodium-DNA (Na-DNA) to chlorhexidine (CHX)-containing mouthwash influenced morphology and viability of a reconstituted human oral epithelium (ROE), and protects ROE against oxidative stress. METHODS: Multi-layered 0.5 cm² ROE specimens were positioned inside a continuous flow bioreactor and grown air-lifted for 24 hours. They were treated with phosphate-buffered saline (PBS) (n= 16) or 1 vol% H2O2 for 1 minute (n= 16). Then, they were treated for 5 (n= 8) or 30 minutes (n= 8) with the experimental mouthwash solutions containing: 0.2 wt% CHX, 0.2 wt% CHX + 0.2 wt% Na-DNA, 0.2 wt% Na-DNA, PBS. After 60 minutes washout specimens were subjected to tetrazolium-based viability assay (MTT) confocal laser-scanning microscopy (CLSM), and histological evaluation using optical microscopy and transmission electron microscopy (TEM). RESULTS: ROE treated with Na-DNA for 30 minutes revealed significantly higher viability than PBS, and CHX + Na-DNA showed higher viability after 30-minute treatment than after 5 minutes, suggesting a significant protective activity of Na-DNA. Moreover, the protective effect of Na-DNA on cell viability was higher after the induction of oxidative stress. After treatment with CHX, CLSM revealed cell stress, leading to cell death in the outer layer. On the contrary, specimens treated with Na-DNA showed a much lower number of dead cells compared to PBS, both in the absence or presence of oxidative stress. Histological examination showed that the protective action of Na-DNA formulations reached more in-depth into the epithelium exposed to oxidative stress, due to intercellular spaces opening in the outer epithelium layers, giving way to Na-DNA to the inner parts of the epithelium. It can be concluded that Na-DNA had a topical protective activity when applied for 30 minutes unless the epithelium barrier is damaged, allowing it to act more in-depth. CLINICAL SIGNIFICANCE: Na-DNA showed a clear and protective action against cellular degeneration due to oxidative stress and, partly, to the exposure to CHX. Its addition to chlorhexidine mouthwash or gels could be clinically helpful in contrasting the detrimental activity of CHX on oral tissues, and in the preservation of cell viability, control of inflammation and wound healing.
Assuntos
Peróxido de Hidrogênio , Antissépticos Bucais/farmacologia , Antissépticos Bucais/toxicidade , Reatores Biológicos , DNA , Humanos , SódioRESUMO
A variety of pathophysiological mechanisms are implicated in Huntington's disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.
Assuntos
Doença de Huntington , Animais , Colesterol , Corpo Estriado , Modelos Animais de Doenças , Proteína Huntingtina/genética , Doença de Huntington/tratamento farmacológico , Camundongos , Camundongos Transgênicos , SinapsesRESUMO
Theranostics based on two-photon excitation of therapeutics in the NIR region is an emerging and powerful tool in cancer therapy since this radiation deeply penetrates healthy biological tissues and produces selective cell death. Aggregates of gold nanoparticles coated with glutathione corona functionalized with the dansyl chromophore (a-DG-AuNPs) were synthesized and found efficient nanodevice for applications in photothermal therapy (PTT). Actually the nanoparticle aggregation enhances the quenching of radiative excitation and the consequent conversion into heat. The a-DG-AuNPs are readily internalized in Hep G2 where the chromophore acts as both antenna and transducer of the NIR radiation under two-photons excitation, determining efficient cell ablation via photothermal effect.
Assuntos
Terapia com Luz de Baixa Intensidade/métodos , Nanopartículas Metálicas/administração & dosagem , Neoplasias/terapia , Terapia Fototérmica/métodos , Nanomedicina Teranóstica/métodos , Animais , Linhagem Celular Tumoral , Terapia Combinada/instrumentação , Terapia Combinada/métodos , Fluorescência , Glutationa/química , Ouro/química , Humanos , Raios Infravermelhos/uso terapêutico , Lasers , Terapia com Luz de Baixa Intensidade/instrumentação , Nanopartículas Metálicas/química , Camundongos , Neoplasias/patologia , Fosfatidilcolinas/química , Fótons/uso terapêutico , Terapia Fototérmica/instrumentação , Nanomedicina Teranóstica/instrumentaçãoRESUMO
A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Disfunção Cognitiva/enzimologia , Doença de Huntington/enzimologia , Proteínas de Membrana/metabolismo , Densidade Pós-Sináptica/enzimologia , Proteína ADAM10/genética , Adulto , Idoso , Secretases da Proteína Precursora do Amiloide/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Masculino , Proteínas de Membrana/genética , Camundongos Transgênicos , Pessoa de Meia-Idade , Densidade Pós-Sináptica/genética , Densidade Pós-Sináptica/patologiaRESUMO
Intellectual disability affects 2-3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1-GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1-GluA2 binding restored synaptic function in Tm4sf2-/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2-/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.
Assuntos
Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/metabolismo , Proteínas de Membrana/deficiência , Proteínas do Tecido Nervoso/deficiência , Psicotrópicos/farmacologia , Receptores de AMPA/metabolismo , Regulação Alostérica , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Deficiência Intelectual/patologia , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Técnicas de Cultura de TecidosRESUMO
AIMS: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. METHODS AND RESULTS: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. CONCLUSION: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.
Assuntos
Potenciais de Ação , Caveolina 3/genética , Caveolina 3/metabolismo , Fibroblastos/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Células 3T3 , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Cavéolas/metabolismo , Caveolina 1/deficiência , Caveolina 1/genética , Simulação por Computador , Fibroblastos/ultraestrutura , Frequência Cardíaca , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico , Cinética , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Camundongos , Camundongos Knockout , Modelos Cardiovasculares , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contração Miocárdica , Miócitos Cardíacos/ultraestrutura , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ratos Sprague-Dawley , TransfecçãoRESUMO
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.