RESUMO
Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.
Assuntos
Fígado Gorduroso , Interleucina-6 , Humanos , Camundongos , Animais , Interleucina-6/metabolismo , Receptor gp130 de Citocina/metabolismo , Gotículas Lipídicas/metabolismo , Hepatócitos/metabolismo , Glicoproteínas , LipídeosRESUMO
The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.
Assuntos
Doença de Alzheimer , Tauopatias , Doença de Alzheimer/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Tauopatias/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.
Assuntos
Hepatócitos , Hepatopatias , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Hepatopatias/metabolismo , Camundongos , RatosRESUMO
The present study was intended to combine three therapeutic approaches in a well-defined rat model of spinal cord injury, a lateral hemisection at thoracic level. A guidance channel was implanted at the lesion site. This channel was seeded with native Schwann cells or Schwann cells that had been previously transduced with a lentiviral vector carrying the GDNF gene. Thereafter, these experiences were reproduced in animals injected with lentiviral vectors carrying a shRNA for GFAP (Lv-shGFAP), which has recently been shown to block glial scar formation. Functional evaluations showed that Lv-shGFAP induced a significant improvement in recovery in animals grafted with Schwann cells. Histological studies demonstrated the outgrowth of axons in the guidance channel containing Schwann cells transduced or not with GDNF. This axonal growth was enhanced in rats receiving Lv-shGFAP vector. Also, a significant increase of serotonergic innervation of the injured hemicord, distal to the lesion, was found only in animals treated with Lv-shGFAP vectors. Importantly, this study confirms that glial scar formation is a major impediment for axonal sprouting after spinal cord injury, and emphasizes the importance of serotonergic innervation for locomotor function. Moreover we show a significant additive effect of a combinatorial approach to axonal regeneration in the injured spinal cord.
Assuntos
Neuroglia/patologia , Células de Schwann/transplante , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Animais , Sobrevivência Celular , Cicatriz/patologia , Feminino , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lentivirus/genética , Locomoção , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Neurônios Serotoninérgicos/fisiologia , Medula Espinal/patologiaRESUMO
In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)-mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral-mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral-mediated RNAi in SCI.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Vimentina/metabolismo , Análise de Variância , Animais , Astrócitos/metabolismo , Axônios/fisiologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/fisiologia , Proteína Glial Fibrilar Ácida/genética , Lentivirus/genética , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serotonina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Vimentina/genéticaRESUMO
Neuropathic pain after peripheral nerve injury, associated with local neuroinflammation in the spinal cord, is a severe incapacitating condition with which clinical treatment remains challenging. Inflammatory molecules signal through various intracellular transduction pathways, activation of which may amplify and cause spreading of the inflammatory response. We showed recently that spinal nerve lesion leads to rapid activation of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signal transduction pathway in dorsal spinal cord microglia in relation with enhanced levels of spinal interleukin-6 (IL-6) protein. Here, we selectively inactivated JAK/STAT3 signaling in rat dorsal spinal cord glia through local, lentiviral-mediated production of the suppressor of cytokine signaling SOCS3, a physiologic inhibitory protein of JAK/STAT3, and analyzed its consequences in a preclinical model of neuropathic pain. The targeted blockade of JAK/STAT3 activity prevented the abnormal expression of IL-6, CC chemokine ligand CCL2, and activating transcription factor ATF3 induced in the spinal cord by chronic constriction injury of the sciatic nerve (CCI) and substantially attenuated mechanical hypersensitivity (allodynia) in rats. In naive rats, intrathecal administration of a proalgesic cytokine IL-6 rapidly activated microglial JAK/STAT3 and induced downstream changes closely resembling CCI-evoked alterations. We identified downstream mechanisms through which JAK/STAT3 pathway activation leads to the spreading of neuroinflammation. Our findings reveal that JAK/STAT3 signaling plays a major role in spinal cord plasticity and mechanical allodynia associated with peripheral nerve injury.
Assuntos
Mediadores da Inflamação/antagonistas & inibidores , Janus Quinases/antagonistas & inibidores , Dor/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Neuropatia Ciática/metabolismo , Medula Espinal/metabolismo , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Humanos , Mediadores da Inflamação/fisiologia , Janus Quinases/fisiologia , Marmota , Dor/etiologia , Medição da Dor/métodos , Estimulação Física/métodos , Ratos , Fator de Transcrição STAT3/fisiologia , Neuropatia Ciática/complicações , Transdução de Sinais/fisiologia , Medula Espinal/patologia , Proteína 3 Supressora da Sinalização de CitocinasRESUMO
BACKGROUND: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). METHODS AND FINDINGS: In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. CONCLUSIONS: Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.
Assuntos
Axônios , Sobrevivência Celular/genética , Técnicas de Transferência de Genes , Neuroglia/fisiologia , Neurônios/citologia , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Primers do DNA , Inativação Gênica , Vetores Genéticos , Proteína Glial Fibrilar Ácida/genética , Imuno-Histoquímica , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/citologia , Neurônios/metabolismo , Interferência de RNA , Vimentina/genéticaRESUMO
Cerebellar Purkinje cells elaborate one of the most complex dendritic arbors among neurons to integrate the numerous signals they receive from the cerebellum circuitry. Their dendritic differentiation undergoes successive, tightly regulated phases of development involving both regressive and growth events. Although many players regulating the late phases of Purkinje cell dendritogenesis have been identified, intracellular factors controlling earlier phases of dendritic development remain mostly unknown. In this study, we explored the biological properties and functions of SCLIP, a protein of the stathmin family, in Purkinje cell dendritic differentiation and cerebellum development. Unlike the other stathmins, SCLIP is strongly expressed in Purkinje cells during cerebellar development and accumulates in their dendritic processes at a critical period of their formation and outgrowth. To reveal SCLIP functions, we developed a lentiviral-mediated approach on cerebellar organotypic cultures to inhibit or increase its expression in Purkinje cells in their tissue environment. Depletion of SCLIP promoted retraction of the Purkinje cell primitive process and then prevented the formation of new dendrites at early stages of postnatal development. It also prevented their elongation and branching at later phases of differentiation. Conversely, SCLIP overexpression promoted dendritic branching and development. Together, our results demonstrate for the first time that SCLIP is crucial for both the formation and proper development of Purkinje cell dendritic arbors. SCLIP appears thus as a novel and specific factor that controls the early phases of Purkinje cell dendritic differentiation during cerebellum development.
Assuntos
Diferenciação Celular/fisiologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Dendritos/metabolismo , Fatores de Crescimento Neural/fisiologia , Células de Purkinje/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Linhagem Celular , Cerebelo/anatomia & histologia , Cerebelo/embriologia , Dendritos/genética , Humanos , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Técnicas de Cultura de Órgãos , Células de Purkinje/citologia , RatosRESUMO
RNA interference (RNAi) mediated by expression of short hairpin RNAs (shRNAs) is a powerful tool for efficiently suppressing target genes. The approach allows studies of the function of individual genes and may also be applied to human therapy. However, in many instances regulation of RNAi by administration of a small inducer molecule will be required. To date, the development of appropriate regulatory systems has been hampered by the few possibilities for modification within RNA polymerase III promoters capable of driving efficient expression of shRNAs. We have developed an inducible minimal RNA polymerase III promoter that is activated by a novel recombinant transactivator in the presence of doxycycline (Dox). The recombinant transactivator and the engineered promoter together form a system permitting regulation of RNAi by Dox-induced expression of shRNAs. Regulated RNAi was mediated by one single lentiviral vector, blocked the expression of green fluorescent protein (GFP) in a GFP-expressing HEK 293T derived cell line and suppressed endogenous p53 in wild-type HEK 293T, MCF-7 and A549 cells. RNA interference was induced in a dose- and time-dependent manner by administration of Dox, silenced the expression of both target genes by 90% and was in particular reversible after withdrawal of Dox.