RESUMO
BACKGROUND: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. METHODS: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters' candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. RESULTS: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. CONCLUSIONS: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , Traumatismos da Medula Espinal , Bexiga Urinária Hiperativa , Animais , Gânglios Espinais/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Ratos , Células Receptoras Sensoriais/metabolismo , Traumatismos da Medula Espinal/metabolismo , Bexiga Urinária Hiperativa/terapiaRESUMO
Friedreich's ataxia is the most common hereditary ataxia for which there is no cure or approved treatment at present. However, therapeutic developments based on the understanding of pathological mechanisms underlying the disease have advanced considerably, with the implementation of cellular models that mimic the disease playing a crucial role. Human olfactory ecto-mesenchymal stem cells represent a novel model that could prove useful due to their accessibility and neurogenic capacity. Here, we isolated and cultured these stem cells from Friedreich´s ataxia patients and healthy donors, characterizing their phenotype and describing disease-specific features such as reduced cell viability, impaired aconitase activity, increased ROS production and the release of cytokines involved in neuroinflammation. Importantly, we observed a positive effect on patient-derived cells, when frataxin levels were restored, confirming the utility of this in vitro model to study the disease. This model will improve our understanding of Friedreich´s ataxia pathogenesis and will help in developing rationally designed therapeutic strategies.
Assuntos
Ataxia de Friedreich/metabolismo , Mucosa Olfatória/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/metabolismo , Aconitato Hidratase/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamação/metabolismoRESUMO
The contribution of the pleiotropic hormone Prolactin (PRL) to several physiological and pathological processes is still unknown. To clarify the role of PRL in these processes during the last decade, different human PRL antagonists have been produced to either partially or fully block the wild type hormone activity. In this work, we have cloned these wild type and antagonist sequences in lentivectors (LV) to express them as recombinant self-processing polypeptides by employing a P2A sequence (hPRL-P2A-GFP). We show that these LVs can efficiently transduce and express the hPRL proteins in different cell types and that the P2A sequence does not affect their activities. Additionally, we have tested their activities in paracrine and autocrine cell culture experiments. Our results demonstrate that these recombinant hPRL-P2A proteins are bioactive in both paracrine and autocrine modes, highlighting the potential usefulness of these hPRL-containing LVs for determining the contribution of hPRL to different biological processes.
Assuntos
Lentivirus/genética , Prolactina/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Animais , Comunicação Autócrina , Técnicas de Cultura de Células , Linhagem Celular , Vetores Genéticos , Células HEK293 , Humanos , Comunicação Parácrina , Prolactina/metabolismo , RatosRESUMO
A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.
Assuntos
Toxinas Botulínicas/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/metabolismo , Herpesvirus Humano 1/genética , Neurotoxinas/genética , Proteínas SNARE/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Células Cultivadas , Feminino , Gânglios Espinais/virologia , Vetores Genéticos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/virologiaRESUMO
BACKGROUND: Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disease caused by mutations in the frataxin gene (FXN), which lead to reduced levels of the essential mitochondrial protein frataxin. Currently, there is no effective cure. METHODS: With the aim of developing a gene therapy for FA neuropathology, we describe the construction and preliminary characterization of a high-capacity nonreplicative genomic herpes simplex virus type 1 vector (H24B-FXNlac vector) carrying a reduced version of the human FXN genomic locus, comprising the 5-kb promoter and the FXN cDNA with the inclusion of intron 1. RESULTS: We show that the transgene cassette contains the elements necessary to preserve physiological neuronal regulation of human FXN expression. Transduction of cultured fetal rat dorsal root ganglia neurons with the H24B-FXNlac vector results in sustained expression of human FXN transcripts and frataxin protein. Rat footpad inoculation with the H24B-FXNlac vector results in human FXN transgene delivery to the dorsal root ganglia, with expression persisting for at least 1 month. CONCLUSIONS: The results of the present study support the feasibility of using this vector for sustained neuronal expression of human frataxin for FA gene therapy.
Assuntos
Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Proteínas de Ligação ao Ferro/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/terapia , Gânglios Espinais/citologia , Terapia Genética/métodos , Células HEK293 , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo , Transgenes/genética , Células Vero , FrataxinaRESUMO
BACKGROUND: Although herpes simplex virus type 1 (HSV-1) has outstanding properties for gene delivery vectors and its genome is available in bacterial artificial chromosomes (BACs) for mutagenesis studies, one impediment is the presence of approximately 15.4 kb of DNA sequences that are duplicated in the HSV-1 genome, complicating vector construction and stability. METHODS: As a useful platform for building HSV-1 vectors, we have constructed a fully haploid HSV-1 genome BAC by deletion of one of these repeats, confirming that viral propagation in culture is not impaired. We used this ΔIR mutant to subsequently investigate whether the insertion of tetracycline-responsive tetO elements into the ICP34.5-ICP0 gene region can be used to control HSV-1 lytic replication. RESULTS: The results of the present study show that ΔIR mutants deleted for ICP34.5 are viable for replication but not when the ICP0 promoter is also disrupted, thus indicating that regulation of infected cell protein 0 (ICP0) levels in the absence of ICP34.5 could be a viable means for controlling growth of HSV-1 vectors. Surprisingly, however, the tetO elements inserted into the ICP0 promoter did not confer ligand responsiveness to growth or ICP0 expression. Further analysis by transfection experiments revealed that ICP0 itself interferes with the tetracycline switch and reduces the the inducibility of this system. CONCLUSIONS: Our new haploid HSV-1 BAC is a useful platform for building multiply deleted HSV-1 vectors. Deletion of the gene for ICP34.5 in this backbone renders viral growth dependent on ICP0, although ICP0 expression could not be regulated by tet-responsive transcriptional regulators.
Assuntos
Vetores Genéticos/genética , Genoma Viral/genética , Haploidia , Herpesvirus Humano 1/genética , Linhagem Celular Tumoral , Deleção de Genes , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Mutação , Regiões Promotoras Genéticas , Elementos de Resposta/genética , Tetraciclinas/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Linckosides are members of the steroid glycoside family isolated from the starfish Linckia laevigata. These natural compounds have notable neuritogenic activity and synergistic effects on NGF-induced neuronal differentiation of PC12 cells. Neurogenic factors or molecules that are able to mimic their activities are known to be involved in the survival, proliferation and migration of neurons and glial cells; however how glial cells respond to specific neurogenic molecules such as linckosides has not been investigated. This study aimed to examine the effect of three different linckosides (linckoside A, B and granulatoside A) on the morphological properties, proliferation and migration of human olfactory ensheathing cells (hOECs). The proliferation rate after all the treatments was higher than control as detected by MTS assay. Additionally, hOECs displayed dramatic morphological changes characterized by a higher number of processes after linckoside treatment. Interestingly changes in microtubule organization and expression levels of some early neuronal markers (GAP43 and ßIII-tubulin) were also observed. An increase in the phosphorylation of ERK 1/2 after addition of the compounds suggests that this pathway may be involved in the linckoside-mediated effects particularly those related to morphological changes. These results are the first description of the stimulating effects of linckosides on hOECs and raise the potential for this natural compound or its derivatives to be used to regulate and enhance the therapeutic properties of OECs, particularly for cell transplantation therapies.
Assuntos
Proliferação de Células , Neuroglia/efeitos dos fármacos , Bulbo Olfatório/citologia , Saponinas/farmacologia , Linhagem Celular , Humanos , Microtúbulos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neuroglia/fisiologiaRESUMO
We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Neuroglia/citologia , Bulbo Olfatório/citologia , Esferoides Celulares/citologia , Animais , Comunicação Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Camundongos , Microfluídica/métodos , Neuroglia/fisiologia , Bulbo Olfatório/fisiologia , Impressão Tridimensional , Esferoides Celulares/fisiologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease which currently has no cure. Research using rodent ALS models transgenic for mutant superoxide dismutase 1 (SOD1) has implicated that glial-neuronal interactions play a major role in the destruction of motor neurons, but the generality of this mechanism is not clear as SOD1 mutations only account for less than 2% of all ALS cases. Recently, this hypothesis was backed up by observation of similar effects using astrocytes derived from post-mortem spinal cord tissue of ALS patients which did not carry SOD1 mutations. However, such necropsy samples may not be easy to obtain and may not always yield viable cell cultures. Here, we have analysed olfactory mucosa (OM) cells, which can be easily isolated from living ALS patients. Disease-specific changes observed when ALS OM cells were co-cultured with human spinal cord neurons included decreased neuronal viability, aberrant neuronal morphology and altered glial inflammatory responses. Our results show the potential of OM cells as new cell models for ALS.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios/patologia , Mucosa Olfatória/patologia , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Western Blotting , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Neurônios/metabolismo , Mucosa Olfatória/metabolismo , Cultura Primária de Células , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Vimentina/metabolismoRESUMO
One of the promising strategies for neural repair therapies is the transplantation of olfactory ensheathing cells (OECs) which are the glial cells of the olfactory system. We evaluated the effects of curcumin on the behaviour of mouse OECs to determine if it could be of use to further enhance the therapeutic potential of OECs. Curcumin, a natural polyphenol compound found in the spice turmeric, is known for its anti-cancer properties at doses over 10 µM, and often at 50 µM, and it exerts its effects on cancer cells in part by activation of MAP kinases. In contrast, we found that low-dose curcumin (0.5 µM) applied to OECs strikingly modulated the dynamic morphology, increased the rate of migration by up to 4-fold, and promoted significant proliferation of the OECs. Most dramatically, low-dose curcumin stimulated a 10-fold increase in the phagocytic activity of OECs. All of these potently stimulated behavioural characteristics of OECs are favourable for neural repair therapies. Importantly, low-dose curcumin gave a transient activation of p38 kinases, which is in contrast to the high dose curcumin effects on cancer cells in which these MAP kinases tend to undergo prolonged activation. Low-dose curcumin mediated effects on OECs demonstrate cell-type specific stimulation of p38 and ERK kinases. These results constitute the first evidence that low-dose curcumin can modulate the behaviour of olfactory glia into a phenotype potentially more favourable for neural repair and thereby improve the therapeutic use of OECs for neural repair therapies.
Assuntos
Movimento Celular/efeitos dos fármacos , Curcumina/farmacologia , Bulbo Olfatório/citologia , Fagocitose/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Separação Celular , Forma Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Imunofluorescência , Ontologia Genética , Masculino , Camundongos Transgênicos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Mucosa Olfatória/citologia , Transdução de Sinais/efeitos dos fármacosRESUMO
This introductory paper gathers general considerations on the biosafety of virus-derived vectors that are used in human gene therapy and/or vaccination. The importance to assess the potential risks for human health and the environment related to the use of genetically modified organisms (GMO) in this case genetically modified viral vectors is highlighted by several examples. This environmental risk assessment is one of the requirements within the European regulatory framework covering the conduct of clinical trials using GMO. Risk assessment methodologies for the environmental risk assessment of genetically modified virus-derived vectors have been developed.
Assuntos
Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Vacinação/efeitos adversos , Vacinação/métodos , Vírus/genética , Animais , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/normas , Substâncias Perigosas/efeitos adversos , Humanos , Organismos Geneticamente Modificados/genética , Medição de RiscoRESUMO
Basic Protocol 1 describes the generation of helper virus stocks. Preparation of recombinant amplicon vector particles by transfection of amplicon and superinfection of helper virus into cells, and harvesting of packaged particles, is delineated in Basic Protocol 3. Thorough characterization of each amplicon viral vector stock involves measuring (1) the helper virus plaque-forming units per ml (pfu/ml) on 2-2 cells and (2) the amplicon stock infectious units per ml (iu/ml) on PC12 cells. The Support Protocols detail methods for determining titers of helper virus by plaque assay, and of amplicon stocks by vector assay.
Assuntos
Vírus Defeituosos/crescimento & desenvolvimento , Vetores Genéticos/fisiologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Cultura de Vírus/métodos , Animais , Linhagem Celular/virologia , Chlorocebus aethiops , Cricetinae , Replicação do DNA , DNA Recombinante/genética , DNA Viral/genética , Vírus Defeituosos/genética , Vírus Defeituosos/isolamento & purificação , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Genoma Viral , Vírus Auxiliares/fisiologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Mesocricetus , Ratos , Origem de Replicação , Superinfecção , Transfecção , Células Vero/virologia , Carga Viral , Ensaio de Placa Viral , Replicação ViralRESUMO
The majority of humans have been infected with Herpes Simplex Virus Type 1 (HSV-1) and harbor its viral DNA in the latent form within neurons for lifetime. This, combined with the absence of serious adverse effects due to HSV-1 derived vectors in clinical trials so far, highlight the potential to use this virus to develop neuronal gene transfer vectors which are transparent to the host, allowing the effects of the transgene to act without interference from the transfer system eg., for functional genomics in basic neuroscience or gene therapy of neurological disorders. On the other hand, other HSV-1 derived vectors which also have a promising perspective in the clinic, are designed to have enhanced cytotoxicity in certain cell types, as in the case of oncolytic vectors. Understanding virus-host interactions is fundamental not only to the success of these gene therapy vectors but also with respect to identifying and minimizing biohazards associated with their use. In this review we discuss characteristics of HSV-1 and gene therapy vectors derived from this virus which are useful to consider in the context of biosafety risk assessment and risk management.
Assuntos
Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Herpesvirus Humano 1/genética , Animais , Ensaios Clínicos como Assunto , Terapia Genética/métodos , Terapia Genética/normas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Medição de RiscoRESUMO
Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hexoquinase/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Mitocôndrias/metabolismo , Rotenona/toxicidade , Desacopladores/toxicidadeRESUMO
Although human olfactory mucosa derived cells (OMC) have been used in animal models and clinical trials with CNS repair purposes, the exact identity of these cells in culture with respect to their tissue of origin is not fully understood and their neuroregenerative capacity in vitro has not yet been demonstrated. In this study we have compared human OMC with human ensheathing glia from olfactory bulb (OB) and human fibroblasts from skin and lung. Our results indicate that these different cultured cell types exhibit considerable overlap of antigenic markers such that it is presently not possible to distinguish them immunocytochemically. However, in rat retinal ganglion neuron coculture assays the axonal regenerative activity of OMC and OB ensheathing glia was dramatically higher than that exhibited by all fibroblast samples, confirming neuroregenerative activity as a unique property shared by cultured cells derived from the human olfactory system.
Assuntos
Axônios/fisiologia , Fibroblastos/fisiologia , Pulmão , Regeneração Nervosa/fisiologia , Mucosa Olfatória/citologia , Células Ganglionares da Retina/citologia , Pele , Animais , Biomarcadores/análise , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Humanos , Pulmão/citologia , Neuroglia/citologia , Bulbo Olfatório/citologia , Mucosa Olfatória/metabolismo , Ratos , Células Ganglionares da Retina/metabolismo , Pele/citologiaRESUMO
Olfactory ensheathing glia (OEG) cells are known to facilitate repair following axotomy of adult neurons, although the molecular mechanisms involved are not fully understood. We previously identified plasminogen activator inhibitor-1 (PAI-1), proteinase-activated receptor-1 (PAR-1), and thrombomodulin (TM) as candidates to regulate rat OEG-dependent axonal regeneration. In this study, we have validated the involvement of these proteins in promoting axonal regeneration by immortalized human OEGs. We studied the effect of silencing these proteins in OEGs on their capacity to promote the regeneration of severed adult retinal ganglion cells (RGCs) axons. Our results support the role of glial PAI-1 as a downstream effector of PAR-1 in promoting axon regeneration. In contrast, we found that TM inhibits OEG induced-axonal regeneration. We also assessed the signaling pathways downstream of PAR-1 that might modulate PAI-1 expression, observing that specifically inhibiting Gα(i), Rho kinase, or PLC and PKC downregulated the expression of PAI-1 in OEGs, with a concomitant reduction in OEG-dependent axon regeneration in adult RGCs. Our findings support an important role for the thrombin system in regulating adult axonal regeneration by OEGs.
Assuntos
Axônios/metabolismo , Regeneração Nervosa/fisiologia , Neuroglia/metabolismo , Bulbo Olfatório/citologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Axônios/efeitos dos fármacos , Axotomia/efeitos adversos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Neuroglia/química , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptor PAR-1/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Trombomodulina/metabolismo , Transdução GenéticaRESUMO
Ensheathing glia have been demonstrated to have neuroregenerative properties but this cell type from human sources has not been extensively studied because tissue samples are not easily obtained, primary cultures are slow growing, and human cell lines are not available. We previously isolated immortalized ensheathing glia by gene transfer of BMI1 and telomerase catalytic subunit into primary cultures derived from olfactory bulbs of an elderly human cadaver donor. These cells escape the replicative senescence characteristic of primary human cells while conserving antigenic and neuroregenerative properties of ensheathing glia, but their low proliferative rate in culture complicates their utility as cell models and their application for preclinical cell therapy experiments. In this study we describe the use of a conditional SV40 T antigen (TAg) transgene to generate human ensheathing glia cell lines, which are easy to maintain due to their robust growth in culture. Although these fast growing clones exhibited polyploid karyotypes frequently observed in cells immortalized by TAg, they did not acquire a transformed phenotype, all of them maintaining neuroregenerative capacity and antigenic markers typical of ensheathing glia. These markers were also retained even after elimination of the TAg transgene using Cre/LoxP technology, although the cells died shortly after, confirming that their survival depended on the presence of the immortalizing genes. We have also demonstrated here the feasibility of using these human cell lines in animal models by genetically marking the cells with GFP and implanting them into the injured spinal cord of immunosuppressed rats. Our conditionally immortalized human ensheathing glia cell lines will thus serve as useful tools for advancing cell therapy approaches and understanding neuroregenerative mechanisms of this unique cell type.
Assuntos
Regeneração Nervosa/fisiologia , Neuroglia/citologia , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Morte Celular , Linhagem Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Transplante de Células , Humanos , Imuno-Histoquímica , Terapia de Imunossupressão , Integrases/metabolismo , Cariotipagem , Camundongos , Camundongos Nus , Neuroglia/transplante , Ratos , Transgenes/genética , Transplante HeterólogoRESUMO
Alzheimer's disease (AD) is the most frequent neurodegenerative disorder leading to dementia in the aged human population. It is characterized by the presence of two main pathological hallmarks in the brain: senile plaques containing beta-amyloid peptide and neurofibrillary tangles (NFTs), consisting of fibrillar polymers of abnormally phosphorylated tau protein. Both of these histological characteristics of the disease have been simulated in genetically modified animals, which today include numerous mouse, fish, worm, and fly models of AD. The objective of this review is to present some of the main animal models that exist for reproducing symptoms of the disorder and their advantages and shortcomings as suitable models of the pathological processes. Moreover, we will discuss the results and conclusions which have been drawn from the use of these models so far and their contribution to the development of therapeutic applications for AD.
RESUMO
A typical feature of Parkinson's disease is the progressive loss of dopaminergic neurons in the substantia nigra, in which inhibition of mitochondrial complex I activity may play an important role. Rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inhibit the mitochondrial complex I and they cause the death of substantia nigra dopaminergic neurons, thereby providing acute murine models of Parkinson's disease. We have found that increasing mitochondrial hexokinase II activity can prevent cell death in neuronal cultures treated with rotenone. As a result, we have studied the effects of hexokinase II gene transfer in vivo using a herpes simplex virus type 1 (HSV-1) amplicon vector. The placHK2 amplicon vector was injected into substantia nigra of mice that were subsequently administered rotenone or MPTP. Overexpression of hexokinase II prevented both rotenone and MPTP-induced dopaminergic neuronal cell death, as well as reducing the associated motor defects. Our results provide the first proof-of-principle that hexokinase II protects against dopaminergic neurodegeneration in vivo, emphasizing the role of this enzyme in promoting neuronal survival. Thus, the increase of hexokinase II expression by gene transfer or other means represents a promising approach to treat Parkinson's and other neurodegenerative diseases.
Assuntos
Morte Celular , Terapia Genética , Hexoquinase/genética , Hexoquinase/uso terapêutico , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/terapia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Catalepsia/induzido quimicamente , Catalepsia/metabolismo , Catalepsia/terapia , Dopamina/metabolismo , Vetores Genéticos , Herpesvirus Humano 1/genética , Hexoquinase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Degeneração Neural/terapia , Neurônios/metabolismo , Neurônios/patologia , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Rotenona , Substância Negra/metabolismo , Substância Negra/patologia , Resultado do TratamentoRESUMO
Reversible immortalization holds great potential for primary tissue expansion to develop cell-based therapies as well as for basic research. Human olfactory ensheathing glia (hOEG) are promising candidates for treating spinal cord injury and for studying extrinsic neuroregenerative mechanisms. We used lentivectors with Cre/loxP technology to achieve reversible gene transfer of BMI1, SV40 large T antigen (TAg), a short hairpin RNA against p53 (shp53), and the catalytic subunit of telomerase (TERT) in primary cultures of hOEG from human donor cadaver olfactory bulbs. Several combinations of these genes were able to immortalize hOEG, conserving their antigenic markers and neuroregenerative properties but only those transduced by BMI1/TERT did not accumulate karyotypic alterations or increase senescence marker levels. Strikingly, these were also the only cells which continued to proliferate after transgene removal by Cre recombinase delivery, whereas hOEG immortalized by shp53 or TAg in combination with TERT entered into growth arrest and died. These data support the idea that immortalization and halting senescent changes are separate processes; hOEG immortalized by BMI1/TERT can revert back to their former primary cell replicative state when deimmortalized, whereas those transduced by the other combinations depend on the presence of these transgenes to maintain their aberrant proliferative state.