RESUMEN

Friedreich ataxia is a hereditary neurodegenerative disorder resulting from reduced levels of the protein frataxin due to an expanded GAA repeat in the FXN gene. This deficiency causes progressive degeneration of specific neuronal populations in the cerebellum and the consequent loss of movement coordination and equilibrium, which are some of the main symptoms observed in affected individuals. Like in other neurodegenerative diseases, previous studies suggest that glial cells could be involved in the neurodegenerative process and disease progression in patients with Friedreich ataxia. In this work, we followed and characterized the progression of changes in the cerebellar cortex in the latest version of Friedreich ataxia humanized mouse model, YG8-800 (Fxnnull:YG8s(GAA)>800), which carries a human FXN transgene containing >800 GAA repeats. Comparative analyses of behavioral, histopathological, and biochemical parameters were conducted between the control strain Y47R and YG8-800 mice at different time points. Our findings revealed that YG8-800 mice exhibit an ataxic phenotype characterized by poor motor coordination, decreased body weight, cerebellar atrophy, neuronal loss, and changes in synaptic proteins. Additionally, early activation of glial cells, predominantly astrocytes and microglia, was observed preceding neuronal degeneration, as was increased expression of key proinflammatory cytokines and downregulation of neurotrophic factors. Together, our results show that the YG8-800 mouse model exhibits a stronger phenotype than previous experimental murine models, reliably recapitulating some of the features observed in humans. Accordingly, this humanized model could represent a valuable tool for studying Friedreich ataxia molecular disease mechanisms and for preclinical evaluation of possible therapies.

Asunto(s)

Corteza Cerebelosa , Modelos Animales de Enfermedad , Frataxina , Ataxia de Friedreich , Ratones Transgénicos , Neuroglía , Ataxia de Friedreich/patología , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/genética , Animales , Neuroglía/metabolismo , Neuroglía/patología , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/patología , Ratones , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Humanos , Degeneración Nerviosa/patología , Degeneración Nerviosa/metabolismo , Masculino

RESUMEN

BACKGROUND: Friedreich's ataxia is a rare hereditary neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Similar to other neurodegenerative pathologies, previous studies suggested that astrocytes might contribute to the progression of the disease. To fully understand the mechanisms underlying neurodegeneration in Friedreich's ataxia, we investigated the reactivity status and functioning of cultured human astrocytes after frataxin depletion using an RNA interference-based approach and tested the effect of pharmacologically modulating the SHH pathway as a novel neuroprotective strategy. RESULTS: We observed loss of cell viability, mitochondrial alterations, increased autophagy and lipid accumulation in cultured astrocytes upon frataxin depletion. Besides, frataxin-deficient cells show higher expression of several A1-reactivity markers and release of pro-inflammatory cytokines. Interestingly, most of these defects were prevented by chronically treating the cells with the smoothened agonist SAG. Furthermore, in vitro culture of neurons with conditioned medium from frataxin-deficient astrocytes results in a reduction of neuronal survival, neurite length and synapse formation. However, when frataxin-deficient astrocytes were chronically treated with SAG, we did not observe these alterations in neurons. CONCLUSIONS: Our results demonstrate that the pharmacological activation of the SHH pathway could be used as a target to modulate astrocyte reactivity and neuron-glia interactions to prevent neurodegeneration in Friedreich's ataxia.

Asunto(s)

Ataxia de Friedreich , Enfermedades Neurodegenerativas , Síndromes de Neurotoxicidad , Astrocitos/metabolismo , Ataxia de Friedreich/tratamiento farmacológico , Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Humanos , Proteínas de Unión a Hierro , Mitocondrias , Enfermedades Neurodegenerativas/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Frataxina

RESUMEN

Friedreich's ataxia is an autosomal recessive neurogenetic disease that is mainly associated with atrophy of the spinal cord and progressive neurodegeneration in the cerebellum. The disease is caused by a GAA-expansion in the first intron of the frataxin gene leading to a decreased level of frataxin protein, which results in mitochondrial dysfunction. Currently, there is no effective treatment to delay neurodegeneration in Friedreich's ataxia. A plausible therapeutic approach is gene therapy. Indeed, Friedreich's ataxia mouse models have been treated with viral vectors en-coding for either FXN or neurotrophins, such as brain-derived neurotrophic factor showing promising results. Thus, gene therapy is increasingly consolidating as one of the most promising therapies. However, several hurdles have to be overcome, including immunotoxicity and pheno-toxicity. We review the state of the art of gene therapy in Friedreich's ataxia, addressing the main challenges and the most feasible solutions for them.

Asunto(s)

Ataxia de Friedreich , Terapia Genética , Proteínas de Unión a Hierro , Animales , Modelos Animales de Enfermedad , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/terapia , Humanos , Proteínas de Unión a Hierro/biosíntesis , Proteínas de Unión a Hierro/genética , Ratones , Frataxina

RESUMEN

Friedreich's ataxia (FRDA) is a hereditary and predominantly neurodegenerative disease caused by a deficiency of the protein frataxin (FXN). As part of the overall efforts to understand the molecular basis of neurodegeneration in FRDA, a new human neural cell line with doxycycline-induced FXN knockdown was established. This cell line, hereafter referred to as iFKD-SY, is derived from the human neuroblastoma SH-SY5Y and retains the ability to differentiate into mature neuron-like cells. In both proliferating and differentiated iFKD-SY cells, the induction of FXN deficiency is accompanied by increases in oxidative stress and DNA damage, reduced aconitase enzyme activity, higher levels of p53 and p21, activation of caspase-3, and subsequent apoptosis. More interestingly, FXN-deficient iFKD-SY cells exhibit an important transcriptional deregulation in many of the genes implicated in DNA repair pathways. The levels of some crucial proteins involved in DNA repair appear notably diminished. Furthermore, similar changes are found in two additional neural cell models of FXN deficit: primary cultures of FXN-deficient mouse neurons and human olfactory mucosa stem cells obtained from biopsies of FRDA patients. These results suggest that the deficiency of FXN leads to a down-regulation of DNA repair pathways that synergizes with oxidative stress to provoke DNA damage, which may be involved in the pathogenesis of FRDA. Thus, a failure in DNA repair may be considered a shared common molecular mechanism contributing to neurodegeneration in a number of hereditary ataxias including FRDA.

Asunto(s)

Daño del ADN , Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Neuronas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ataxia de Friedreich/genética , Humanos , Proteínas de Unión a Hierro/genética , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Proteína p53 Supresora de Tumor/metabolismo , Frataxina

RESUMEN

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by recessive mutations in the frataxin gene that lead to a deficiency of the mitochondrial frataxin (FXN) protein. Alternative forms of frataxin have been described, with different cellular localization and tissue distribution, including a cerebellum-specific cytosolic isoform called FXN II. Here, we explored the functional roles of FXN II in comparison to the mitochondrial FXN I isoform, highlighting the existence of potential cross-talk between cellular compartments. To achieve this, we transduced two human cell lines of patient and healthy subjects with lentiviral vectors overexpressing the mitochondrial or the cytosolic FXN isoforms and studied their effect on the mitochondrial network and metabolism. We confirmed the cytosolic localization of FXN isoform II in our in vitro models. Interestingly, both cytosolic and mitochondrial isoforms have an effect on mitochondrial dynamics, affecting different parameters. Accordingly, increases of mitochondrial respiration were detected after transduction with FXN I or FXN II in both cellular models. Together, these results point to the existence of a potential cross-talk mechanism between the cytosol and mitochondria, mediated by FXN isoforms. A more thorough knowledge of the mechanisms of action behind the extra-mitochondrial FXN II isoform could prove useful in unraveling FRDA physiopathology.

Asunto(s)

Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales/genética , Adolescente , Adulto , Estudios de Casos y Controles , Células Cultivadas , Niño , Citosol/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patología , Humanos , Masculino , Mitocondrias/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribución Tisular , Adulto Joven , Frataxina

RESUMEN

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.

Asunto(s)

Ataxia de Friedreich/metabolismo , Mucosa Olfatoria/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Madre/metabolismo , Aconitato Hidratasa/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamación/metabolismo

RESUMEN

Friedreich´s ataxia (FRDA) is an autosomal recessive disease caused by an abnormally expanded Guanine-Adenine-Adenine (GAA) repeat sequence within the first intron of the frataxin gene (FXN). The molecular mechanisms associated with FRDA are still poorly understood and most studies on FXN gene regulation have been focused on the region around the minimal promoter and the region in which triplet expansion occurs. Nevertheless, since there could be more epigenetic changes involved in the reduced levels of FXN transcripts, the aim of this study was to obtain a more detailed view of the possible regulatory elements by analyzing data from ENCODE and Roadmap consortia databases. This bioinformatic analysis indicated new putative regulatory regions within the FXN genomic locus, including exons, introns, and upstream and downstream regions. Moreover, the region next to the end of intron 4 is of special interest, since the enhancer signals in FRDA-affected tissues are weak or absent in this region, whilst they are strong in the rest of the analyzed tissues. Therefore, these results suggest that there could be a direct relationship between the absence of enhancer sequences in this specific region and their predisposition to be affected in this pathology.

Asunto(s)

Epigénesis Genética , Ataxia de Friedreich/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de Unión a Hierro/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Expansión de Repetición de Trinucleótido/genética , Animales , Epigenómica/métodos , Genómica/métodos , Humanos , Frataxina

RESUMEN

Herpes simplex virus 1 (HSV-1)-derived amplicon vectors are unique in their ability to accommodate large DNA molecules allowing whole genomic loci to be included with all of their regulatory elements. Additional advantages of these amplicons include their minimal toxicity and ability to persist as episomes, with negligible risk of insertional mutagenesis, being particularly well-suited for gene therapy of neurological disorders due to their outstanding ability to deliver genes into neurons and other neural cells. However, extensive gene therapy application has been hindered by difficulties in vector production. This work improved HSV-1 amplicons production by genetic modification of the packaging cell line and optimization of the culture medium. A stably-transfected Vero 2-2 cell line overexpressing the anti-apoptotic Bcl-2 protein was generated, exhibiting an increased resistance to apoptosis, prolonged culture duration, and a significant improvement in viral vector production. Additionally, supplementation of the growth medium with antioxidants, polyamines, amino acids, and reduced glutathione further increased the yield of packaged amplicon vectors. With these modifications, HSV-1 amplicons could be isolated from culture supernatants instead of cell lysates, leading to vector preparations with higher titer and purity and paving the way for generation of stable cell lines that are capable of continuous herpesviral vector production.

RESUMEN

Friedreich's ataxia is a predominantly neurodegenerative disease caused by recessive mutations that produce a deficiency of frataxin (FXN). Here, we have used a herpesviral amplicon vector carrying a gene encoding for brain-derived neurotrophic factor (BDNF) to drive its overexpression in neuronal cells and test for its effect on FXN-deficient neurons both in culture and in the mouse cerebellum in vivo. Gene transfer of BDNF to primary cultures of mouse neurons prevents the apoptosis which is triggered by the knockdown of FXN gene expression. This neuroprotective effect of BDNF is also observed in vivo in a viral vector-based knockdown mouse cerebellar model. The injection of a lentiviral vector carrying a minigene encoding for a FXN-specific short hairpin ribonucleic acid (shRNA) into the mouse cerebellar cortex triggers a FXN deficit which is accompanied by significant apoptosis of granule neurons as well as loss of calbindin in Purkinje cells. These pathological changes are accompanied by a loss of motor coordination of mice as assayed by the rota-rod test. Coinjection of a herpesviral vector encoding for BDNF efficiently prevents both the development of cerebellar neuropathology and the ataxic phenotype. These data demonstrate the potential therapeutic usefulness of neurotrophins like BDNF to protect FXN-deficient neurons from degeneration.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/genética , Ataxia de Friedreich/prevención & control , Terapia Genética/métodos , Proteínas de Unión a Hierro/genética , Neuronas/patología , Animales , Apoptosis/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Ataxia de Friedreich/genética , Técnicas de Silenciamiento del Gen , Vectores Genéticos/administración & dosificación , Herpesviridae/genética , Humanos , Ratones , Neuronas/efectos de los fármacos , Frataxina

RESUMEN

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.

Asunto(s)

Técnicas de Cultivo Celular por Lotes/métodos , Neuroglía/citología , Bulbo Olfatorio/citología , Esferoides Celulares/citología , Animales , Comunicación Celular/fisiología , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Ratones , Microfluídica/métodos , Neuroglía/fisiología , Bulbo Olfatorio/fisiología , Impresión Tridimensional , Esferoides Celulares/fisiología

RESUMEN

Friedreich's ataxia (FA) is a recessive, predominantly neurodegenerative disorder caused in most cases by mutations in the first intron of the frataxin (FXN) gene. This mutation drives the expansion of a homozygous GAA repeat that results in decreased levels of FXN transcription and frataxin protein. Frataxin (Fxn) is a ubiquitous mitochondrial protein involved in iron-sulfur cluster biogenesis, and a decrease in the levels of this protein is responsible for the symptoms observed in the disease. Although the pathological manifestations of FA are mainly observed in neurons of both the central and peripheral nervous system, it is not clear if changes in non-neuronal cells may also contribute to the pathogenesis of FA, as recently suggested for other neurodegenerative disorders. Therefore, the aims of this study were to generate and characterize a cell model of Fxn deficiency in human astrocytes (HAs) and to evaluate the possible involvement of non-cell autonomous processes in FA. To knockdown frataxin in vitro, we transduced HAs with a specific shRNA lentivirus (shRNA37), which produced a decrease in both frataxin mRNA and protein expression, along with mitochondrial superoxide production, and signs of p53-mediated cell cycle arrest and apoptotic cell death. To test for non-cell autonomous interactions we cultured wild-type mouse neurons in the presence of frataxin-deficient astrocyte conditioned medium, which provoked a delay in the maturation of these neurons, a decrease in neurite length and enhanced cell death. Our findings confirm a detrimental effect of frataxin silencing, not only for astrocytes, but also for neuron-glia interactions, underlining the need to take into account the role of non-cell autonomous processes in FA.

Asunto(s)

Astrocitos/metabolismo , Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Animales , Apoptosis , Puntos de Control del Ciclo Celular , Muerte Celular , Supervivencia Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Unión a Hierro/genética , Ratones , Mitocondrias/metabolismo , Neuronas/fisiología , Superóxidos/metabolismo , Frataxina

RESUMEN

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.

Asunto(s)

Movimiento Celular/efectos de los fármacos , Curcumina/farmacología , Bulbo Olfatorio/citología , Fagocitosis/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Separación Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Técnica del Anticuerpo Fluorescente , Ontología de Genes , Masculino , Ratones Transgénicos , Neuroglía/citología , Neuroglía/efectos de los fármacos , Mucosa Olfatoria/citología , Transducción de Señal/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Hexoquinasa/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Muerte Celular/fisiología , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Mitocondrias/metabolismo , Rotenona/toxicidad , Desacopladores/toxicidad

RESUMEN

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.

Asunto(s)

Axones/metabolismo , Regeneración Nerviosa/fisiología , Neuroglía/metabolismo , Bulbo Olfatorio/citología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Axones/efectos de los fármacos , Axotomía/efectos adversos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa/efectos de los fármacos , Neuroglía/química , Inhibidor 1 de Activador Plasminogénico/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptor PAR-1/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Trombomodulina/metabolismo , Transducción Genética

RESUMEN

Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by mutations that produce a deficiency in frataxin. Despite the importance of neurodegeneration in FRDA, little is known about the consequences of frataxin deficiency in neuronal cells. Here we describe a neuronal cell model for FRDA based on the use of lentiviral vectors that carry minigenes encoding frataxin-specific shRNAs that silence the expression of this gene. These lentivectors can knockdown frataxin expression in human neuroblastoma SH-SY5Y cells, which results in large-scale cell death in differentiated neuron-like cells but not in undifferentiated neuroblastoma cells. Frataxin-deficient neuron-like cells appear to die through apoptosis that is accompanied by up-regulation of p53, PUMA and Bax and activation of caspase-3. No significant autophagy is observed in frataxin-deficient neuron-like cells and the pharmacological activation of autophagy does not significantly increase neuronal cell death in response to the frataxin deficiency. Cell death triggered by frataxin knockdown can be impaired by interference with p53, caspase inhibitors and gene transfer of FXN. These results suggest that frataxin gene silencing in human neuron-like cells may constitute a useful cell model to characterize the molecular changes triggered by frataxin deficiency in neurons, as well as to search for therapies that may protect against neurodegeneration.

Asunto(s)

Apoptosis , Silenciador del Gen , Proteínas de Unión a Hierro/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/terapia , Humanos , Proteínas de Unión a Hierro/genética , Modelos Biológicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Frataxina

RESUMEN

Artificial chromosomes and minichromosome-like episomes are large DNA molecules capable of containing whole genomic loci, and be maintained as nonintegrating, replicating molecules in proliferating human somatic cells. Authentic human artificial chromosomes are very difficult to engineer because of the difficulties associated with centromere structure, so they are not widely used for gene-therapy applications. However, OriP/EBNA1-based episomes, which they lack true centromeres, can be maintained stably in dividing cells as they bind to mitotic chromosomes and segregate into daughter cells. These episomes are more easily engineered than true human artificial chromosomes and can carry entire genes along with all their regulatory sequences. Thus, these constructs may facilitate the long-term persistence and physiological regulation of the expression of therapeutic genes, which is crucial for some gene therapy applications. In particular, they are promising vectors for gene therapy in inherited diseases that are caused by recessive mutations, for example haemophilia A and Friedreich's ataxia. Interestingly, the episome carrying the frataxin gene (deficient in Friedreich's ataxia) has been demonstrated to rescue the susceptibility to oxidative stress which is typical of fibroblasts from Friedreich's ataxia patients. This provides evidence of their potential to treat genetic diseases linked to recessive mutations through gene therapy.

Asunto(s)

Cromosomas Artificiales Humanos/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Expresión Génica , Terapia Genética/métodos , Plásmidos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ataxia de Friedreich/terapia , Hemofilia A/terapia , Herpesvirus Humano 4/genética , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/uso terapéutico , Frataxina

RESUMEN

Parkinson's disease (PD) is a neurodegenerative disease affecting nigrostriatal dopaminergic neurons. Dopamine depletion in the striatum leads to functional changes in several deep brain nuclei, including the subthalamic nucleus (STN), which becomes disinhibited and perturbs the control of body movement. Although there is no cure for PD, some pharmacological and surgical treatments can significantly improve the functional ability of patients, particularly in the early stages of the disease. Among neurodegenerative diseases, PD is a particularly suitable target for gene therapy because the neuropathology is largely confined to a relatively small region of the brain. Neurologix Inc is developing NLX-P101 (AAV2-GAD), an adeno-associated viral vector encoding glutamic acid decarboxylase (GAD), for the potential therapy of PD. As GAD potentiates inhibitory neurotransmission from the STN, sustained expression of GAD in the STN by direct delivery of NLX-P101 decreases STN overactivation. This procedure was demonstrated to be a safe and efficient method of reducing motor deficits in animal models of PD. A phase I clinical trial has demonstrated that NLX-P101 was safe and indicated the efficacy of this approach in patients with PD. Results from an ongoing phase II clinical trial of NLX-P101 are awaited to establish the clinical efficacy of this gene therapy.

Asunto(s)

Dependovirus/enzimología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Glutamato Descarboxilasa/uso terapéutico , Enfermedad de Parkinson/terapia , Animales , Dependovirus/genética , Vectores Genéticos , Glutamato Descarboxilasa/genética , Humanos , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Patentes como Asunto , Núcleo Subtalámico/enzimología

RESUMEN

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.

Asunto(s)

Muerte Celular , Terapia Genética , Hexoquinasa/genética , Hexoquinasa/uso terapéutico , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/terapia , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Catalepsia/inducido químicamente , Catalepsia/metabolismo , Catalepsia/terapia , Dopamina/metabolismo , Vectores Genéticos , Herpesvirus Humano 1/genética , Hexoquinasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/terapia , Neuronas/metabolismo , Neuronas/patología , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Rotenona , Sustancia Negra/metabolismo , Sustancia Negra/patología , Resultado del Tratamiento

RESUMEN

A continuous normal function of olfactory ensheathing glia (OEG) is to promote axonal regeneration from the olfactory neuroepithelium to the brain, and their neuroregenerative potential in other CNS sites such as the injured spinal cord has been studied for over a decade. However, human OEG are difficult to obtain in large amounts directly from tissues, and the derived primary cultures have a limited duplication capacity. Thus, although auto-transplantation may be an obvious option for initial proof-of-concept trials, alternatives must be explored to obtain large quantities of homogeneous, pre-characterized OEG for wide-scale therapeutic use. We have cultured primary human OEG derived from olfactory bulbs (OB) obtained by necropsy and successfully extended the replicative lifespan of these cells using lentivectors encoding Bmi-1 and TERT transgenes flanked by loxP sites. In contrast to the primary cells which could only be expanded for a limited number of passages (approximately 12), adult human OEG immortalized Bmi-1/TERT divided indefinitely in culture. Clonal lines were isolated and the floxed transgenes could be excised by lentivector-mediated Cre recombinase delivery. Primary, immortalized, and deimmortalized human OEG all expressed typical markers of this cell type and importantly, were all able to promote axonal regeneration of adult rat retinal ganglion neurons (RGN) in co-culture assays.

Asunto(s)

Regeneración Nerviosa/fisiología , Neuroglía/fisiología , Bulbo Olfatorio/citología , Adolescente , Adulto , Animales , Animales Recién Nacidos , Células Cultivadas , Células Clonales , Técnicas de Cocultivo/métodos , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/trasplante , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Ganglionares de la Retina/metabolismo , Traumatismos de la Médula Espinal/cirugía , Telomerasa/genética , Telomerasa/metabolismo , Transducción Genética/métodos

RESUMEN

Gene therapy has been a clinical possibility since 1989 and the steadily increasing number of clinical trials now includes strategies targeting neurodegenerative conditions such as lysosomal storage disease, multiple sclerosis, Alzheimer's and, Parkinson's disease. In spite of lack of knowledge of the molecular causes of these diseases, results so far in these trials have been promising. Thus there is gaining confidence in the potential to develop effective treatments based on gene transfer for neurological diseases in the near future. Furthermore, the accelerating progress in knowledge of the molecular pathologies of neurogenetic disorders, including rare diseases such as the ataxias, makes them even more amenable to gene therapy. Here we review recent preclinical studies relevant to gene therapy of ataxias and discuss developments needed to bring these strategies into the clinic.

Asunto(s)

Ataxia/terapia , Terapia Genética/métodos , Animales , Ataxia/genética , Humanos