RESUMEN
Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson's disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson's disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson's disease, providing novel opportunities for therapeutic intervention.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Técnicas Biosensibles , Células HEK293 , Humanos , Transporte de ProteínasRESUMEN
Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering.
Asunto(s)
Neuronas Adrenérgicas/metabolismo , Encéfalo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Hipocalcina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Espectrometría de Masas , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Técnicas de Placa-Clamp , Ubiquitina-Proteína Ligasas/genética , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-ß, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.
Asunto(s)
Apoptosis/genética , GTP Fosfohidrolasas/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Mitocondrias/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular , Fibroblastos/metabolismo , Fibroblastos/patología , GTP Fosfohidrolasas/genética , Regulación del Desarrollo de la Expresión Génica , Quinasa I-kappa B/biosíntesis , Quinasa I-kappa B/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , FN-kappa B/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-ß hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Encéfalo/metabolismo , Mitocondrias/fisiología , Enfermedad de Parkinson/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/genética , Animales , Encéfalo/fisiopatología , Regulación de la Expresión Génica , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Recambio Mitocondrial , Mutación , Enfermedad de Parkinson/fisiopatología , Ratas , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Neurodegenerative disorders (ND) include a wide spectrum of diseases characterized by progressive neuronal dysfunctions or degeneration. With an estimated cost of 135 billion in 2010 in the European Union (Olesen et al., 2012), they put an enormous economic as well as social burden on modern societies. Hence, they have been the subject of a huge amount of research for the last fifty years. For many of these diseases, our understanding of their profound causes is incomplete and this hinders the discovery of efficient therapies. ND form a highly heterogeneous group of diseases affecting various neuronal subpopulations reflecting different origins and different pathological mechanisms. However, some common themes in the physiopathology of these disorders are emerging. There is growing evidence that mitochondrial dysfunctions play a pivotal role at some point in the course of neurodegeneration. In some cases (e.g. Alzheimer's disease, amyotrophic lateral sclerosis), impairment of mitochondrial functions probably occurs late in the course of the disease. In a subset of ND, current evidence suggests that mitochondrial dysfunctions play a more seminal role in neuronal demise. Parkinson's disease (PD) presents one of the strongest cases based in part on post-mortem studies that have shown mitochondrial impairment (e.g. reduced complex I activity) and oxidative damage in idiopathic PD brains. The occurrence of PD is largely sporadic, but clinical syndromes resembling sporadic PD have been linked to specific environmental insults or to mutations in at least 5 distinct genes (α-synuclein, parkin, DJ-1, PINK1 and LRRK2). It is postulated that the elucidation of the pathogenic mechanisms underlying the selective dopaminergic degeneration in familial and environmental Parkinsonism should provide important clues to the pathogenic mechanisms responsible for idiopathic PD. Hence, numerous cellular and animal models of the disease have been generated that mimic these environmental or genetic insults. The study of these models has yielded valuable information regarding the pathogenic mechanisms underlying dopaminergic degeneration in PD, many of which point towards an involvement of mitochondrial dysfunction. In this short review we will analyze critically the experimental evidence for the mitochondrial origin of PD and evaluate its relevance for our general understanding of the disease.
Asunto(s)
Mitocondrias/fisiología , Enfermedades Mitocondriales/etiología , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/fisiopatología , Predisposición Genética a la Enfermedad , Humanos , Enfermedad de Parkinson/genéticaRESUMEN
Introducción: Los agonistas LHRH son de elección en bloqueo androgénico por cáncer prostático. En base a pacientes castrados se considera un bloqueo adecuado una testosterona total plasmática < 50 ng/dl. Se ha sugerido controlar la testosterona total por la posibilidad de no lograr una supresión adecuada. Material y método: Entre junio de 2008 a marzo de 2009 se midió el nivel de testosterona total al tercer mes de administrada una dosis de leuprolide 11,25 mg IM. Los exámenes se realizaron en la mañana y en el mismo laboratorio. En un grupo de pacientes se estimó peso, talla e índice de masa corporal (IMC) y se evaluó su asociación con los niveles de testosterona alcanzados. Resultados: Se evaluaron 81 pacientes, la edad promedio fue 76,4 años. La testosterona total plasmática promedio fue 33,9 ng/dl. En 12/81 pacientes (14,8 por ciento) el nivel de testosterona fue menos 50 ng/dl. No se observó asociación entre la duración de hormonoterapia y los niveles de testosterona. En 40 pacientes se estimó peso, talla e IMC sin encontrarse asociación de estas variables con el nivel de testosterona. En los pacientes en que se aumentó la dosis a leuprolide 22,5 mg se obtuvo una adecuada supresión de testosterona. Conclusión: En los pacientes en tratamiento con agonistas LHRH se debe medir el nivel de testosterona plasmática dada la posibilidad de un bloqueo inadecuado. En pacientes en tratamiento con leuprolide 11,25 mg y testosterona menos 50 ng/dl el aumento de la dosis a 22,5 mg lograría un nivel de testosterona en rangos de castración quirúrgica.
Introduction: The agonistas are LHRH of election in blockage androgenic for prostatic cancer. On the basis of castrated patients considers an adequate blockage a total plasmatic testosterone < 50 ng/dl. The total testosterone for the possibility to not to achieve an adequate suppression has been suggested to control. Material and method: Between June 2008 to March 2009 leuprolides dose measured the level of total testosterone itself to person under administrations third month 11.25 mg IM. The exams had done in the morning and at the same laboratory. Weight, size and index of muscle mass (IMC) were estimated in patients group and his association with the levels of testosterone caught up with was evaluated. Results: We evaluated 81 patients, the mean age was 76.4 years. The total testosterone the average plasmatic was 33.9 ng/dl. In 12/81 patient (14.8 percent) the level of testosterone was > 50 ng/dl. we did not observe association between hormonoterapias duration and the levels of testosterone. Weight, size and IMC without finding association of these variables with the level of testosterone were estimated in 40 patients. We got an adequate suppression from testosterone in the patients that 22.5 mg increased itself the dose in to leuprolide. Conclusion: LHRH must try on in the patients in treatment with agonistic the level of plasmatic testosterone once the possibility of an inadequate blockage was given. In patients in treatment with leuprolide 11.25 mg and testosterone > 50 ng/dl the increase of the dose to 22.5 mg would achieve a level of testosterone in ranges of surgical castration.
Asunto(s)
Humanos , Masculino , Persona de Mediana Edad , Anciano de 80 o más Años , Antineoplásicos Hormonales/administración & dosificación , Leuprolida/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Testosterona/sangre , Neoplasias de la Próstata/sangre , Índice de Masa CorporalRESUMEN
Introducción: El tumor de células pequeñas redondas desmoplásico (TCPRD), es una neoplasia agresiva maligna poco frecuente que afecta a adolescentes y adultos jóvenes. Usualmente son intraabdominales asociados con un mal pronóstico. Existen algunas publicaciones donde incluyen manifestaciones paratesticulares. Caso: Reportamos un caso de un joven de 29 años que consulta por aumento de volumen del hemiescroto izquierdo de 3 meses de evolución. Al examen destacaba 2 nódulos duros uno en relación al polo inferior y el otro al epidídimo de aproximadamente 2 cm. Alfafetoproteína y beta HCG eran normales. Se decide exploración quirúrgica donde biopsia rápida confirma tumor que se origina en las envolturas y que infiltra hacia el testículo. La biopsia definitiva informó tumor desmoplásico de células pequeñas redondas. El estudio de extensión con tomografía axial computarizada y radiografía de tórax no mostró metástasis. El paciente recibió 2 ciclos de QMT con ciclofosfamida, etoposido, adriamicina y cisplatino, con buena tolerancia, evolucionando con depresión medular moderada que se recupera. Actualmente 6 años después del diagnóstico el paciente se encuentra en remisión completa. Discusión: Al parecer y según los últimos reportes de la literatura, la ubicación paratesticular ha mostrado mejor pronóstico en comparación con los tumores abdominales. Se debe incluir este diagnóstico diferencial al enfrentarse con tumores paratesticulares.
Introduction: Small round cell desmoplastic tumor (SRCDT) is an infrequent malignant tumor that affects adolescents and young adults. Usually they occur in the abdomen. Paratesticular manifestations have been reported. Case report: A 29 year old male presented with a 3 month history of a mass in the left scrotum. Physical exam showed 2 hard nodules in the scrotum. Serum levels of alpha-fetoprotein and beta-HCG were normal. The patient was submitted to surgery. Frozen section confirmed a tumor arising in the paratesticular area with involvement of the testis. Permanent sections showed a SRCDT. CT scans and chest x-rays showed no metastases. The patient received two courses of ciclophosphamide, etoposide, adryamicin and cisplatinum. Treatment was well tolerated. The patient is in complete remission at 6 years following the diagnosis. Discussion: Paratesticular location seems to have a better prognosis compared to intraabdominal tumors. SRCDT should be included in the differential diagnosis of paratesticular tumors.
Asunto(s)
Humanos , Masculino , Adulto , Fibroma Desmoplásico/tratamiento farmacológico , Neoplasias Testiculares/tratamiento farmacológicoRESUMEN
El cáncer de pene es una enfermedad de baja incidencia. Su tratamiento se basa en la cirugía y no está establecido si el control tumoral local debe o no ser complementado con linfadenectomía inguinal y pélvica. La linfadenectomía en el cáncer de pene se asocia a elevadas tasas de complicaciones y no está establecido a qué pacientes se les debe realizar. Éste trabajo retrospectivo analiza la expresión de marcadores tumorales en cáncer de pene y su correlación con otros factores pronósticos. La expresión del marcador Ciclina D1 fue alta en todas las muestras, la de Ki-67 y P16 fue alta en los tumores de alto grado y la de BCL-2 fue negativa en todos los casos.
Penile carcinoma is a low incidence disease. Treatment is based in surgery and controversy persists regarding the need of inguinal and pelvic lymphadenectomy. To date, no specific inclusion criteria have been established to indicate surgery. This retrospective work analyses the expression of tumoral markers in penile cancer and correlation with other prognostic factors. The expression of the Cicline D1 marker was high in all the samples, Ki-67, P16 was high in high grade tumors and BCL-2 was negative in all cases.
Asunto(s)
Humanos , Masculino , /metabolismo , Ciclina D1/metabolismo , Neoplasias del Pene/metabolismo , Neoplasias del Pene/patología , /metabolismo , Estadificación de Neoplasias , Estudios Retrospectivos , Inmunohistoquímica , Biomarcadores de TumorRESUMEN
Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Cisteína Endopeptidasas/química , ADN Complementario/metabolismo , Humanos , Inmunoprecipitación , Modelos Genéticos , Complejos Multienzimáticos/química , Mutación , Células PC12 , Plásmidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Ubiquitina/química , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Developments of technologies for delivery of foreign genes to the central nervous system are opening the field to promising treatments for human neurodegenerative diseases. Gene delivery vectors need to fulfill several criteria of efficacy and safety before being applied to humans. The ability to drive expression of a therapeutic gene in an adequate number of cells, to maintain long-term expression, and to allow exogenous control over the transgene product are essential requirements for clinical application. We describe the use of an adenovirus vector encoding human tyrosine hydroxylase (TH) 1 under the negative control of the tetracycline-sensitive gene regulatory system for direct injection into the dopamine-depleted striatum of a rat model of Parkinson's disease. This vector mediated synthesis of TH in numerous striatal cells and transgene expression was observed in a large proportion of them for at least 17 weeks. Furthermore, doxycyline, a tetracycline analog, allowed efficient and reversible control of transgene expression. Thus, the insertion of a tetracycline-sensitive regulatory cassette into a single adenovirus vector provides a promising system for the development of successful and safe therapies for human neurological diseases. Our results also confirm that future effective gene replacement approaches to Parkinson's disease will have to consider the concomitant transfer of TH and GTP-cyclohydrolase transgenes because the synthesis of the TH cofactor tetrahydrobiopterin may be crucial for restoration of the dopaminergic deficit.
Asunto(s)
Adenoviridae/genética , Antibacterianos/farmacología , Doxiciclina/farmacología , Técnicas de Transferencia de Gen , Enfermedad de Parkinson/terapia , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Animales , Antiparkinsonianos/farmacología , Apomorfina/farmacología , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Oxidopamina , Enfermedad de Parkinson/genética , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
Neurotrophic factors may be valuable for improving the survival and the functional efficacy of fetal nigral grafts to treat Parkinson's disease (PD). However, further characterization of their effects is required. New methods of protein delivery also need to be explored to supply sustained and regulated levels of these molecules. Gene transfer via adenoviral vectors is a promising strategy for this purpose. We show herein the effect of adenovirus-mediated transforming growth factor beta1 (TGFbeta1) gene transfer on fetal nigral grafts in a rat model of PD. Direct injection of AdTGFbeta1 into the dopamine-depleted striatum decreased the survival of the transplanted tyrosine hydroxylase-positive (TH+) neurons and impaired the functional efficacy of grafts. Viral toxicity to the graft was avoided by separating the site of viral infection from the transplant by a distance that allowed TGFbeta1 effect on the graft. This infection protocol may be useful for delivering secreted molecules with neurotrophic effects to dopaminergic grafts.
Asunto(s)
Adenoviridae/genética , Cuerpo Estriado/metabolismo , Trasplante de Tejido Fetal , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , Factor de Crecimiento Transformador beta/genética , Animales , Supervivencia Celular/fisiología , Dopamina/fisiología , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Ratas , Ratas Sprague-Dawley , Sustancia Negra/embriología , Sustancia Negra/trasplanteRESUMEN
Ex vivo gene transfer is emerging as a promising therapeutic approach to human neurodegenerative diseases. By combining efficient methodologies for cell amplification and gene delivery, large numbers of cells can be generated with the capacity to synthesize therapeutic molecules. These cells can then be transplanted into the degenerating central nervous system (CNS). Applying this approach to human diseases will require the development of suitable cellular vehicles, as well as safe gene delivery systems capable of tightly controlled transgene expression. For such brain repair technologies, human neural progenitors may be extremely valuable, because of their human CNS origin and developmental potential. We have used these cells to develop a system for the regulated expression of a gene of therapeutic potential. We report the construction of a single adenovirus encoding human tyrosine hydroxylase 1 (hTH-1) under the negative control of the tetracycline-based gene regulatory system. Human neural progenitors infected with this vector produced large amounts of hTH-1. Most importantly, doxycycline allowed a reversible switch of transgene transcription both in vitro and in vivo. This system may be applied to the development of therapies for human neurodegenerative diseases.
Asunto(s)
Adenoviridae/genética , Trasplante de Tejido Encefálico , Encéfalo/citología , Técnicas de Transferencia de Gen , Trasplante de Células Madre , Tirosina 3-Monooxigenasa/genética , Animales , Encéfalo/embriología , Encéfalo/enzimología , Trasplante de Células , Doxiciclina/farmacología , Trasplante de Tejido Fetal , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Ratas , Células Madre/virología , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
The combination of gene transfer techniques and cell transplantation is a promising approach to deliver therapeutic molecules into the CNS. To optimize gene transfer systems, several neural and nonneural cell types are currently under investigation. Among these cells, astrocytes are particularly well suited because of their CNS origin, their efficient secretory mechanisms, and their role as neuronal support. Most importantly, the use of human adult astrocytes as cellular vehicles for ex vivo gene transfer may open the way to autologous transplantation, thus obviating immunological rejection and the side effects of immunosuppressors. In the present study, we report the ability of these cells to be expanded and genetically modified in vitro. Astrocytes derived from human adult cerebral cortex were grown and maintained in vitro as pure primary cultures for at least 10 months. In addition, cells were efficiently transduced by an adenoviral vector encoding human tyrosine hydroxylase (hTH) under the negative control of the tetracycline-based regulatory system (tet-off). The infected cells synthesized large amounts of active hTH and released L-dopa. In addition, doxycycline, a potent analog of tetracycline, efficiently regulated transgene expression. This work is a first step toward the development of therapeutic strategies based on the use of genetically engineered human adult astrocytes for autologous transplantation in human neurodegenerative diseases and CNS trauma.
Asunto(s)
Astrocitos/trasplante , Trasplante de Células , Corteza Cerebral/citología , Terapia Genética , Adenoviridae/genética , Adulto , Anciano , Astrocitos/enzimología , División Celular , Células Cultivadas , Congelación , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Tetraciclinas/farmacología , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Adenovirus is an efficient vector for neuronal gene therapy due to its ability to infect post-mitotic cells, its high efficacy of cell transduction and its low pathogenicity. Recombinant adenoviruses encoding for therapeutical agents can be delivered in vivo after direct intracerebral injection into specific brain areas. They can be transported in a retrograde manner from the injection site to the projection cell bodies offering promising applications for the specific targeting of selected neuronal populations not easily accessible by direct injection, such as the motor neurons in the spinal cord. Adenoviral vectors are also efficient tools for the ex vivo gene therapy, that is, the genetical modification of cells prior to their transplantation into the nervous system. Recently, the efficacy of the adenovirus as a gene vector system has been demonstrated in several models of neurodegenerative diseases including Parkinson's disease (PD) and motor neuron diseases. In rat models of PD, adenoviruses encoding for either tyrosine hydroxylase, superoxide dismutase or glial-derived neurotrophic factor improved the survival and the functional efficacy of dopaminergic cells. Similarly, the intramuscular injection of an adenovirus encoding for neurotrophin-3 had substantial therapeutic effects in a mutant mouse model of motor neuron degenerative disease. However, although adenoviruses are highly attractive for neuronal gene transfer, they can trigger a strong inflammatory reaction leading in particular to the destruction of infected cells. The recent development of new generations of adenoviral vectors could shed light on the nature of the immune reaction caused by adenoviral vectors in the brain. The use of these new vectors, combined with that of neurospecific and regulatable promoters, should improve adenovirus gene transfer into the central nervous system.
Asunto(s)
Adenoviridae , Encefalopatías/terapia , Terapia Genética/tendencias , Enfermedades Neurodegenerativas/terapia , Animales , HumanosRESUMEN
Tight control of the activity of a therapeutic gene introduced in vivo is a major issue in gene therapy research. Appropriate levels of expression may be crucial for gene correction. The tetracycline-sensitive regulatory system is highly effective for transcriptional regulation of foreign genes in mammalian cells. Here we report tight tetracycline-dependent regulation of a luciferase reporter gene transferred into the rat brain in the genetically modified neural precursor cell line ST14A as early as 2 days and until at least 6 days after transplantation. This is the first demonstration of the potential of this regulatory system for the modulation of the expression of therapeutic genes introduced into the central nervous system.
Asunto(s)
Encéfalo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Tetraciclina/farmacología , Animales , Trasplante de Tejido Encefálico , Línea Celular , Trasplante de Células , Femenino , Técnicas de Transferencia de Gen , Ratas , Ratas Sprague-DawleyRESUMEN
We identified two partially overlapping cDNAs containing divergent 5' sequences of human carnitine acetyltransferase (CAT). cDNA lambda SM-1400 extends the sequence of peroxisomal CAT, whereas cDNA lambda SM-1200 creates a new open reading frame encoding a putative mitochondrial leader peptide. An intron is located where sequences diverge, suggesting that mitochondrial, peroxisomal and possibly endoplasmic reticulum CAT mRNAs derive from alternative splicing of the CAT gene.
Asunto(s)
Empalme Alternativo , Carnitina O-Acetiltransferasa/biosíntesis , ADN Complementario/química , Isoenzimas/biosíntesis , Músculos/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cartilla de ADN , ADN Complementario/metabolismo , Biblioteca de Genes , Humanos , Intrones , Mitocondrias Musculares/enzimología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/biosíntesis , Fracciones Subcelulares/enzimología , Transcripción GenéticaRESUMEN
Using a combination of PCR screening of cDNA libraries and reverse transcription PCR, we have cloned three overlapping DNA fragments that encode human carnitine acetyltransferase (CAT), a key enzyme for metabolic pathways involved with the control of the acyl-CoA/CoA ratio in mitochondria, peroxisomes, and endoplasmic reticulum. The resulting cDNA (2436 bp) hybridizes to a mRNA species of approximately 2.9 kb that is particularly abundant in skeletal muscle and encodes a 68-kDa protein containing a peroxisomal targeting signal. The sequence matches those of several tryptic peptides obtained from purified human liver CAT and shows striking similarities with other members of the carnitine/choline acetyltransferase family very distant throughout evolution. CAT cDNA has also been used for fluorescence in situ hybridization on metaphase spreads of human chromosomes, and the corresponding gene, CAT1, has been mapped to chromosome 9q34.1.
Asunto(s)
Carnitina O-Acetiltransferasa/genética , Cromosomas Humanos Par 9 , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Genes , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Se analizan 120 casos de tuberculosis urogenital, diagnosticados a través de una pesquisa precoz, en el Hospital de Valdivia entre febrero de 1982 y abril de 1987. Todos fueron tratados con los esquemas abreviados TA-81 A y TA-81 B del Programa Nacional de Control de la Tuberculosis. El 88,3% de los casos tuvo confirmación bacteriológica y el 11,7% histológica. En 97 pacientes se efectuó una serie de 6 muestras de orina para estudio de Koch y en 13 se requirió más de una serie; en 32,8% la positividad se obtuvo en la 4ta., 5ta. o 6ta. muestra de la serie. Sólo 21 pacientes (18,5%) necesitaron algún tipo de cirugía, 9 de ellos nefectomía. El tratamiento fue exitoso en el 88,3%, hubo 13 fracasos y recidivas y sólo una de estas últimas persistió con cultivo de Koch positivo al finalizar el retratamiento