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1.
Cell ; 172(5): 897-909.e21, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29474918

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Genoma Humano , Transcriptoma/genética , Empalme Alternativo/genética , Elementos Alu/genética , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Estudios de Cohortes , Familia , Femenino , Sitios Genéticos , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Intrones/genética , Masculino , Repeticiones de Minisatélite/genética , Modelos Genéticos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo
2.
Am J Hum Genet ; 108(11): 2145-2158, 2021 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-34672987

RESUMEN

Dystonia is a neurologic disorder associated with an increasingly large number of genetic variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is critical in understanding the pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations. We used this strategy to investigate the molecular consequences of genetic variation in THAP1, which encodes a transcription factor linked to neural differentiation. Multiple pathogenic mutations associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter DNA-binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight alterations in a common induced pluripotent stem cell background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling followed by joint analysis of the most robust signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. On the basis of these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al ADN/genética , Distonía/genética , Trastornos Distónicos/genética , Mutación , Vaina de Mielina/genética , Alelos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Ratones
3.
Proc Natl Acad Sci U S A ; 114(51): E11020-E11028, 2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-29229810

RESUMEN

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1 This unique insertion coincides with six additional noncoding sequence changes in TAF1, the gene that encodes TATA-binding protein-associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n = 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)n The number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference in TAF1 expression.


Asunto(s)
Expansión de las Repeticiones de ADN , Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Histona Acetiltransferasas/genética , Retroelementos , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Orden Génico , Estudios de Asociación Genética , Sitios Genéticos , Humanos , Masculino , Modelos Biológicos , Linaje , Fenotipo , Regiones Promotoras Genéticas , Activación Transcripcional
4.
Stem Cells ; 36(6): 932-942, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29451340

RESUMEN

Stem cells are emerging as promising treatment strategies for several brain disorders and pathologies. In this study, we explored the potential of creating induced pluripotent stem cell-derived neural stem cells (ipNSC) by using either unmodified or gene-modified somatic cells and tested their fate and therapeutic efficacies in vitro and in vivo. We show that cells engineered in somatic state lose transgene-expression during the neural induction process, which is partially restored by histone deacetylase inhibitor treatment whereas cells engineered at the ipNSC state have sustained expression of transgenes. In vivo, bimodal mouse and human ipNSCs engineered to express tumor specific death-receptor ligand and suicide-inducing therapeutic proteins have profound anti-tumor efficacy when encapsulated in synthetic extracellular matrix and transplanted in mouse models of resected-glioblastoma. This study provides insights into using somatic cells for treating CNS disorders and presents a receptor-targeted cancer therapeutic approach for brain tumors. Stem Cells 2018;36:932-942.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Ingeniería Celular/métodos , Reprogramación Celular/fisiología , Células-Madre Neurales/fisiología , Animales , Humanos , Ratones
5.
Neurobiol Dis ; 100: 108-118, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28017799

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a progressive neurodegenerative disease involving the loss of medium spiny neurons within the striatum. An XDP-specific haplotype has been identified, consisting of seven sequence variants which cluster around the human TAF1 gene, but a direct relationship between any of these variants and disease pathogenesis has not yet been demonstrated. Because the pathogenic gene lesion remains unclear, it has been difficult to predict cellular pathways which are affected in XDP cells. To address that issue, we assayed expression of defined gene sets in XDP vs. control fibroblasts to identify networks of functionally-related transcripts which may be dysregulated in XDP patient cells. That analysis derived a 51-gene signature distinguishing XDP vs. control fibroblasts which mapped strongly to nuclear factor-kappa B (NFκB), a transcription factor pathway also implicated in the pathogenesis of other neurodegenerative diseases, including Parkinson's (PD) and Huntington's disease (HD). Constitutive and TNFα-evoked NFκB signaling was further evaluated in XDP vs. control fibroblasts based on luciferase reporter activity, DNA binding of NFκB subunits, and endogenous target gene transcription. Compared to control cells, XDP fibroblasts exhibited decreased basal NFκB activity and decreased levels of the active NFκB p50 subunit, but increased target gene expression in response to TNFα. NFκB signaling was further examined in neural stem cells differentiated from XDP and control induced pluripotent stem cell (iPSC) lines, revealing a similar pattern of increased TNFα responses in the patient lines compared to controls. These data indicate that an NFκB signaling phenotype is present in both patient fibroblasts and neural stem cells, suggesting this pathway as a site of dysfunction in XDP.


Asunto(s)
Núcleo Celular/metabolismo , Trastornos Distónicos/genética , Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , FN-kappa B/metabolismo , Células-Madre Neurales/metabolismo , Transducción de Señal , Cuerpo Estriado/metabolismo , Distonía/genética , Distonía/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Fenotipo , Factor de Transcripción TFIID/genética
6.
eNeuro ; 2022 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-35868859

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. The mechanisms underlying regional differences in degeneration and adult onset are unknown. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due, in part, to a partial loss of TAF1 function. A disease-specific SINE-VNTR-Alu (SVA) retrotransposon insertion occurs within intron 32 of TAF1, a subunit of TFIID involved in transcription initiation. While all XDP males are usually clinically affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight iPSC lines from three XDP female carrier individuals for X chromosome inactivation status and identified clonal lines that express either the wild-type X or XDP haplotype. Furthermore, we characterized XDP-relevant transcript expression in neurotypical humans, and found that SVA-F expression decreases after 30 years of age in the brain and that TAF1 is decreased in most female samples. Uniquely in the caudate nucleus, TAF1 expression is not sexually dimorphic and decreased after adolescence. These findings indicate that regional-, age- and sex-specific mechanisms regulate TAF1, highlighting the importance of disease-relevant models and postmortem tissue. We propose that the decreased TAF1 expression in the adult caudate may synergize with the XDP-specific partial loss of TAF1 function in patients, thereby passing a minimum threshold of TAF1 function, and triggering degeneration in the neostriatum.Significance StatementXDP is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due to a partial loss of TAF1 function. While all XDP males are usually affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight stem cell lines from XDP female carrier individuals. Furthermore, we found that, uniquely in the caudate nucleus, TAF1 expression decreases after adolescence in healthy humans. We hypothesize that the decrease of TAF1 after adolescence in human caudate, in general, may underlie the vulnerability of the adult neostriatum in XDP.

7.
Glia ; 59(10): 1529-39, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21656857

RESUMEN

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.


Asunto(s)
Axones/fisiología , Regeneración Nerviosa/fisiología , Ribosomas/metabolismo , Células de Schwann/ultraestructura , Neuropatía Ciática/cirugía , Animales , Axones/metabolismo , Axones/patología , Transporte Biológico/fisiología , Lateralidad Funcional , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Compresión Nerviosa/métodos , Proteínas de Neurofilamentos/metabolismo , Ratas , Ratas Endogámicas Lew , Ribosomas/ultraestructura , Células de Schwann/patología , Células de Schwann/trasplante , Neuropatía Ciática/etiología , Factores de Tiempo , Transducción Genética/métodos
8.
J Neurosci ; 29(49): 15542-50, 2009 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-20007478

RESUMEN

Successful regeneration of damaged neurons depends on the coordinated expression of neuron-intrinsic genes. At present however, there is no comprehensive view of the transcriptional regulatory mechanisms underlying neuronal regeneration. We used high-content cellular screening to investigate the functional contribution of 62 transcription factors to regenerative neuron outgrowth. Ten transcription factors are identified that either increase or decrease neurite outgrowth. One of these, NFIL3, is specifically upregulated during successful regeneration in vivo. Paradoxically however, knockdown of NFIL3 and overexpression of dominant-negative NFIL3 both increase neurite outgrowth. Our data show that NFIL3, together with CREB, forms an incoherent feedforward transcriptional regulatory loop in which NFIL3 acts as a negative regulator of CREB-induced regeneration-associated genes.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Transcripción Genética/fisiología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ganglios Espinales/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Neuritas/fisiología , Neuronas/fisiología , Ratas , Ratas Wistar , Transcripción Genética/genética
9.
PLoS One ; 15(12): e0243655, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33315879

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Histona Acetiltransferasas/genética , Histonas/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Acetilación , Células Cultivadas , Trastornos Distónicos/metabolismo , Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Humanos , Intrones , Retroelementos
10.
J Neurosci ; 28(43): 11024-9, 2008 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-18945910

RESUMEN

Schwann cells play pivotal roles in the development and maintenance of the peripheral nervous system. Here, we show that intact sciatic nerve axons of mice contain a small population of ribosomes, which increases by several orders of magnitude when axons are desomatized (severed from their cell bodies). We furthermore demonstrate, using the Wallerian degeneration slow mouse as a model, that Schwann cells transfer polyribosomes to desomatized axons. These data indicate that Schwann cells have the propensity to control axonal protein synthesis by supplying ribosomes on local basis.


Asunto(s)
Axones/fisiología , Neuroglía/citología , Ribosomas/metabolismo , Células de Schwann/fisiología , Animales , Axones/ultraestructura , Transporte Biológico/genética , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Transmisión/métodos , Proteína Básica de Mielina/metabolismo , Proteína P0 de la Mielina/metabolismo , Proteínas del Tejido Nervioso/genética , Neuroglía/fisiología , Polirribosomas/metabolismo , Neuropatía Ciática/genética , Neuropatía Ciática/patología , Neuropatía Ciática/fisiopatología , Transfección/métodos
11.
Mol Cell Neurosci ; 39(1): 105-17, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18585464

RESUMEN

Traumatic avulsion of spinal nerve roots causes complete paralysis of the affected limb. Reimplantation of avulsed roots results in only limited functional recovery in humans, specifically of distal targets. Therefore, root avulsion causes serious and permanent disability. Here, we show in a rat model that lentiviral vector-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) in reimplanted nerve roots completely prevents motoneuron atrophy after ventral root avulsion and stimulates regeneration of axons into reimplanted roots. However, over the course of 16 weeks neuroma-like structures are formed in the reimplanted roots, and regenerating axons are trapped at sites with high levels of GDNF expression. A high local concentration of GDNF therefore impairs long distance regeneration. These observations show the feasibility of combining neurosurgical repair of avulsed roots with gene-therapeutic approaches. Our data also point to the importance of developing viral vectors that allow regulated expression of neurotrophic factors.


Asunto(s)
Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Lentivirus , Regeneración Nerviosa/fisiología , Radiculopatía/cirugía , Raíces Nerviosas Espinales , Animales , Atrofia/prevención & control , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Ganglios Espinales/citología , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Radiculopatía/patología , Ratas , Ratas Wistar , Recuperación de la Función , Células de Schwann/citología , Células de Schwann/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Raíces Nerviosas Espinales/fisiología , Raíces Nerviosas Espinales/cirugía , Transgenes
12.
Eur J Neurosci ; 28(8): 1467-79, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18973572

RESUMEN

Even after reconstructive surgery, major functional impairments remain in the majority of patients with peripheral nerve injuries. The application of novel emerging therapeutic strategies, such as lentiviral (LV) vectors, may help to stimulate peripheral nerve regeneration at a molecular level. In the experiments described here, we examined the effect of LV vector-mediated overexpression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on regeneration of the rat peripheral nerve in a transection/repair model in vivo. We showed that LV vectors can be used to locally elevate levels of NGF and GDNF in the injured rat peripheral nerve and this has profound and differential effects on regenerating sensory and motor neurons. For sensory neurons, increased levels of NGF and GDNF do not affect the number of regenerated neurons 1 cm distal to a lesion at 4 weeks post-lesion but do cause changes in the expression of markers for different populations of nociceptive neurons. These changes are accompanied by significant alterations in the recovery of nociceptive function. For motoneurons, overexpression of GDNF causes trapping of regenerating axons, impairing both long-distance axonal outgrowth and reinnervation of target muscles, whereas NGF has no effect on these parameters. These observations show the feasibility of combining surgical repair of the transected nerve with the application of viral vectors. Furthermore, they show a difference between the regenerative responses of motor and sensory neurons to locally increased levels of NGF and GDNF.


Asunto(s)
Vectores Genéticos/uso terapéutico , Lentivirus/genética , Factores de Crecimiento Nervioso/genética , Regeneración Nerviosa/genética , Traumatismos de los Nervios Periféricos , Nervios Periféricos/metabolismo , Animales , Axones/metabolismo , Biomarcadores/metabolismo , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Neuronas Motoras/metabolismo , Factor de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nociceptores/metabolismo , Nervios Periféricos/citología , Enfermedades del Sistema Nervioso Periférico/terapia , Ratas , Ratas Wistar , Recuperación de la Función/genética , Células Receptoras Sensoriales/metabolismo , Resultado del Tratamiento , Regulación hacia Arriba/genética
13.
Restor Neurol Neurosci ; 25(5-6): 585-99, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18418947

RESUMEN

PURPOSE: Spinal root avulsions result in paralysis of the upper and/or lower extremities. Implanting a peripheral nerve bridge or reinsertion of the avulsed roots in the spinal cord are surgical strategies that lead to some degree of functional recovery. In the current study lentiviral (LV) vector-mediated gene transfer of a green fluorescent protein (GFP) reporter gene was used to study the feasibility of gene therapy in the reimplanted root to further promote regeneration of motor axons. METHODS: A total of 68 female Wistar rats underwent unilateral root avulsion of the L4, L5 and L6 ventral lumbar roots. From 23 rats intercostal nerves were dissected before ventral root avulsion surgery, injected with a lentiviral vector encoding GFP (LV-GFP) and inserted between the spinal cord and avulsed rootlet. In the remaining 45 rats, the avulsed ventral root was injected with either LV-GFP or a lentiviral vector encoding a fusion between a GlyAla repeat and GFP (LV-GArGFP), and reinserted into the spinal cord. Expression of GFP was evaluated at 1,2, 4 and 10 weeks, and one group at 4 months. RESULTS: LV-GFP transduction of either nerve implants or reimplanted ventral roots revealed high GFP expression during the first 2 post-lesion weeks, but virtually no expression at 4 weeks. Since this reduction coincided with the appearance of mononuclear cells at the repair site, an immune response against GFP may have occurred. In a subsequent experiment reimplanted ventral roots were transduced with a vector encoding GFP fused with the GlyAla repeat of Epstein-Barr virus Nuclear Antigen 1 known to prevent generation of antigenic peptides from transgene products. Expression of this "stealth" gene persisted for at least 4 months in the reimplanted root. CONCLUSION: Thus persistent transgene expression can be achieved with non-immunogenic transgene products in reimplanted ventral roots. This demonstrates the feasibility of combining neurosurgical repair with LV vector-mediated gene therapy. The current approach will be used in future experiments with LV vectors encoding neurotrophic factors to enhance the regeneration of spinal motor neurons after traumatic avulsion of spinal nerve roots.


Asunto(s)
Expresión Génica/fisiología , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Lentivirus/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de la Médula Espinal/patología , Raíces Nerviosas Espinales/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/terapia , Raíces Nerviosas Espinales/lesiones , Factores de Tiempo
14.
J Neurotrauma ; 23(1): 18-35, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16430370

RESUMEN

The purpose of this study was to compare spontaneous functional recovery after different spinal motor tract lesions in the rat spinal cord using three methods of analysis, the BBB, the rope test, and the CatWalk. We transected the dorsal corticospinal tract (CSTx) or the rubrospinal tract (RSTx) or the complete dorsal half of the spinal cord (Hx) at thoracic level T8. Functional recovery was monitored for 31 weeks. We found no recovery of consistent inter limb coordination in any experimental group over time using the BBB locomotor rating scale. Quantitative CatWalk analysis revealed significant differences between experimental groups for inter limb coordination (RI). RSTx and Hx animals showed a significant decrease in the RI, and only in the RSTx group did the RI improve from 6 weeks post-lesion onward. Significant differences between experimental groups in step sequence patterns and base of support were also observed. In the rope test all experimental groups had significantly higher error percentages compared to control animals. Tracing of the CST revealed enhanced collateral formation rostral to the lesion in the CSTx group, not in other groups. The results presented here show that locomotor function in all, but CSTx groups gradually improved over time. This is important for studies that employ pharmacological, cell-, and/or gene therapy- based interventions to improve axonal regeneration and functional recovery after spinal cord injury.


Asunto(s)
Vías Eferentes/fisiopatología , Trastornos Neurológicos de la Marcha/fisiopatología , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Animales , Biotina/análogos & derivados , Desnervación , Dextranos , Evaluación de la Discapacidad , Modelos Animales de Enfermedad , Vías Eferentes/patología , Femenino , Trastornos Neurológicos de la Marcha/diagnóstico , Trastornos Neurológicos de la Marcha/etiología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Locomoción/fisiología , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Tractos Piramidales/lesiones , Tractos Piramidales/patología , Tractos Piramidales/fisiopatología , Ratas , Ratas Wistar , Núcleo Rojo/lesiones , Núcleo Rojo/patología , Núcleo Rojo/fisiopatología , Médula Espinal/patología , Traumatismos de la Médula Espinal/diagnóstico , Tiempo , Factores de Tiempo
15.
Cell Stem Cell ; 18(1): 53-65, 2016 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-26748756

RESUMEN

Human pluripotent stem cells (hPSCs) with knockout or mutant alleles can be generated using custom-engineered nucleases. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases are the most commonly employed technologies for editing hPSC genomes. In this Protocol Review, we provide a brief overview of custom-engineered nucleases in the context of gene editing in hPSCs with a focus on the application of TALENs and CRISPR/Cas9. We will highlight the advantages and disadvantages of each method and discuss theoretical and technical considerations for experimental design.


Asunto(s)
Sistemas CRISPR-Cas , Técnicas Genéticas , Genoma Humano , Células Madre Pluripotentes/citología , Alelos , ADN/análisis , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Vectores Genéticos , Genotipo , Humanos , Mutación , Mutación Puntual , Homología de Secuencia de Ácido Nucleico
16.
Dis Model Mech ; 9(4): 451-62, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26769797

RESUMEN

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells, XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34'), was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration.


Asunto(s)
Trastornos Distónicos/genética , Trastornos Distónicos/patología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Histona Acetiltransferasas/genética , Células-Madre Neurales/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Adulto , Anciano , Anciano de 80 o más Años , Empalme Alternativo/genética , Secuencia de Aminoácidos , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Marcadores Genéticos , Genotipo , Haplotipos/genética , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Persona de Mediana Edad , Células-Madre Neurales/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo
17.
Curr Protoc Stem Cell Biol ; 34: 5B.3.1-5B.3.25, 2015 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-26237572

RESUMEN

Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications.


Asunto(s)
Sistemas CRISPR-Cas , Desoxirribonucleasas , Vectores Genéticos , Células Madre Pluripotentes , Transfección/métodos , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo
18.
Prog Brain Res ; 146: 451-76, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-14699980

RESUMEN

Injuries to the adult mammalian spinal cord often lead to severe damage to both ascending (sensory) pathways and descending (motor) nerve pathways without the perspective of complete functional recovery. Future spinal cord repair strategies should comprise a multi-factorial approach addressing several issues, including optimalization of survival and function of spared central nervous system neurons in partial lesions and the modulation of trophic and inhibitory influences to promote and guide axonal regrowth. Neurotrophins have emerged as promising molecules to augment neuroprotection and neuronal regeneration. Although intracerebroventricular, intrathecal and local protein delivery of neurotrophins to the injured spinal cord has resulted in enhanced survival and regeneration of injured neurons, there are a number of drawbacks to these methods. Viral vector-mediated transfer of neurotrophin genes to the injured spinal cord is emerging as a novel and effective strategy to express neurotrophins in the injured nervous system. Ex vivo transfer of neurotrophic factor genes is explored as a way to bridge lesions cavities for axonal regeneration. Several viral vector systems, based on herpes simplex virus, adenovirus, adeno-associated virus, lentivirus, and moloney leukaemia virus, have been employed. The genetic modification of fibroblasts, Schwann cells, olfactory ensheathing glia cells, and stem cells, prior to implantation to the injured spinal cord has resulted in improved cellular nerve guides. So far, neurotrophic factor gene transfer to the injured spinal cord has led to results comparable to those obtained with direct protein delivery, but has a number of advantages. The steady advances that have been made in combining new viral vector systems with a range of promising cellular platforms for ex vivo gene transfer (e.g., primary embryonic neurons, Schwann cells, olfactory ensheating glia cells and neural stem cells) holds promising perspectives for the development of new neurotrophic factor-based therapies to repair the injured nervous system.


Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética/métodos , Factores de Crecimiento Nervioso/uso terapéutico , Regeneración Nerviosa , Traumatismos de la Médula Espinal/terapia , Animales , Vías de Administración de Medicamentos , Genes Virales , Vectores Genéticos , Humanos , Factores de Crecimiento Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/virología , Recuperación de la Función , Traumatismos de la Médula Espinal/virología , Factores de Tiempo
19.
Cell Stem Cell ; 12(2): 238-51, 2013 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-23246482

RESUMEN

Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.


Asunto(s)
Desoxirribonucleasas/genética , Células Madre/enzimología , Alelos , Genoma Humano/genética , Humanos , Mutación
20.
PLoS One ; 5(3): e9541, 2010 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-20221404

RESUMEN

BACKGROUND: Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts. METHODOLOGY/PRINCIPAL FINDINGS: Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Dicroismo Circular , Dimerización , Variación Genética , Humanos , Neuronas Motoras/metabolismo , Mutación , Sistemas de Lectura Abierta , Células PC12 , Estructura Terciaria de Proteína , Ratas , Superóxido Dismutasa-1
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