Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Más filtros











Base de datos
Intervalo de año de publicación
1.
Cereb Cortex ; 25(11): 4259-72, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25596590

RESUMEN

Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1(nm3419) (UCHL1(-/-)) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1(-/-) mice as a tool to study CSMN pathology.


Asunto(s)
Estrés del Retículo Endoplásmico/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Médula Espinal/citología , Ubiquitina Tiolesterasa/deficiencia , Vías Aferentes/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Corteza Motora/metabolismo , Fuerza Muscular/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina Tiolesterasa/genética
2.
Brain Res ; 1550: 47-60, 2014 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-24463035

RESUMEN

BACKGROUND: Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson's disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Furthermore, the shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. OBJECTIVE: Examine hSNCA gene silencing using mir30-hSNCA in vivo. METHODS: Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. RESULTS: AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2 m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2 m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2 m in hSNCA-silenced SN. CONCLUSION: hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD.


Asunto(s)
MicroARNs/metabolismo , Interferencia de ARN , Sustancia Negra/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Dependovirus/genética , Dopamina/metabolismo , Miembro Anterior/fisiopatología , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Masculino , MicroARNs/genética , Actividad Motora/fisiología , Trastornos del Movimiento/metabolismo , Trastornos del Movimiento/patología , Neuronas/metabolismo , Neuronas/patología , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/patología , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo
3.
Gene ; 534(2): 400-7, 2014 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-23954875

RESUMEN

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.


Asunto(s)
Diferenciación Celular/genética , Neuronas Dopaminérgicas/fisiología , Células Madre Embrionarias/fisiología , Células Madre Pluripotentes/fisiología , Formación de Roseta/métodos , Neuronas Serotoninérgicas/fisiología , Animales , Biomarcadores/metabolismo , Línea Celular , Neuronas Dopaminérgicas/metabolismo , Regulación hacia Abajo/genética , Células Madre Embrionarias/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Corteza Motora/metabolismo , Corteza Motora/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Células Madre Pluripotentes/metabolismo , Neuronas Serotoninérgicas/metabolismo
4.
Exp Neurol ; 247: 25-38, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23542820

RESUMEN

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by a progressive and extensive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and their terminals in the striatum, which results in debilitating movement disorders. This devastating disease affects over 1 million individuals in the United States and is increasing in incidence worldwide. Currently available pharmacological and surgical therapies ameliorate clinical symptoms in the early stages of disease, but they cannot stop or reverse degeneration of DA neurons. Stem cell therapies have come to the forefront of the PD research field as promising regenerative therapies. The majority of preclinical stem cell studies in experimental models of PD are focused on the idea that stem cell-derived DA neurons could be developed for replacement of diseased neurons. Alternatively, our studies and the studies from other groups suggest that stem cells also have the potential to protect and stimulate regeneration of compromised DA neurons. This review is focused on strategies based on the therapeutic potential for PD of the neurotrophic and neuroregenerative properties of a subclass of stem cells, mesenchymal stem cells (MSCs).


Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Degeneración Nerviosa/terapia , Enfermedad de Parkinson/terapia , Animales , Humanos , Degeneración Nerviosa/epidemiología , Degeneración Nerviosa/etiología , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/epidemiología
5.
Endocrinology ; 154(6): 2166-73, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23584855

RESUMEN

There is substantial evidence that age-related ovarian failure in rats is preceded by abnormal responsiveness of the neuroendocrine axis to estrogen positive feedback. Because IGF-I seems to act as a permissive factor for proper GnRH neuronal response to estrogen positive feedback and considering that the hypothalamic content of IGF-I declines in middle-aged (M-A) rats, we assessed the effectiveness of long-term IGF-I gene therapy in the mediobasal hypothalamus (MBH) of M-A female rats to extend regular cyclicity and preserve ovarian structure. We used 3 groups of M-A rats: 1 group of intact animals and 2 groups injected, at 36.2 weeks of age, in the MBH with either a bicistronic recombinant adeno-associated virus (rAAV) harboring the genes for IGF-I and the red fluorescent protein DsRed2, or a control rAAV expressing only DsRed2. Daily vaginal smears were taken throughout the study, which ended at 49.5 weeks of age. We measured serum levels of reproductive hormones and assessed ovarian histology at the end of the study. Although most of the rats injected with the IGF-I rAAV had, on the average, well-preserved estrous cyclicity as well as a generally normal ovarian histology, the intact and control rAAV groups showed a high percentage of acyclic rats at the end of the study and ovaries with numerous enlarged cysts and scarce corpora lutea. Serum LH was higher and hyperprolactinemia lower in the treated animals. These results suggest that overexpression of IGF-I in the MBH prolongs normal ovarian function in M-A female rats.


Asunto(s)
Ciclo Estral/fisiología , Terapia Genética/métodos , Hipotálamo/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Ovario/fisiología , Factores de Edad , Animales , Dependovirus/genética , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Hormona Luteinizante/sangre , Microscopía Fluorescente , Ovario/anatomía & histología , Prolactina/sangre , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
6.
Neurobiol Dis ; 47(2): 174-83, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22521461

RESUMEN

Corticospinal motor neurons (CSMN) are the cortical component of motor neuron circuitry, which controls voluntary movement and degenerates in diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. By using dual labeling combined with molecular marker analysis, we identified AAV2-2 mediated retrograde transduction as an effective approach to selectively target CSMN without affecting other neuron populations both in wild-type and hSOD1(G93A) transgenic ALS mice. This approach reveals very precise details of cytoarchitectural defects within vulnerable neurons in vivo. We report that CSMN vulnerability is marked by selective degeneration of apical dendrites especially in layer II/III of the hSOD1(G93A) mouse motor cortex, where cortical input to CSMN function is vastly modulated. While our findings confirm the presence of astrogliosis and microglia activation, they do not lend support to their direct role for the initiation of CSMN vulnerability. This study enables development of targeted gene replacement strategies to CSMN in the cerebral cortex, and reveals CSMN cortical modulation defects as a potential cause of neuronal vulnerability in ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/virología , Dendritas/patología , Dependovirus/fisiología , Neuronas Motoras/patología , Tractos Piramidales/patología , Transducción de Señal/fisiología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Dendritas/química , Dendritas/virología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/virología , Tractos Piramidales/metabolismo , Tractos Piramidales/virología
7.
Dev Neurobiol ; 72(6): 906-17, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22021082

RESUMEN

Experimental models of Parkinson's disease (PD) created by aberrant expression of the alpha-synuclein (SNCA) coding region have been reported. However, noncoding regions function in normal physiology and recent in vitro studies have shown that microRNAs-7 and -153 regulate SNCA expression by binding the 3'UTR. Here, effects of different hSNCA forms were examined in vivo. Adult, male rats were injected into one substantia nigra (SN) with AAV-wtSNCA, AAV-S129A hSNCA, or AAV-S129D hSNCA either with or without a portion of the native 3'UTR. DA neurons in SN that maintained striatal (ST) projections at the end of treatment were retrogradely labeled by bilateral ST fluorogold (FG) injections and FG-positive DA neurons in SN were counted. At 5 weeks, hSNCA coding vectors reduced numbers of FG-positive neurons in injected SN compared with uninjected SN (wtSNCA, p = 0.05; S129A/D hSNCA, p = 0.01). At 7 and 9 weeks, wtSNCA- and S129D hSNCA-treated rats exhibited recovery, but S129A hSNCA-injected rats did not (p = 0.01). In contrast, numbers of FG-positive neurons were unaffected by hSNCA expression when the 3'UTR was included. When FG-positive neurons were expressed as the ratio of numbers in injected to uninjected sides, the S129A hSNCA coding vector resulted in the highest decrease at 9 weeks versus wtSNCA (p = 0.05) or S129D hSNCA (p = 0.01). Inclusion of the 3'UTR resulted in no significant differences in FG-positive neuron ratios. These data suggest that inclusion of the 3'UTR protects against S129A hSNCA-induced loss of nigrostriatal-projecting DA neurons in vivo and that mis-regulation of hSNCA expression and function at noncoding regions contribute to PD pathogenesis.


Asunto(s)
Regiones no Traducidas 3'/genética , Cuerpo Estriado/metabolismo , Neuronas Dopaminérgicas/metabolismo , Degeneración Nerviosa/metabolismo , Sustancia Negra/metabolismo , alfa-Sinucleína/genética , Animales , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Terapia Genética , Masculino , Degeneración Nerviosa/patología , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fosforilación , Ratas , Ratas Sprague-Dawley , Sustancia Negra/patología , alfa-Sinucleína/metabolismo
8.
Brain Res ; 1395: 94-107, 2011 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-21565333

RESUMEN

Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson's disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9weeks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.


Asunto(s)
Dependovirus/genética , Silenciador del Gen/fisiología , Terapia Genética/métodos , Vectores Genéticos/farmacología , Vectores Genéticos/toxicidad , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/terapia , alfa-Sinucleína/genética , Animales , Modelos Animales de Enfermedad , Dopamina/fisiología , Miembro Anterior/fisiopatología , Células HEK293 , Humanos , Masculino , Neuronas/metabolismo , Neuronas/patología , Trastornos Parkinsonianos/fisiopatología , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/toxicidad , Ratas , Ratas Sprague-Dawley , Resultado del Tratamiento , alfa-Sinucleína/fisiología
9.
Brain Res ; 1386: 15-24, 2011 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-21338582

RESUMEN

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson's disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.


Asunto(s)
Dependovirus/genética , Dopamina/metabolismo , Silenciador del Gen/fisiología , Vectores Genéticos/toxicidad , MicroARNs/toxicidad , Neuronas/metabolismo , alfa-Sinucleína/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Vectores Genéticos/fisiología , Células HEK293 , Humanos , Ratones , MicroARNs/genética , Neuronas/patología , Células PC12 , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Plásmidos/genética , Ratas , alfa-Sinucleína/genética
10.
J Neurosci Res ; 88(12): 2669-81, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20544825

RESUMEN

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive degeneration of nigrostriatal dopaminergic (DA) neurons. The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF), the most potent neurotrophic factor for DA neurons, has been demonstrated in many experimental models of PD. However, chronic delivery of GDNF to DA neurons in the brain remains an unmet challenge. Here, we report the effects of GDNF-releasing Notch-induced human bone marrow-derived mesenchymal stem cells (MSC) grafted into striatum of the 6-hydroxydopamine (6-OHDA) progressively lesioned rat model of PD. Human MSC, obtained from bone marrow aspirates of young, healthy adult volunteers, were transiently transfected with the intracellular domain of the Notch1 gene (NICD) to generate SB623 cells. SB623 cells expressing GDNF and/or humanized Renilla green fluorescent protein (hrGFP) following lentiviral transduction or nontransduced cells were stereotaxically placed into rat striatum 1 week after a unilateral partial 6-OHDA striatal lesion. At 4 weeks, rats that had received GDNF-transduced SB623 cells had significantly decreased amphetamine-induced rotation compared with control rats, although this effect was not observed in rats that received GFP-transduced or nontransduced SB623 cells. At 5 weeks, rejuvenated tyrosine hydroxylase-immunoreactive (TH-IR) fibers that appeared to be host DA axons were observed in and around grafts. This effect was more prominent in rats that received GDNF-secreting cells and was not observed in controls. These observations suggest that human bone-marrow derived MSC, genetically modified to secrete GDNF, hold potential as an allogeneic or autologous stem cell therapy for PD.


Asunto(s)
Trasplante de Médula Ósea/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/terapia , Recuperación de la Función/fisiología , Adulto , Animales , Diferenciación Celular/genética , Línea Celular , Modelos Animales de Enfermedad , Ingeniería Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Supervivencia de Injerto/fisiología , Humanos , Masculino , Regeneración Nerviosa/fisiología , Trastornos Parkinsonianos/fisiopatología , Ratas , Ratas Endogámicas F344 , Trasplante Autólogo , Trasplante Heterólogo/métodos , Trasplante Homólogo
11.
Restor Neurol Neurosci ; 28(3): 293-309, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20479525

RESUMEN

PURPOSE: The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) gene delivery was examined in a rodent model of traumatic brain injury (TBI), the controlled cortical impact (CCI). METHODS: An adenoviral vector harboring human GDNF (AdGDNF) or green fluorescent protein (AdGFP) was injected unilaterally into the forelimb sensorimotor cortex (FL-SMC) of the rat one week prior to a unilateral CCI. Tests of forelimb function and asymmetry were administered for 2 weeks post-injury. At 2 weeks post-injury, animals were sacrificed and contusion size, neuronal survival, neurodegeneration, and virally-mediated GDNF and GFP protein expression were measured. RESULTS: Rats injected with AdGDNF had significantly smaller contusions, more surviving neurons, and less neurodegeneration than AdGFP injected and uninjected injured animals. GDNF gene delivery also resulted in significantly faster recovery of forelimb coordination and a smaller initial preference for the uninjured forelimb during exploration of the walls of a platform. However, overall recovery of symmetrical forelimb use was not achieved. CONCLUSIONS: The discrepancy between neural protection and behavioral recovery suggests that while GDNF gene delivery provided a high degree of protection of damaged cortical neurons in this model of TBI, it may not have fully protected their terminals and synaptic functioning, resulting in only mild protection against behavioral deficits.


Asunto(s)
Lesiones Encefálicas/terapia , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Fármacos Neuroprotectores/administración & dosificación , Adenoviridae/genética , Animales , Lesiones Encefálicas/genética , Lesiones Encefálicas/patología , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Cojera Animal/etiología , Cojera Animal/fisiopatología , Cojera Animal/terapia , Masculino , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Degeneración Nerviosa/terapia , Fármacos Neuroprotectores/metabolismo , Ratas , Ratas Endogámicas F344 , Recuperación de la Función/genética
12.
Cell Transplant ; 18(7): 801-14, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19796495

RESUMEN

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the selective loss of dopaminergic (DA) neurons in the midbrain. Various types of stem cells that have potential to differentiate into DA neurons are being investigated as cellular therapies for PD. Stem cells also secrete growth factors and therefore also may have therapeutic effects in promoting the health of diseased DA neurons in the PD brain. To address this possibility in an experimental model of PD, bone marrow-derived neuroprogenitor-like cells were generated from bone marrow procured from healthy human adult volunteers and their potential to elicit recovery of damaged DA axons was studied in a partial lesion rat model of PD. Following collection of bone marrow, mesenchymal stem cells (MSC) were isolated and then genetically modified to create SB623 cells by transient transfection with the intracellular domain of the Notch1 gene (NICD), a modification that upregulates expression of certain neuroprogenitor markers. Ten deposits of 0.5 microl of SB623 cell suspension adjusted from 6,000 to 21,000 cells/microl in PBS or PBS alone were stereotaxically placed in the striatum 1 week after the nigrostriatal projection had been partially lesioned in adult F344 rats by injection of 6-hydroxydopamine (6-OHDA) into the striatum. At 3 weeks, a small number of grafted SB623 cells survived in the lesioned striatum as visualized by expression of the human specific nuclear matrix protein (hNuMA). In rats that received SB623 cells, but not in control rats, dense tyrosine hydroxylase immunoreactive (TH-ir) fibers were observed around the grafts. These fibers appeared to be rejuvenated host DA axons because no TH-ir in soma of surviving SB623 cells or coexpression of TH and hNuMA-ir were observed. In addition, dense serotonin immunoreactive (5-HT-ir) fibers were observed around grafted SB623 cells and these fibers also appeared to be of the host origin. Also, in some SB623 grafted rats that were sacrificed within 2 h of dl-amphetamine injection, hot spots of c-Fos-positive nuclei that coincided with rejuvenated dense TH fibers around the grafted SB623 cells were observed, suggesting increased availability of DA in these locations. Our observations suggest that NICD-transfected MSC hold potential as a readily available autologous or allogenic cellular therapy for ameliorating the degeneration of DA and 5-HT neurons in PD patients.


Asunto(s)
Dopamina , Trasplante de Células Madre Mesenquimatosas , Degeneración Nerviosa/terapia , Neuronas/fisiología , Trastornos Parkinsonianos/terapia , Anfetamina/metabolismo , Anfetamina/farmacología , Animales , Células de la Médula Ósea/citología , Comunicación Celular , Línea Celular , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patología , Neostriado/citología , Fibras Nerviosas/metabolismo , Neuronas/citología , Trastornos Parkinsonianos/patología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Endogámicas F344 , Serotonina/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/patología , Transfección , Tirosina 3-Monooxigenasa/metabolismo
13.
Tissue Eng Part C Methods ; 14(3): 207-19, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18721070

RESUMEN

Recent advances in tissue engineering that combine an extracellular matrix-like scaffold with therapeutic molecules, cells, DNA encoding therapeutic proteins, or a combination of the three hold promise for treating defects in the brain resulting from a penetrating injury or tumor resection. The purpose of this study was to investigate a porous sponge-like collagen scaffold for non-viral delivery of a plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) to rat marrow stromal stem cells (also referred to as mesenchymal stem cells, MSCs). The effects of the following parameters on GDNF synthesis in the three-dimensional (3D) constructs were evaluated and compared with results in monolayer culture: initial plasmid load (2-50 microg pGDNF), ratio of a lipid transfection reagent to plasmid (5:10), culture environment during the transfection (static and dynamic), and cell density. The level of gene expression in the collagen scaffolds achieved therapeutic levels that had previously been found to support survival of dopaminergic and trigeminal neurons in vitro. For the highest loading of plasmid (50 microg), the level of GDNF protein remained six to seven times above the control level after 2 weeks, a significant difference. Cell density in the scaffold was of importance for an early increase in GDNF production, with accumulated GDNF being approximately 60% greater after 9 days of culture when scaffolds were initially seeded with 2 million rat MSCs compared to 500,000 cells. Application of orbital shaking during the 4 h of transfection had a positive effect on the production of GDNF on 3D constructs but not of the same magnitude as reported in monolayer studies. Overall, these results demonstrate that the combination of tissue engineering and non-viral transfection of MSCs for the over-expression of GDNF is a promising approach for the long-term production of GDNF and probably for neurotrophic factors in general.


Asunto(s)
Colágeno/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Células Madre Mesenquimatosas/citología , Animales , Línea Celular , Proliferación Celular , Células Cultivadas , ADN/metabolismo , Técnicas de Transferencia de Gen , Técnicas In Vitro , Modelos Biológicos , Modelos Genéticos , Neuroglía/citología , Plásmidos/metabolismo , Ratas , Ratas Endogámicas Lew
14.
J Neurosci Res ; 86(2): 317-25, 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17787016

RESUMEN

6-Hydroxydopamine (6-OHDA), a neurotoxin that causes the death of dopamine (DA) neurons, is commonly used to produce experimental models of Parkinson's disease (PD) in rodents. In the rat model of PD first described by Sauer and Oertel, DA neurons progressively die over several weeks following a striatal injection of 6-OHDA. It is generally assumed that DA neurons die through apoptosis after exposure to 6-OHDA, but data supporting activation of a caspase enzymatic cascade are lacking. In this study, we sought to determine if caspases involved in the intrinsic apoptotic cascade play a role in the initial stages of 6-OHDA-induced death of DA neurons in the progressively lesioned rat model of PD. We found that injection of 6-OHDA into adult rat striatum did not activate caspase-9 or caspase-3 or increase levels of caspase-dependent cleavage products in the substantia nigra at various survival times up to 7 days after the lesion, even though this paradigm produced DA neuronal loss. These data suggest that in the adult rat brain DA neurons whose terminals are challenged with 6-OHDA do not die through a classical caspase-dependent apoptotic mechanism.


Asunto(s)
Adrenérgicos/farmacología , Caspasas/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Neuronas/patología , Oxidopamina/farmacología , Trastornos Parkinsonianos/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Encéfalo/metabolismo , Encéfalo/patología , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Caspasas/metabolismo , Dopamina/metabolismo , Activación Enzimática/fisiología , Inmunohistoquímica , Masculino , Degeneración Nerviosa/patología , Trastornos Parkinsonianos/metabolismo , Ratas , Ratas Endogámicas F344
15.
Mol Ther ; 14(1): 69-78, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16650807

RESUMEN

We investigated the movement of interstitially infused macromolecules within the central nervous system (CNS) in rats with high and low blood pressure (BP)/heart rate and in rats euthanized immediately before infusion (no heart action). Adeno-associated virus 2 (AAV2), fluorescent liposomes, or bovine serum albumin was infused into rat striatum (six hemispheres per group) by convection-enhanced delivery (CED). After infusion, distribution volumes were evaluated. The rats with high BP/heart rate displayed a significantly larger distribution of the infused molecules within the injected site and more extensive transport of those molecules to the globus pallidus. This difference was particularly apparent for AAV2, for which a 16.5-fold greater distribution of viral capsids was observed in the rats with high BP/heart rate than in the rats with no heartbeat. Similar results were observed for liposomes, despite their larger diameter. The distribution of all infused molecules in all rats that had low or no blood flow was confined to the space around brain blood vessels. These findings show that fluid circulation within the CNS through the perivascular space is the primary mechanism by which viral particles and other therapeutic agents administered by CED are spread within the brain and that cardiac contractions power this process.


Asunto(s)
Vasos Sanguíneos/fisiología , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Animales , Presión Sanguínea/fisiología , Vasos Sanguíneos/metabolismo , Convección , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/farmacocinética , Frecuencia Cardíaca/fisiología , Liposomas/administración & dosificación , Liposomas/farmacocinética , Angiografía por Resonancia Magnética , Masculino , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/farmacocinética
16.
Exp Neurol ; 198(2): 382-90, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16455076

RESUMEN

Human alpha-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson's disease and other related neurodegenerative synucleinopathies. Several single point mutations in the alpha-synuclein gene, as well as gene duplication and triplication, have been linked to familial Parkinson's disease. Moreover, genetic variability of the alpha-synuclein gene promoter is associated with idiopathic Parkinson's disease. Silencing of the human alpha-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human alpha-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human alpha-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human alpha-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an alpha-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human alpha-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human alpha-synuclein in vivo in rat brain. Our results demonstrate potent silencing of human alpha-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson's disease.


Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Lentivirus/fisiología , Interferencia de ARN/fisiología , alfa-Sinucleína/metabolismo , Alelos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/virología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Masculino , Proteínas Mutantes/fisiología , Fosfopiruvato Hidratasa/metabolismo , Interferencia de ARN/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Transfección/métodos , alfa-Sinucleína/genética
17.
Exp Neurol ; 191 Suppl 1: S80-94, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15629764

RESUMEN

Caspase-9 is a critical downstream effector molecule involved in apoptosis, a cell death process thought to be involved in the demise of dopamine (DA) neurons in the substantia nigra (SN) affected by Parkinson's disease (PD). In this study, we determined that a tetracycline-regulated adenovirus harboring a dominant-negative form of caspase-9 (Casp9DN) and the marker gene, enhanced green fluorescent protein (EGFP), under the control of a bidirectional promoter could each be regulated in vitro and in vivo by doxycycline. We next observed that Casp9DN gene delivery significantly protected against TNFalpha and cycloheximide-induced chromatin condensation in HeLa cells and prevented chromatin condensation and the appearance of the early apoptotic marker annexin V in 6-hydroxydopamine (6-OHDA) treated MN9D cells, a dopaminergic cell line. Effects of Casp9DN on DA neurons in vivo were also assessed. DA neurons were retrogradely labeled with fluorogold (FG) and transduced with Casp9DN and EGFP or EGFP alone. A progressive lesion of DA neurons was induced by striatal injection of 6-OHDA 1 week later. At 2 weeks post-lesion, a morphometric analysis of FG+ neurons in the SN revealed that the mean cell diameter of FG labeled neurons in the Casp9DN group was 8% and 21% larger than the EGFP and PBS groups, respectively (P <0.05). However, there was no difference among the treatment groups in the number of neurons remaining in the lesioned SN. These results suggest that while inhibiting apoptosis at the level of caspase-9 is protective in vitro, it is not protective against 6-OHDA-induced cell death in vivo.


Asunto(s)
Adenoviridae/genética , Encéfalo/metabolismo , Caspasas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Trastornos Parkinsonianos/metabolismo , Tetraciclina/farmacología , Adenoviridae/efectos de los fármacos , Animales , Apoptosis , Encéfalo/efectos de los fármacos , Encéfalo/patología , Caspasa 9 , Caspasas/metabolismo , Línea Celular , Técnicas de Transferencia de Gen , Genes Dominantes , Genes Reporteros , Vectores Genéticos/efectos de los fármacos , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Riñón/citología , Riñón/metabolismo , Masculino , Neostriado/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Neurotoxinas , Oxidopamina , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/terapia , Ratas , Ratas Endogámicas F344
18.
Exp Neurol ; 190(2): 263-75, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15530868

RESUMEN

This is a commentary on the developmental and therapeutic relevance of recent studies in the glial fibrillary acid protein (GFAP)-glial cell line-derived neurotrophic factor (GDNF) transgenic mouse reported by Zhao et al. (2004). This interesting study demonstrated that increased expression of GDNF in astrocytes increases the number of neighboring motoneurons of certain motoneuron subpopulations by diminishing programmed cell death during development. In addition, astrocyte-derived GDNF was shown to protect facial motoneurons from injury-induced cell death. Since this is the first direct demonstration that secretion of GDNF from astrocytes in the CNS can affect motoneuron development in utero and motoneuron survival after axotomy, novel approaches for motor neuron disease are suggested. The known target neurons that respond to GDNF are reviewed, as are studies using GDNF gene delivery in animal models of amyotrophic lateral sclerosis (ALS). It is postulated that GDNF is a factor to which many motoneurons respond along their whole extent from soma to axon to terminal.


Asunto(s)
Neuronas Motoras/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Animales , Astrocitos/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Ratones , Ratones Transgénicos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA