RESUMEN

BACKGROUND: Acute and chronic brain damage in type 2 diabetes mellitus (DM) determines the need to investigate the neuroprotective potential of glucose-lowering drugs. The purpose was to directly compare the neuroprotective effects of glucagon-like peptide-1 receptor agonists (GLP-1RAs) with different duration of action and sodium-glucose cotransporter-2 inhibitors (SGLT-2i) in type 2 diabetic rats with and without stroke. METHODS: DM was modelled using high-fat diet and nicotinamide+streptozotocin protocol. The following groups (n = 15 each) were formed: DM without treatment, treatment with liraglutide, dulaglutide, canagliflozin as well as control group without DM and treatment. After 8 weeks, 10 rats from each group underwent middle cerebral artery occlusion. In the reperfusion period neurological deficit, neuroglial damage markers and brain necrosis were evaluated. Brain slices from the remaining 5 animals in each group were histologically examined for microglial activation and neuronal damage. RESULTS: Brain damage was similar in "DM" and "Control" (17.53 [14.23; 26.58] and 15.87 [13.40; 22.68] % of total brain volume, respectively). All study drugs diminished damage volume comparing with "DM" and "Control" whereas the necrosis volume in "DM+Liraglutide" was smaller than in "DM+Canagliflozin" and did not significantly differ from "DM+Dulaglutide" (2.9 [1.83; 4.71], 6.17 [3.88; 8.88] and 4.57 [3.27; 7.90] %). The neurological deficit was more prominent in "DM" than in "Control", while all the drugs demonstrated similar positive effect. Neurofilament light chains (NLC) did not differ between "DM" and "Control". Dulaglutide and canagliflozin caused a marked decrease in NLC. Protein S100BB level was similar in "DM" and "Control". Liraglutide caused the largest S100BB decrease, while canagliflozin did not influence it. In chronic brain ischaemia, all drugs increased the number of normal neurons, but GLP-1RAs had a more pronounced effect. DM was accompanied by increased number of activated microglial cells in Cornu Ammonis (CA)1 hippocampal region. Both GLP-1RAs reduced the number of Iba-1-positive cells, with dulaglutide being more effective than liraglutide, whereas canagliflozin did not affect this parameter. CONCLUSIONS: GLP-1RAs and SGLT-2i have neuroprotective properties against acute and chronic brain damage in diabetic rats, although the infarct-limiting effect of GLP-1RAs may be more pronounced. GLP-1RAs and SGLT-2i exert their protective effects by directly influencing neuronal survival, whereas GLP-1RAs also affect microglia.

Asunto(s)

Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón , Microglía , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Masculino , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Ratas , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Ratas Wistar , Daño Encefálico Crónico/prevención & control , Daño Encefálico Crónico/etiología , Daño Encefálico Crónico/patología , Liraglutida/farmacología , Liraglutida/uso terapéutico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico

RESUMEN

The most common cause of autosomal recessive familial Parkinson's disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus. The established line displays typical human primed iPSC morphology and expression of pluripotency-associated markers, normal karyotype without SNP array-detectable copy number variations and can give rise to derivatives of all three embryonic germ layers. We envisage the usefulness of this iPSC line, carrying a common and well-studied missense mutation in the RING1 domain of the PARKIN protein, for the elucidation of PARKIN-dependent mechanisms of PD using in vitro and in vivo models.

Asunto(s)

Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Masculino , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Variaciones en el Número de Copia de ADN , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

AIMS: Metabolic dysfunction is involved in modulating the disease process in Huntington disease (HD) but the underlying mechanisms are not known. The aim of this study was to investigate if the metabolic regulators sirtuins are affected in HD. METHODS: Quantitative real-time polymerase chain reactions were used to assess levels of SIRT1-3 and downstream targets in post mortem brain tissue from HD patients and control cases as well as after selective hypothalamic expression of mutant huntingtin (HTT) using recombinant adeno-associated viral vectors in mice. RESULTS: We show that mRNA levels of the metabolic regulator SIRT1 are increased in the striatum and the cerebral cortex but not in the less affected cerebellum in post mortem HD brains. Levels of SIRT2 are only increased in the striatum and SIRT3 is not affected in HD. Interestingly, mRNA levels of SIRT1 are selectively increased in the lateral hypothalamic area (LHA) and ventromedial hypothalamus (VMH) in HD. Further analyses of the LHA and VMH confirmed pathological changes in these regions including effects on SIRT1 downstream targets and reduced mRNA levels of orexin (hypocretin), prodynorphin and melanin-concentrating hormone (MCH) in the LHA and of brain-derived neurotrophic factor (BDNF) in the VMH. Analyses after selective hypothalamic expression of mutant HTT suggest that effects on BDNF, orexin, dynorphin and MCH are early and direct, whereas changes in SIRT1 require more widespread expression of mutant HTT. CONCLUSIONS: We show that SIRT1 expression is increased in HD-affected brain regions and that metabolic pathways are altered in the HD hypothalamus.

Asunto(s)

Encéfalo/metabolismo , Enfermedad de Huntington/metabolismo , Hipotálamo/metabolismo , Sirtuina 1/metabolismo , Anciano , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología

RESUMEN

Neuropathological and cell culture studies suggest that tau and α-synuclein pathologies may promote each other. To study the relevance and functional implications of these findings in vivo, we transduced hippocampal neurons of wild-type or human A30P α-synuclein transgenic mice with wild-type or P301S mutated human tau using an adeno-associated virus vector. Green fluorescent protein transduction was used as a control. We assessed spontaneous exploratory activity, anxiety and spatial learning and memory 11 weeks after the transduction and perfused the mice for histology. The transduced tau was mainly found in axon terminals and largely restricted within the hippocampi. In addition, neurons around the injection site showed cytoplasmic staining for human tau in both wild-type and A30P mice. Of these tau-positive neurons, 44% in A30P mice but only 3% in wild-type mice receiving human wild-type tau transduction formed paired helical filament-1 (PHF-1)-positive cytoplasmic densities. In contrast, only 1% of tau-positive neurons were also PHF-1 positive after transduction with P301S tau in mice of either genotype. Transduction of P301S tau reduced swimming speed but otherwise tau transduction had no significant behavioral consequences. Cytoplasmic PHF-1 densities were associated with poor spatial memory in wild-type mice but slightly improved memory in A30P mice, indicating that also tau hyperphosphorylation does not necessarily compromise neural functions. These data demonstrate that α-synuclein promotes tau hyperphosphorylation depending on the amino acids on the 301 site.

Asunto(s)

Mutación , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Animales , Humanos , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Fosforilación/fisiología , Transducción Genética , alfa-Sinucleína/genética , Proteínas tau/genética

RESUMEN

We have previously shown that persistent α-synuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) α-synuclein. However, A53T α-synuclein overexpression induced neurodegeneration that resulted in nigral dopaminergic cell death. Here, we analyze the microglia population in the midbrain of these animals by stereological quantification of Iba1+ cells. Our data here show that monkeys overexpressing A53T α-synuclein showed a long-term increase in microglia presenting macrophagic morphology. However, wt α-synuclein overexpression, despite the absence of dopaminergic cell death, resulted in a permanent robust increase of the microglia population characterized by a range of distinct morphological types that persisted after 1 year. These results confirm that the microglial response differs depending on the type of α-synuclein (wt/A53T) and/or whether α-synuclein expression results in cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, the microglial response is modulated by events related to α-synuclein expression in substantia nigra and persists in the long term. The data presented here is in agreement with that previously observed in a recombinant adeno-associated virus (rAAV) α-synuclein rat model, thereby validating both the findings and the model, and highlighting the translational potential of the rodent model to higher species closer to humans.

Asunto(s)

Polaridad Celular/efectos de los fármacos , Microglía/fisiología , alfa-Sinucleína/fisiología , Animales , Callithrix , Núcleo Caudado/citología , Núcleo Caudado/efectos de los fármacos , Núcleo Caudado/fisiología , Recuento de Células , Muerte Celular/efectos de los fármacos , Dependovirus/genética , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Femenino , Vectores Genéticos , Gliosis/inducido químicamente , Gliosis/patología , Antígenos HLA-DR/biosíntesis , Inmunohistoquímica , Activación de Macrófagos/efectos de los fármacos , Masculino , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Mutación/fisiología , Enfermedades Neurodegenerativas/patología , Enfermedad de Parkinson Secundaria/patología , Células Madre Pluripotentes/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Putamen/citología , Putamen/efectos de los fármacos , Putamen/fisiología , alfa-Sinucleína/biosíntesis , alfa-Sinucleína/genética

RESUMEN

Modeling Parkinson's disease remains a major challenge for preclinical researchers, as existing models fail to reliably recapitulate all of the classic features of the disease, namely, the progressive emergence of a bradykinetic motor syndrome with underlying nigrostriatal α-synuclein protein accumulation and nigrostriatal neurodegeneration. One limitation of the existing models is that they are normally induced by a single neuropathological insult, whereas the human disease is thought to be multifactorial with genetic and environmental factors contributing to the disease pathogenesis. Thus, in order to develop a more relevant model, we sought to determine if administration of the Parkinson's disease-associated pesticide, rotenone, into the substantia nigra of rats overexpressing the Parkinson's disease-associated protein, α-synuclein, could reliably model the triad of classic features of the human disease. To do so, rats underwent stereotaxic surgery for unilateral delivery of the adeno-associated virus (AAV)-α-synuclein into the substantia nigra. This was followed 13 weeks later by delivery of rotenone into the same site. The effect of the genetic and environmental insults alone or in combination on lateralised motor performance (Corridor, Stepping, and Whisker Tests), nigrostriatal integrity (tyrosine hydroxylase immunohistochemistry), and α-synucleinopathy (α-synuclein immunohistochemistry) was assessed. We found that rats treated with either AAV-α-synuclein or rotenone developed significant motor dysfunction with underlying nigrostriatal neurodegeneration. However, when the genetic and environmental insults were sequentially administered, the detrimental impact of the combined insults on motor performance and nigrostriatal integrity was significantly greater than the impact of either insult alone. This indicates that sequential exposure to relevant genetic and environmental insults is a valid approach to modeling human Parkinson's disease in the rat.

Asunto(s)

Neuronas/efectos de los fármacos , Enfermedad de Parkinson Secundaria/fisiopatología , Rotenona/farmacología , Sustancia Negra/fisiopatología , alfa-Sinucleína/farmacología , Animales , Conducta Animal/efectos de los fármacos , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Masculino , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo

RESUMEN

Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.

Asunto(s)

Hidrazonas/farmacología , Microglía/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , alfa-Sinucleína/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Humanos , Masculino , Microglía/enzimología , Microglía/patología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Neuronas/enzimología , Neuronas/patología , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Functional recovery following intrastriatal transplantation of fetal dopaminergic neurons in animal models of Parkinson's disease is, at least in part, dependent on the number of surviving dopaminergic neurons and the degree of graft-derived dopaminergic reinnervation of the host striatum. In the present study, we analyzed whether continuous exposure of glial cell line-derived neurotrophic factor (GDNF) to mature dopaminergic grafts could further boost the functional outcome of widespread intrastriatal dopaminergic grafts. Rats with dopamine-denervating lesions received multiple intrastriatal transplants of fetal dopaminergic cells and graft-induced behavioral effects were analyzed in drug-induced and spontaneous motor behaviors. At three months after grafting, animals received intrastriatal injections of recombinant lentiviral vectors encoding for either human GDNF or the green fluorescent protein. Continuous exposure of GDNF to the grafts did not boost the functional recovery beyond what was observed in the control animals. Rather, in some of the spontaneous motor behaviors, animals in the GDNF-group showed deterioration as compared with control animals, and this negative effect of GDNF was associated with a down-regulation of the tyrosine hydroxylase enzyme. Based on these and our earlier results, we propose that intrastriatal administration of GDNF at the time of or shortly after grafting is highly effective in initially promoting the cell survival and fiber outgrowth from the grafts. However, once the grafts are mature, GDNF's ability to boost dopaminergic neurotransmission follows the same dynamics as for the native nigral dopaminergic neurons, which appears to be dependent on the concentration of GDNF. Since rather low doses of glial cell line-derived neurotrophic factor at nanogram levels appear to saturate these effects, it may be critical to adjust GDNF levels using tightly regulated gene expression systems.

Asunto(s)

Trasplante de Células/métodos , Dopamina/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Actividad Motora/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/cirugía , Análisis de Varianza , Animales , Apomorfina/farmacología , Conducta Animal , Recuento de Células/métodos , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/trasplante , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Actividad Motora/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Prueba de Desempeño de Rotación con Aceleración Constante/métodos , Trasplantes , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo

Asunto(s)

Terapia Genética/métodos , Enfermedad de Parkinson/terapia , Animales , Modelos Animales de Enfermedad , Lentivirus/genética , Enfermedad de Parkinson/genética , Ratas , Ubiquitina-Proteína Ligasas/genética

RESUMEN

In an attempt to define clinically relevant models of akinesia and dyskinesia in 6-hydroxydopamine (6-OHDA)-lesioned rats, we have examined the effects of drugs with high (L-DOPA) vs. low (bromocriptine) dyskinesiogenic potential in Parkinson's disease on three types of motor performance, namely: (i) abnormal involuntary movements (AIMs) (ii) rotational behaviour, and (iii) spontaneous forelimb use (cylinder test). Rats with unilateral 6-OHDA lesions received single daily i.p. injections of L-DOPA or bromocriptine at therapeutic doses. During 3 weeks of treatment, L-DOPA but not bromocriptine induced increasingly severe AIMs affecting the limb, trunk and orofacial region. Rotational behaviour was induced to a much higher extent by bromocriptine than L-DOPA. In the cylinder test, the two drugs initially improved the performance of the parkinsonian limb to a similar extent. However, L-DOPA-treated animals showed declining levels of performance in this test because the drug-induced AIMs interfered with physiological limb use, and gradually replaced all normal motor activities. L-DOPA-induced axial, limb and orolingual AIM scores were significantly reduced by the acute administration of compounds that have antidyskinetic efficacy in parkinsonian patients and/or nonhuman primates (-91%, yohimbine 10 mg/kg; -19%, naloxone 4-8 mg/kg; -37%, 5-methoxy 5-N,N-dimethyl-tryptamine 2 mg/kg; -30%, clozapine 8 mg/kg; -50%, amantadine 40 mg/kg). L-DOPA-induced rotation was, however, not affected. The present results demonstrate that 6-OHDA-lesioned rats do exhibit motor deficits that share essential functional similarities with parkinsonian akinesia or dyskinesia. Such deficits can be quantified using novel and relatively simple testing procedures, whereas rotometry cannot discriminate between dyskinetic and antiakinetic effects of antiparkinsonian treatments.

Asunto(s)

Conducta Animal/efectos de los fármacos , Discinesia Inducida por Medicamentos/psicología , Enfermedad de Parkinson Secundaria/psicología , Animales , Antidiscinéticos/farmacología , Antiparkinsonianos/farmacología , Bromocriptina/farmacología , Discinesia Inducida por Medicamentos/tratamiento farmacológico , Femenino , Marcha/efectos de los fármacos , Levodopa/farmacología , Actividad Motora/efectos de los fármacos , Oxidopamina/farmacología , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Sprague-Dawley , Rotación , Conducta Estereotipada/efectos de los fármacos , Simpaticolíticos/farmacología

RESUMEN

Here we studied the effects of glial cell line-derived neurotrophic factor (GDNF) in a rat model that represents the symptomatic stages of Parkinson's disease. GDNF was infused starting 2 weeks after an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in order to halt the ongoing degeneration of the nigrostriatal dopaminergic neurons. GDNF or vehicle was infused in the striatum or the lateral ventricle via an osmotic minipump over a total 4-week period (2-6 weeks postlesion). Motor function was evaluated by the stepping, paw reaching and drug-induced motor asymmetry tests before the pump infusion was initiated, and was repeated once during (5 weeks postlesion) and twice after the withdrawal of the minipumps (7 and 11 weeks postlesion). We found that within two weeks following the lesion approximately 40% of the nigral TH-positive neurons were lost. In the vehicle infusion groups there was an additional 20% cell loss between 2 and 12 weeks after the lesion. This latter cell loss occurred mainly in the caudal part of the SN whereas the cell loss in the rostral SN was almost complete within the first two weeks. Ventricular GDNF infusion completely blocked the late degenerating neurons in the caudal SN and had long lasting behavioural effects on the stepping test and amphetamine rotation, extending to 6 weeks after withdrawal of the factor. Striatal infusion affected the motor behaviour transiently during the infusion period but the motor performance of these animals returned to baseline upon cessation of the GDNF delivery, and the delayed nigral cell loss was marginally affected. We conclude that intraventricular GDNF can successfully block the already initiated degenerative process in the substantia nigra, and that the effects achieved via the striatal route, when GDNF is given acutely after the lesion, diminish as the fibre terminal degeneration proceeds.

Asunto(s)

Actividad Motora/efectos de los fármacos , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Enfermedad de Parkinson/fisiopatología , Anfetamina/farmacología , Animales , Cuerpo Estriado/enzimología , Cuerpo Estriado/patología , Dopamina/metabolismo , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial , Bombas de Infusión , Inyecciones Intraventriculares , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Oxidopamina , Enfermedad de Parkinson/patología , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Sprague-Dawley , Rotación , Conducta Estereotipada/fisiología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

Previous studies have shown that the functional efficacy of intrastriatal transplants of fetal dopamine (DA) neurons in the rat Parkinson model depends on their ability to establish a new functional innervation of the denervated striatum. Here we report that the survival, growth, and function of the grafted DA neurons greatly depend on the severity of the lesion of the host nigrostriatal system. Fiber outgrowth, and to a lesser extent also cell survival, were significantly reduced in animals in which part of the intrinsic DA system was left intact. Moreover, graft-induced functional recovery, as assessed in the stepping, paw-use, and apomorphine rotation tests, was obtained only in severely lesioned animals, i.e., in rats with >70% DA denervation of the host striatum. Functional recovery seen in these animals in which the 6-hydroxydopamine (6-OHDA) lesion was confined to the striatum was more pronounced than that previously obtained in rats with complete lesions of the mesencephalic DA system, indicating that spared portions of the host DA system, particularly those innervating nonstriatal forebrain areas, may be necessary for the grafts to exert their optimal functional effect. These data have implications for the optimal use of fetal nigral transplants in Parkinson patients in different stages of the disease.

Asunto(s)

Cuerpo Estriado/patología , Supervivencia de Injerto , Enfermedad de Parkinson Secundaria/terapia , Sustancia Negra/trasplante , Animales , Conducta Animal , Trasplante de Tejido Encefálico/métodos , Recuento de Células , División Celular , Supervivencia Celular , Corteza Cerebral , Cuerpo Estriado/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Trasplante de Tejido Fetal/métodos , Sistema Límbico , Actividad Motora , Oxidopamina , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/citología , Sustancia Negra/embriología , Sustancia Negra/enzimología , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson's disease.

Asunto(s)

Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/administración & dosificación , Enfermedad de Parkinson Secundaria/terapia , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Supervivencia Celular/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial , Haplorrinos , Inflamación/etiología , Inflamación/inmunología , Lentivirus/genética , Microinyecciones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Oxidopamina , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/metabolismo , Ratas , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , Resultado del Tratamiento

RESUMEN

Here we studied whether glial cell line-derived neurotrophic factor (GDNF), given as a single bolus injection before an intrastriatal 6-hydroxydopamine (6-OHDA) lesion, can protect the nigrostriatal dopamine neurons against the toxin-induced damage and preserve normal motor functions in the lesioned animals. GDNF or vehicle was injected in the striatum (25 microg), substantia nigra (25 microg) or lateral ventricle (50 microg) 6 h before the 6-OHDA lesion (20 microg/3 microL). Motor function was evaluated by the stepping and drug-induced motor asymmetry tests. Lesioned animals given vehicle alone showed a clear ipsilateral-side bias in response to amphetamine (13 turns/min), a moderate contralateral-side bias to apomorphine (4.5 turns/min) and a moderate to severe stepping deficit on the contralateral forepaw (three to four steps, as compared with 11-13 steps on the unimpaired side). Injection of GDNF into the striatum had a significant protective effect both on nigrostriatal function (1-2 turns/min in the rotation tests and seven to eight steps in the stepping test), and the integrity of the nigrostriatal pathway, seen as a protection of both the cell bodies in the substantia nigra and the dopamine innervation in the striatum. Injection of GDNF in the nigra had a protective effect on the nigral cell bodies, but not the striatal innervation, and failed to provide any functional benefit. In contrast, intranigral GDNF had deleterious effects on both the striatal TH-positive fibre density and on drug-induced rotation tests. Intraventricular injection had no effect. We conclude that preservation of normal motor functions in the intrastriatal 6-OHDA lesion model requires protection of striatal terminal innervation, and that this can be achieved by intrastriatal, but not nigral or intraventricular, administration of GDNF.

Asunto(s)

Ventrículos Cerebrales/fisiología , Cuerpo Estriado/fisiología , Dopamina/fisiología , Fibras Nerviosas/fisiología , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Neuronas/fisiología , Fármacos Neuroprotectores/farmacología , Sustancia Negra/fisiología , Animales , Apomorfina/administración & dosificación , Apomorfina/farmacología , Ventrículos Cerebrales/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Femenino , Lateralidad Funcional , Factor Neurotrófico Derivado de la Línea Celular Glial , Inyecciones Intraventriculares , Microinyecciones , Actividad Motora/efectos de los fármacos , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/ultraestructura , Proteínas del Tejido Nervioso/administración & dosificación , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Oxidopamina , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Tirosina 3-Monooxigenasa/análisis

RESUMEN

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Trasplante de Tejido Fetal , Células Madre/efectos de los fármacos , Animales , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Cuerpo Estriado/fisiología , Factor de Crecimiento Epidérmico/fisiología , Femenino , Trasplante de Tejido Fetal/fisiología , Ventrículos Laterales/efectos de los fármacos , Ventrículos Laterales/fisiología , Ratones , Ratones Transgénicos , Ratas , Ratas Sprague-Dawley , Trasplante de Células Madre , Células Madre/fisiología

RESUMEN

Previous studies have used recombinant adeno-associated viral (rAAV) vectors to deliver glial cell line-derived neurotrophic factor (GDNF) in the substantia nigra to protect the nigral dopamine (DA) neurons from 6-hydroxydopamine-induced damage. However, no regeneration or functional recovery was observed in these experiments. Here, we have used an rAAV-GDNF vector to express GDNF long-term (6 months) in either the nigral DA neurons themselves, in the striatal target cells, or in both of these structures. The results demonstrate that both nigral and striatal transduction provide significant protection of nigral DA neurons against the toxin-induced degeneration. However, only the rats receiving rAAV-GDNF in the striatum displayed behavioral recovery, accompanied by significant reinnervation of the lesioned striatum, which developed gradually over the first 4-5 months after the lesion. GDNF transgene expression was maintained at high levels throughout this period. These results provide evidence that rAAV is a highly efficient vector system for long-term expression of therapeutic proteins in the nigrostriatal system.

Asunto(s)

Cuerpo Estriado/fisiopatología , Técnicas de Transferencia de Gen , Terapia Genética , Factores de Crecimiento Nervioso , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/genética , Fármacos Neuroprotectores , Trastornos Parkinsonianos/terapia , Sustancia Negra/fisiopatología , Animales , Benzazepinas/farmacología , Cuerpo Estriado/efectos de los fármacos , Dependovirus , Agonistas de Dopamina/farmacología , Femenino , Genes Reporteros , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas del Tejido Nervioso/fisiología , Oxidopamina/toxicidad , Trastornos Parkinsonianos/fisiopatología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Sustancia Negra/efectos de los fármacos , Tirosina 3-Monooxigenasa/análisis

Asunto(s)

Sistema Nervioso Central/lesiones , Regeneración Nerviosa , Adulto , Diferenciación Celular , Humanos , Mesencéfalo/trasplante , Enfermedades Neurodegenerativas/terapia , Neuronas/metabolismo , Neuronas/trasplante , Trasplante de Células Madre , Células Madre/metabolismo

RESUMEN

Glial cell line-derived neurotrophic factor (GDNF) has prominent survival-promoting effects on lesioned nigrostriatal dopamine neurons, but understanding of the conditions under which functional recovery can be obtained remains to be acquired. We report here the time course of nigrostriatal axon degeneration in the partial lesion model of Parkinson's disease and the morphological and functional effects of sequential administration of GDNF in the substantia nigra (SN) and striatum during the first 5 weeks postlesion. By 1 day postlesion, the nigrostriatal axons had retracted back to the level of the caudal globus pallidus. Over the next 6 days axonal retraction progressed down to the SN, and during the following 7 weeks 74% of tyrosine hydroxylase-positive (TH(+)) and 84% of retrogradely labeled nigral neurons were lost, with a more pronounced loss in the rostral part of the SN. GDNF administration protected 70 and 72% of the nigral TH(+) and retrogradely labeled cell bodies, respectively, but did not prevent the die-back of the lesioned nigrostriatal axons. Although clear signs of sprouting were observed close to the injection site in the striatum as well as in the globus pallidus, the overall DA innervation of the striatum [as measured by [(3)H]-N-[1-(2-benzo(b)thiopenyl)cyclohexyl]piperidine-binding autoradiography] was not improved by the GDNF treatment. Moreover, the lesion-induced deficits in forelimb akinesia and drug-induced rotation were not attenuated. We conclude that functional recovery in the partial lesion model depends not only on preservation of the nigral cell bodies, but more critically on the ability of GDNF to promote significant reinnervation of the denervated striatum.

Asunto(s)

Axones/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Sustancia Negra/efectos de los fármacos , Animales , Transporte Axonal , Axones/fisiología , Axones/ultraestructura , Peso Corporal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Femenino , Miembro Anterior/inervación , Factor Neurotrófico Derivado de la Línea Celular Glial , Microinyecciones , Actividad Motora/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Regeneración Nerviosa , Proteínas del Tejido Nervioso/administración & dosificación , Neuronas/citología , Neuronas/fisiología , Fármacos Neuroprotectores/administración & dosificación , Oxidopamina/toxicidad , Prosencéfalo/efectos de los fármacos , Prosencéfalo/fisiología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/citología , Sustancia Negra/fisiología , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

The glial cell line-derived neurotrophic factor (GDNF)-family of neurotrophic factors consisted until recently of three members, GDNF, neurturin, and persephin. We describe here the cloning of a new GDNF-family member, neublastin (NBN), identical to artemin (ART), recently published (Baloh et al., 1998). Addition of NBN/ART to cultures of fetal mesencephalic dopamine (DA) neurons increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons by approximately 70%, similar to the maximal effect obtained with GDNF. To investigate the neuroprotective effects in vivo, lentiviral vectors carrying the cDNA for NBN/ART or GDNF were injected into the striatum and ventral midbrain. Three weeks after an intrastriatal 6-hydroxydopamine lesion only about 20% of the nigral DA neurons were left in the control group, while 80-90% of the DA neurons remained in the NBN/ART and GDNF treatment groups, and the striatal TH-immunoreactive innervation was partly spared. We conclude that NBN/ART, similarly to GDNF, is a potent neuroprotective factor for the nigrostriatal DA neurons in vivo.

Asunto(s)

Cuerpo Estriado/citología , Dopamina/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/citología , Neuronas/fisiología , Sustancia Negra/citología , Tirosina 3-Monooxigenasa/análisis , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Clonación Molecular , Técnicas de Transferencia de Gen , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Lentivirus , Ratones , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fármacos Neuroprotectores , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido

Asunto(s)

Trasplante de Tejido Encefálico/métodos , Supervivencia de Injerto/fisiología , Trastornos Parkinsonianos/cirugía , Trasplante Heterotópico/métodos , Animales , Trasplante de Tejido Encefálico/tendencias , Evaluación de la Discapacidad , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Neostriado/patología , Neostriado/fisiopatología , Neostriado/cirugía , Neuronas/citología , Neuronas/fisiología , Neuronas/trasplante , Trastornos Parkinsonianos/diagnóstico , Trastornos Parkinsonianos/fisiopatología , Ratas , Recuperación de la Función/fisiología , Sustancia Negra/citología , Sustancia Negra/fisiología , Sustancia Negra/trasplante , Trasplante Heterotópico/tendencias , Resultado del Tratamiento , Ácido gamma-Aminobutírico/metabolismo