RESUMEN
Cell-replacement therapies have long been an attractive prospect for treating Parkinson disease. However, the outcomes of fetal tissue-derived cell transplants in individuals with Parkinson disease have been variable, in part owing to the limitations of fetal tissue as a cell source, relating to its availability and the lack of possibility for standardization and to variation in methods. Advances in developmental and stem cell biology have allowed the development of cell-replacement therapies that comprise dopamine neurons derived from human pluripotent stem cells, which have several advantages over fetal cell-derived therapies. In this Review, we critically assess the potential trajectory of this line of translational and clinical research and address its possibilities and current limitations and the broader range of Parkinson disease features that dopamine cell replacement based on generating neurons from human pluripotent stem cells could effectively treat in the future.
Asunto(s)
Encéfalo/fisiopatología , Enfermedad de Parkinson/terapia , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Neuronas Dopaminérgicas/fisiología , Humanos , Células Madre Pluripotentes/fisiología , Células Madre Pluripotentes/trasplante , Trasplante de Células Madre/tendencias , Investigación Biomédica TraslacionalRESUMEN
Cell replacement is currently being explored as a therapeutic approach for neurodegenerative disease. Using stem cells as a source, transplantable progenitors can now be generated under conditions compliant with clinical application in patients. In this study, we elucidate factors controlling target-appropriate innervation and circuitry integration of human embryonic stem cell (hESC)-derived grafts after transplantation to the adult brain. We show that cell-intrinsic factors determine graft-derived axonal innervation, whereas synaptic inputs from host neurons primarily reflect the graft location. Furthermore, we provide evidence that hESC-derived dopaminergic grafts transplanted in a long-term preclinical rat model of Parkinson's disease (PD) receive synaptic input from subtypes of host cortical, striatal, and pallidal neurons that are known to regulate the function of endogenous nigral dopamine neurons. This refined understanding of how graft neurons integrate with host circuitry will be important for the design of clinical stem-cell-based replacement therapies for PD, as well as for other neurodegenerative diseases.
Asunto(s)
Ganglios Basales/fisiopatología , Células Madre Embrionarias Humanas/metabolismo , Enfermedad de Parkinson/genética , Animales , Modelos Animales de Enfermedad , Humanos , Ratones Desnudos , RatasRESUMEN
Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non-human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC-derived progenitors were grafted into the midbrain of 6-hydroxydopamine-lesioned rats, and analyzed at 6, 18, and 24 weeks for a time-course evaluation of specificity and extent of graft-derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies-based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft-derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine-induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC-derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC-derived DA neurons to the midbrain.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Neuronas Dopaminérgicas/trasplante , Mesencéfalo/cirugía , Vías Nerviosas/fisiología , Células-Madre Neurales/fisiología , Células-Madre Neurales/trasplante , Trastornos Parkinsonianos/cirugía , Prosencéfalo/fisiología , Sinapsis/fisiología , Anfetamina/farmacología , Animales , Inhibidores de Captación de Dopamina/farmacología , Femenino , Humanos , Hidroxidopaminas , Ratones , Fibras Nerviosas/fisiología , Trastornos Parkinsonianos/inducido químicamente , Ratas Desnudas , Trasplante de Células Madre , Conducta Estereotipada/efectos de los fármacosRESUMEN
Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.
Asunto(s)
Antígeno CD47/metabolismo , Dopamina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células Madre Pluripotentes/citología , Trasplante de Células Madre , Animales , Biomarcadores , Diferenciación Celular , Supervivencia Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Supervivencia de Injerto , Humanos , Separación Inmunomagnética , Inmunofenotipificación , Mesencéfalo/metabolismo , Ratas , RegeneraciónRESUMEN
Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson's disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.
Asunto(s)
Biomarcadores/metabolismo , Diferenciación Celular , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/trasplante , Enfermedad de Parkinson/terapia , Trasplante de Células Madre , Investigación Biomédica Traslacional , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Células Cultivadas , Dopamina/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Laminina/farmacología , Mesencéfalo/metabolismo , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Núcleo Subtalámico/citología , Núcleo Subtalámico/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.
Asunto(s)
Lesiones Encefálicas/cirugía , Potenciales Evocados Somatosensoriales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Sinapsis/fisiología , Potenciales de Acción , Vías Aferentes/fisiología , Animales , Encéfalo/citología , Encéfalo/ultraestructura , Lesiones Encefálicas/etiología , Línea Celular Transformada , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Modelos Animales de Enfermedad , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Neuronas/fisiología , Neuronas/ultraestructura , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Accidente Cerebrovascular/complicaciones , Sinapsis/ultraestructura , Núcleos Talámicos Ventrales/citologíaRESUMEN
In a recent issue of Nature,Falkner et al. (2016) use chronic two-photon imaging, virus-based transsynaptic tracing, and dynamic calcium indicators to elegantly demonstrate extensive in vivo functional maturation and target-specific functional integration of transplanted embryonic mouse cortical progenitors into adult lesioned visual cortical circuits.
Asunto(s)
Encéfalo , Neuronas , Animales , Proteínas Fluorescentes VerdesRESUMEN
The possibility of directly converting non-neuronal cells into neurons in situ in the brain would open therapeutic avenues aimed at repairing the brain after injury or degenerative disease. We have developed an adeno-associated virus (AAV)-based reporter system that allows selective GFP labeling of reprogrammed neurons. In this system, GFP is turned on only in reprogrammed neurons where it is stable and maintained for long time periods, allowing for histological and functional characterization of mature neurons. When combined with a modified rabies virus-based trans-synaptic tracing methodology, the system allows mapping of 3D circuitry integration into local and distal brain regions and shows that the newly reprogrammed neurons are integrated into host brain.
Asunto(s)
Antígenos/fisiología , Cuerpo Estriado/fisiología , Red Nerviosa/fisiología , Neurogénesis/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Proteoglicanos/fisiología , Animales , Antígenos/biosíntesis , Antígenos/genética , Cuerpo Estriado/citología , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Red Nerviosa/citología , Red Nerviosa/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteoglicanos/biosíntesis , Proteoglicanos/genéticaRESUMEN
Human embryonic stem cell (hESC)-derived dopamine neurons are currently moving toward clinical use for Parkinson's disease (PD). However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models.
Asunto(s)
Rastreo Celular/métodos , Neuronas Dopaminérgicas/citología , Células Madre Embrionarias Humanas/citología , Virus de la Rabia/genética , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/trasplante , Femenino , Citometría de Flujo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Histonas/genética , Humanos , Inmunohistoquímica , Microscopía Confocal , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Virus de la Rabia/fisiología , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Integración Viral/fisiologíaRESUMEN
Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson's disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/trasplante , Células Madre Embrionarias/citología , Feto/citología , Enfermedad de Parkinson/terapia , Animales , Axones/metabolismo , Supervivencia Celular , Modelos Animales de Enfermedad , Humanos , Masculino , Mesencéfalo/embriología , Neostriado/patología , Factores de Transcripción Otx/metabolismo , Enfermedad de Parkinson/patología , Ratas Desnudas , Sustancia Negra/patología , Transmisión Sináptica , Factores de TiempoRESUMEN
Induced neurons (iNs) offer a novel source of human neurons that can be explored for applications of disease modelling, diagnostics, drug screening and cell replacement therapy. Here we present a protocol for highly efficient generation of functional iNs from fetal human fibroblasts, and also demonstrate the ability of these converted human iNs (hiNs) to survive transplantation and maintain their phenotype in the adult rat brain. The protocol encompasses a delay in transgene activation after viral transduction that resulted in a significant increase in conversion efficiency. Combining this approach with treatment of small molecules that inhibit SMAD signalling and activate WNT signalling provides a further increase in the conversion efficiency and neuronal purity, resulting in a protocol that provides a highly efficient method for the generation of large numbers of functional and transplantable iNs from human fibroblasts without the use of a selection step. When transplanting the converted neurons from different stages of in vitro culture into the brain of adult rats, we observed robust survival and maintenance of neuronal identity four weeks post-transplantation. Interestingly, the positive effect of small molecule treatment observed in vitro did not result in a higher yield of iNs surviving transplantation.
Asunto(s)
Encéfalo/citología , Reprogramación Celular/fisiología , Células Madre Pluripotentes Inducidas/citología , Neuronas/trasplante , Trasplante Heterólogo/métodos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/fisiología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Dopamina/biosíntesis , Femenino , Fibroblastos/citología , Proteínas de Homeodominio/genética , Humanos , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Factores del Dominio POU/genética , Ratas , Ratas Sprague-Dawley , Proteínas Smad/antagonistas & inhibidores , Factores de Transcripción/genética , Transgenes/genética , Tubulina (Proteína)/biosíntesis , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Human foetal brain tissue has been used in experimental and clinical trials to develop cell replacement therapy in neurodegenerative disorders such as Parkinson's disease and Huntington's disease. These pioneering clinical studies have shown proof of principle that cell replacement therapy can be effective and is worthwhile to develop as a therapeutic strategy for repairing the damaged brain. However, because of the limited availability of foetal brain material, and difficulties in producing standardized and quality-tested cell preparations from this source, there have been extensive efforts in investigating the potential use of alternative cell sources for generating a large number of transplantable, authentic neural progenitors and neurons. In this review, we highlight the value of using human foetal tissue as a reference material for quality control of acquired cell fate of in vitro generated neurons before and after transplantation.
Asunto(s)
Trasplante de Tejido Encefálico/normas , Células Madre Embrionarias , Trasplante de Tejido Fetal/normas , Enfermedades Neurodegenerativas/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Humanos , Control de CalidadRESUMEN
Intrastriatal transplantation of dopaminergic neurons can restore striatal dopamine levels and improve parkinsonian deficits, but the mechanisms underlying these effects are poorly understood. Here, we show that transplants of dopamine neurons partially restore activity-dependent synaptic plasticity in the host striatal neurons. We evaluated synaptic plasticity in regions distal or proximal to the transplant (i.e., dorsolateral and ventrolateral striatum) and compared the effects of dopamine- and serotonin-enriched grafts using a rat model of Parkinson disease. Naïve rats showed comparable intrinsic membrane properties in the two subregions but distinct patterns of long-term synaptic plasticity. The ventrolateral striatum showed long-term potentiation using the same protocol that elicited long-term depression in the dorsolateral striatum. The long-term potentiation was linked to higher expression of postsynaptic AMPA and N2B NMDA subunits (GluN2B) and was dependent on the activation of GluN2A and GluN2B subunits and the D1 dopamine receptor. In both regions, the synaptic plasticity was abolished after a severe dopamine depletion and could not be restored by grafted serotonergic neurons. Solely, dopamine-enriched grafts could restore the long-term potentiation and partially restore motor deficits in the rats. The restoration could only be seen close to the graft, in the ventrolateral striatum where the graft-derived reinnervation was denser, compared with the distal dorsolateral region. These data provide proof of concept that dopamine-enriched transplants are able to functionally integrate into the host brain and restore deficits in striatal synaptic plasticity after experimental parkinsonism. The region-specific restoration might impose limitations in symptomatic improvement following neural transplantation.
Asunto(s)
Cuerpo Estriado/fisiología , Neuronas Dopaminérgicas/trasplante , Plasticidad Neuronal/fisiología , Trastornos Parkinsonianos/fisiopatología , Trastornos Parkinsonianos/terapia , Análisis de Varianza , Animales , Western Blotting , Dopamina/metabolismo , Embrión de Mamíferos/citología , Femenino , Inmunohistoquímica , Potenciación a Largo Plazo/fisiología , Actividad Motora/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D1/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Cellular reprogramming is a new and rapidly emerging field in which somatic cells can be turned into pluripotent stem cells or other somatic cell types simply by the expression of specific combinations of genes. By viral expression of neural fate determinants, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons. The resulting cells are nonproliferating and present an alternative to induced pluripotent stem cells for obtaining patient- and disease-specific neurons to be used for disease modeling and for development of cell therapy. In addition, because the cells do not pass a stem cell intermediate, direct neural conversion has the potential to be performed in vivo. In this study, we show that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprogramming genes are activated after transplantation. Using a transgenic mouse model to specifically direct expression of reprogramming genes to parenchymal astrocytes residing in the striatum, we also show that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ. Taken together, our data provide proof of principle that direct neural conversion can take place in the adult rodent brain when using transplanted human cells or endogenous mouse cells as a starting cell for neural conversion.
Asunto(s)
Astrocitos/trasplante , Diferenciación Celular/fisiología , Reprogramación Celular/fisiología , Fibroblastos/trasplante , Neuronas/citología , Animales , Astrocitos/citología , Reprogramación Celular/efectos de los fármacos , Cuerpo Estriado/citología , Doxiciclina/farmacología , Fibroblastos/citología , Citometría de Flujo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Humanos , Lentivirus , Ratones , Ratones Transgénicos , Neuronas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Células-Madre Neurales/citología , Neuronas/citología , Envejecimiento/patología , Animales , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Actividad Motora/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/trasplante , Tubo Neural/efectos de los fármacos , Tubo Neural/embriología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Telencéfalo/citología , Telencéfalo/efectos de los fármacos , Telencéfalo/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Despite the widespread use of mice as models of Parkinson's disease there is a surprising lack of validation and characterisation of unilateral lesion models in mice and the extent of behavioural impairments induced by such lesions. The aim of the present study was to characterise the behavioural deficits observed after injection of 6-hydroxydopamine unilaterally into the substantia nigra, and correlate the behavioural impairments with the extent of damage to the mesostriatal dopaminergic pathway. We found that a recently introduced test for assessment of sensorimotor impairment, the corridor task, was particularly useful in determining lesion severity, and that this test, in combination with standard drug-induced rotation tests, can be used to select animals with profound (> or = 80%) dopaminergic lesions that are stable over time. Based on these data we propose criteria that can be used to predict the extent of lesion, classified as severe, intermediate or mild lesions of the mesostriatal pathway. The correlation of cell loss and striatal innervation with the performance in each test provides a useful tool for the assessment of functional recovery in neurorestoration and cell transplantation studies, and for the evaluation of the in vivo efficacy and performance of stem cell-derived dopamine neuron preparations.
Asunto(s)
Conducta Animal/efectos de los fármacos , Oxidopamina/farmacología , Enfermedad de Parkinson , Sustancia Negra , Simpaticolíticos/farmacología , Anfetamina/farmacología , Animales , Apomorfina/farmacología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Dopaminérgicos/farmacología , Agonistas de Dopamina/farmacología , Femenino , Ratones , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Pruebas Neuropsicológicas , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/fisiopatologíaRESUMEN
Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson's disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson's disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson's disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.
Asunto(s)
Modelos Animales de Enfermedad , Dopamina/fisiología , Mesencéfalo/trasplante , Neuronas/trasplante , Enfermedad de Parkinson/cirugía , Recuperación de la Función/fisiología , Trasplantes , Animales , Femenino , Técnicas de Sustitución del Gen , Mesencéfalo/citología , Mesencéfalo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/citología , Neuronas/fisiología , Enfermedad de Parkinson/patología , Embarazo , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Transplants of fetal dopamine neurons can be used to restore dopamine neurotransmission in animal models of Parkinson's disease, as well as in patients with advanced Parkinson's disease. In these studies the cells are placed in the striatum rather than in the substantia nigra where they normally reside, which may limit their ability to achieve full restoration of motor function. Using a microtransplantation approach, which allows precise placement of small cell deposits directly into the host substantia nigra, and fetal donor cells that express green fluorescent protein under the control of the tyrosine hydroxylase promoter, we show that dopamine neuroblasts implanted into the substantia nigra of adult mice are capable of generating a new nigrostriatal pathway with an outgrowth pattern that matches the anatomy of the intrinsic system. This target-directed regrowth was closely aligned with the intrinsic striatonigral fibre projection and further enhanced by over-expression of glial cell line-derived neurotrophic factor in the striatal target. Results from testing of amphetamine-induced rotational behaviour suggest, moreover, that dopamine neurons implanted into the substantia nigra are also capable of integrating into the host circuitry at the functional level.