RESUMO
Modeling neurological diseases using human embryonic or patient-derived induced pluripotent stem cells (iPSCs) improves the understanding of molecular and cellular changes underlying these diseases and can lead to new, potentially personalized therapies. Changes in expression of axon guidance cues and altered cytoskeletal maintenance have been implicated in neurodegenerative and neuropsychiatric disorders. To date, most of the iPSC patient-derived cellular dysfunction and phenotypic studies have been performed in vitro. To study the intrinsic axonal impairments and neuronal connectivity deficits in human disease iPSC-derived neurons we propose to graft these cells into the physiological three-dimensional multi-structural environment of the central nervous system of rodent models to obtain relevant in vivo data. Such human iPSC in vivo chimeric models can allow for neuronal maturation, capture neuropathological phenotypes of axonal and connectivity impairments, and serve as target engagement and drug validation studies using human cells, thus highly relevant for advancement of the drug development process in the late pre-clinical stages.
Assuntos
Axônios/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Transtornos Mentais/terapia , Doenças Neurodegenerativas/terapia , Transplante de Células-Tronco/métodos , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Regeneração Nervosa , Transplante de Células-Tronco/efeitos adversosRESUMO
Clinical trials are under way using fetal cells to repair damaged neuronal circuitry. However, little is known about how transplanted immature neurons can grow anatomically correct connections in the adult central nervous system (CNS). We transplanted embryonic porcine neural cells in vivo into adult rat brains with neuronal and axonal loss typical of Parkinson's or Huntington's disease. Using complementary species-specific cellular markers, we found donor axons and CD44+ astroglial fibres in host white matter tracts up to 8 mm from CNS transplant sites, although only donor axons were capable of reaching correct gray matter target regions. This work demonstrates that adult host brain can orient growth of transplanted neurons and that there are differences in transplant donor glial and axonal growth patterns in cellular repair of the mature CNS.
Assuntos
Axônios/ultraestrutura , Doenças dos Gânglios da Base/cirurgia , Encéfalo/citologia , Neuroglia/citologia , Neurônios/transplante , Transplante Heterólogo , Animais , Biomarcadores/análise , Encéfalo/patologia , Encéfalo/cirurgia , Células Cultivadas , Modelos Animais de Doenças , Doença de Huntington/cirurgia , Imuno-Histoquímica , Masculino , Doença de Parkinson/cirurgia , Ratos , Ratos Sprague-Dawley , SuínosRESUMO
The movement disorder in Parkinson's disease results from the selective degeneration of a small group of dopaminergic neurons in the substantia nigra pars compacta region of the brain. A number of exploratory studies using human fetal tissue allografts have suggested that transplantation of dopaminergic neurons may become an effective treatment for patients with Parkinson's disease and the difficulty in obtaining human fetal tissue has generated interest in finding corresponding non-human donor cells. Here we report a post-mortem histological analysis of fetal pig neural cells that were placed unilaterally into the caudate-putamen brain region of a patient suffering from Parkinson's disease. Long-term (over seven months) graft survival was found and the presence of pig dopaminergic neurons and other pig neural and glial cells is documented. Pig neurons extended axons from the graft sites into the host brain. Furthermore, other graft derived cells were observed several millimeters from the implantation sites. Markers for human microglia and T-cells showed only low reactivity in direct proximity to the grafts. This is the first documentation of neural xenograft survival in the human brain and of appropriate growth of non-human dopaminergic neurons for a potential therapeutic response in Parkinson's disease.
Assuntos
Transplante de Tecido Fetal , Neurônios/transplante , Doença de Parkinson/terapia , Idoso , Animais , Sobrevivência de Enxerto , Humanos , Masculino , Suínos , Transplante HeterólogoRESUMO
We used brain imaging to study long-term neurodegenerative and bioadaptive neurochemical changes in a primate model of Parkinson disease. We gradually induced a selective loss of nigrostriatal dopamine neurons, similar to that of Parkinson disease, by creating oxidative stress through infusion of the mitochondrial complex 1 inhibitor MPTP for 14+/-5 months. Repeated evaluations over 3 years by positron emission tomography (PET) demonstrated progressive and persistent loss of neuronal dopamine pre-synaptic re-uptake sites; repeated magnetic resonance spectroscopy (MRS) studies indicated a 23-fold increase in lactate and macromolecules in the striatum region of the brain for up to 10 months after the last administration of MPTP. By 2 years after the MPTP infusions, these MRS striatal lactate and macromolecule values had returned to normal levels. In contrast, there were persistent increases in striatal choline and decreases in N-acetylaspartate. Thus, these combined PET/MRS studies demonstrate patterns of neurochemical changes that are both dynamic and persistent long after selective dopaminergic degeneration.
Assuntos
Encéfalo/fisiopatologia , Doença de Parkinson Secundária/fisiopatologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Radioisótopos de Carbono , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dopamina/metabolismo , Lactatos/metabolismo , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Degeneração Neural/patologia , Neurônios/patologia , Neurônios/fisiologia , Doença de Parkinson Secundária/diagnóstico por imagem , Doença de Parkinson Secundária/patologia , Tomografia Computadorizada de EmissãoRESUMO
Target cells are thought to regulate the survival of afferent neurons during development by supplying limiting amounts of neurotrophic factors, but the degree to which afferent neurons remain dependent on target-derived support in the adult is uncertain. In this study, uninjured basal forebrain cholinergic neurons did not die after excitotoxic ablation of their target neurons in young adult rats, indicating that they are either not dependent on neurotrophic factors for survival or can obtain trophic support from other sources after target neurons are lost. This finding suggests that cholinergic cell death in neurodegenerative conditions such as Alzheimer's disease is not due solely to a loss of target neurons or factors provided by them.
Assuntos
Colina/fisiologia , Diencéfalo/citologia , Neurônios Aferentes/fisiologia , Telencéfalo/citologia , Acetilcolinesterase/análise , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Transporte Axonal , Sobrevivência Celular , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , N-Metilaspartato , Fatores de Crescimento Neural/fisiologia , Ratos , Núcleos Septais/citologiaRESUMO
OFF-L-dopa dyskinesias have been a surprising side-effect of intrastriatal foetal ventral mesencephalic transplantation in patients with Parkinson's disease. It has been proposed that excessive and unregulated dopaminergic stimulation of host post-synaptic striatal neurons by the grafts could be responsible for these dyskinesias. To address this issue we transplanted foetal dopaminergic neurons from mice lacking the dopamine transporter (DATKO) or from wild-type mice, into a rat model of Parkinson's disease and L-dopa-induced dyskinesias. Both wild-type and DATKO grafts reinnervated the host striatum to a similar extent, but DATKO grafts produced a greater and more diffuse increase in extra-cellular striatal dopamine levels. Interestingly, grafts containing wild-type dopaminergic neurons improved parkinsonian signs to a similar extent as DATKO grafts, but provided a more complete reduction of L-dopa induced dyskinesias. Neither DATKO nor wild-type grafts induced OFF-L-dopa dyskinesias. Behavioural and receptor autoradiography analyses demonstrated that DATKO grafts induced a greater normalization of striatal dopaminergic receptor supersensitivity than wild-type grafts. Both graft types induced a similar downregulation and normalization of PEnk and fosb/Deltafosb in striatal neurons. In summary, DATKO grafts causing high and diffuse extra-cellular dompamine levels do not per se alter graft-induced recovery or produce OFF-L-dopa dyskinesias. Wild-type dopaminergic neurons appear to be the most effective neuronal type to restore function and reduce L-dopa-induced dyskinesias.
Assuntos
Transplante de Tecido Encefálico , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Discinesia Induzida por Medicamentos/etiologia , Levodopa/efeitos adversos , Doença de Parkinson/terapia , Animais , Antiparkinsonianos/efeitos adversos , Antiparkinsonianos/uso terapêutico , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/fisiologia , Discinesia Induzida por Medicamentos/terapia , Encefalinas/biossíntese , Encefalinas/genética , Feminino , Transplante de Tecido Fetal , Regulação da Expressão Gênica , Sobrevivência de Enxerto , Levodopa/uso terapêutico , Mesencéfalo/transplante , Microdiálise/métodos , Neurônios/transplante , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The interplay between dopamine and glutamate in the basal ganglia regulates critical aspects of motor learning and behavior. Metabotropic glutamate receptors (mGluR) are increasingly regarded as key modulators of neuroadaptation in these circuits, in normal and disease conditions. Using PET, we demonstrate a significant upregulation of mGluR type 5 in the striatum of MPTP-lesioned, parkinsonian primates, providing the basis for therapeutic exploration of mGluR5 antagonists in Parkinson disease.
Assuntos
Corpo Estriado/diagnóstico por imagem , Corpo Estriado/metabolismo , Aumento da Imagem/métodos , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Macaca fascicularis , Masculino , Ligação Proteica , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Glutamato Metabotrópico 5RESUMO
Parkinson's disease is caused by a complex interplay of genetic and environmental factors. Although a number of independent molecular pathways and processes have been associated with familial Parkinson's disease, a common mechanism underlying especially sporadic Parkinson's disease is still largely unknown. In order to gain further insight into the etiology of Parkinson's disease, we here conducted genetic network and literature analyses to integrate the top-ranked findings from thirteen published genome-wide association studies of Parkinson's disease (involving 13.094 cases and 47.148 controls) and other genes implicated in (familial) Parkinson's disease, into a molecular interaction landscape. The molecular Parkinson's disease landscape harbors four main biological processes-oxidative stress response, endosomal-lysosomal functioning, endoplasmic reticulum stress response, and immune response activation-that interact with each other and regulate dopaminergic neuron function and death, the pathological hallmark of Parkinson's disease. Interestingly, lipids and lipoproteins are functionally involved in and influenced by all these processes, and affect dopaminergic neuron-specific signaling cascades. Furthermore, we validate the Parkinson's disease -lipid relationship by genome-wide association studies data-based polygenic risk score analyses that indicate a shared genetic risk between lipid/lipoprotein traits and Parkinson's disease. Taken together, our findings provide novel insights into the molecular pathways underlying the etiology of (sporadic) Parkinson's disease and highlight a key role for lipids and lipoproteins in Parkinson's disease pathogenesis, providing important clues for the development of disease-modifying treatments of Parkinson's disease.
RESUMO
Many recent studies of the degeneration, neuroprotection and regeneration of CNS neurons have departed from previous dichotomous descriptions of neurons as either dead or alive. In this brief article aspects of neuronal health are examined by outlining ways to assess both neuronal resilience and vulnerability to common forms of structural brain insults. According to this theory of neuronal health, neurons exist in a dynamic equilibrium that spans a spectrum of cellular existence, constantly influenced by both extracellular physiological changes and intracellular mechanisms designed to react to external stimuli while maintaining structural integrity. The spectrum between particularly resilient and vulnerable neuronal states is illustrated by experiments in vivo that examine trophic and metabolic fluctuations influencing the likelihood of neuronal death after neuronal insults. Studies show that adult CNS neurons can be protected in vivo by trophic agents or other pharmacological interventions against structural and toxic damage. Conversely, low-level neuronal impairment due to genetic or physiological perturbations can predispose neurons to demise by insults that normally would not cause cell death. The experimental approaches described may help in the study of neuronal pathophysiology, and in investigations towards new treatments for the neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease.
Assuntos
Saúde , Doenças do Sistema Nervoso/fisiopatologia , Humanos , Degeneração Neural/fisiologiaRESUMO
Basic research using cell transplantation indicates that structural developmental mechanisms seen in immature brains can also function in the adult brain. As the brain matures, cellular migration and axonal growth is impeded. However, fetal neural transplantation studies have shown that directional cues are available for fetal axons to find specific host neurons in the adult brain. By reaching specific and distant CNS target zones, donor tissue with extended axonal growth periods demonstrate both an abundance and specificity of CNS neurotropic signals. The presence of specific guidance cues, despite strong inhibition of regenerative long-distance axonal growth, suggests that these cues play other physiological roles in the adult CNS, and could be utilized therapeutically for reconnection of neuronal pathways.
Assuntos
Transplante de Tecido Encefálico/fisiologia , Encéfalo/fisiologia , Regeneração Nervosa/fisiologia , Adulto , Animais , Transplante de Células/fisiologia , Transplante de Tecido Fetal/fisiologia , HumanosRESUMO
The adult CNS provides a poor environment for axonal growth and regeneration. The question of to what extent the loss of axonal growth occurring as the brain matures is dependent on factors intrinsic or extrinsic to the growing neuron is still unanswered. Examination of axonal growth from neural transplants provides insight into the roles of growth factors, inhibitory molecules, growth-promoting substrates and the differences between CNS and PNS environments in the regulation of neurite extension. The data that imply a role for BCL2 and related molecules in such processes are reviewed in this article, which analyzes the factors intrinsic to the neuron that control its capacity for axonal growth.
Assuntos
Axônios/fisiologia , Encéfalo/fisiologia , Genes bcl-2/fisiologia , Substâncias de Crescimento/metabolismo , Regeneração Nervosa/fisiologia , Animais , Regulação da Expressão Gênica , Genes bcl-2/genética , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , RatosRESUMO
Degeneration of the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease. To facilitate the study of the differentiation and maintenance of this population of dopaminergic neurons both in vivo and in vitro, we generated a knock-in reporter line in which the yellow fluorescent protein (YFP) replaced the first exon and the first intron of the tyrosine hydroxylase (TH) gene in one allele by homologous recombination. Expression of YFP under the direct control of the entire endogenous 5' upstream region of the TH gene was predicted to closely match expression of TH from the wild type allele, thus marking functional dopaminergic neurons. We found that YFP was expressed in dopaminergic neurons differentiated in vitro from the knock-in mouse embryonic stem cell line and in dopaminergic brain regions in knock-in mice. Surprisingly, however, YFP expression did not overlap completely with TH expression, and the degree of overlap varied in different TH-expressing brain regions. Thus, the reporter gene did not identify functional TH-expressing cells with complete accuracy. A DNaseI hypersensitivity assay revealed a cluster of hypersensitivity sites in the first intron of the TH gene, which was deleted by insertion of the reporter gene, suggesting that this region may contain cis-acting regulatory sequences. Our results suggest that the first intron of the rodent TH gene may be important for accurate expression of TH.
Assuntos
Dopamina/metabolismo , Íntrons/fisiologia , Neurônios/metabolismo , Células-Tronco/fisiologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Southern Blotting/métodos , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Genes Reporter/fisiologia , Humanos , Imuno-Histoquímica/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , RatosAssuntos
Transplante de Tecido Encefálico/efeitos adversos , Transplante de Tecido Encefálico/métodos , Transplante Heterólogo , Animais , Neoplasias Encefálicas/terapia , Transplante de Tecido Encefálico/fisiologia , Ensaios Clínicos como Assunto , Transplante de Tecido Fetal/efeitos adversos , Transplante de Tecido Fetal/métodos , Transplante de Tecido Fetal/fisiologia , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Humanos , Camundongos , Doença de Parkinson/fisiopatologia , Doença de Parkinson/terapia , Retroviridae/genética , Retroviridae/patogenicidade , Fatores de Risco , Suínos , Timidina Quinase/genéticaAssuntos
Neurônios/transplante , Doença de Parkinson/terapia , Animais , Humanos , Suínos , Transplante HeterólogoRESUMO
Huntington's disease (HD) is neurologically characterized by involuntary movements, associated with degeneration of the medium-sized spiny neurons (MSNs) and ubiquitin-positive neuronal intranuclear inclusions (NIIs). It has been reported that the proteolytic activities of the ubiquitin-proteasome system (UPS) are generally inhibited in HD patient's brain. We previously discovered that a proteasome activator (PA), PA28γ enhances proteasome activities and cell survival in in vitro HD model. In this study, we aimed to find whether PA28γ gene transfer improves the proteasome activities and pathological symptoms in in vivo HD model. We stereotaxically injected lenti-PA28γ virus into the striatum of mutant (MT) YAC128 HD mice and littermate (LM) controls at 14-18months of age, and validated their behavioral and biochemical changes at 12weeks after the injection. YAC128 mice showed a significant increase in their peptidyl-glutamyl preferring hydrolytic (PGPH) proteasome activity and the mRNA or protein levels of brain-derived neurotrophic factor (BDNF) and pro-BDNF after lenti-PA28γ injection. The number of ubiquitin-positive inclusion bodies was reduced in the striatum of YAC128 mice after lenti-PA28γ injection. YAC128 mice showed significant improvement of latency to fall on the rota-rod test after lenti-PA28γ injection. These data demonstrate that the gene therapy with PA, PA28γ can improve UPS function as well as behavioral abnormalities in HD model mice.
Assuntos
Autoantígenos/genética , Autoantígenos/metabolismo , Corpo Estriado/fisiopatologia , Terapia Genética/métodos , Doença de Huntington/fisiopatologia , Doença de Huntington/terapia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/patologia , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Camundongos Transgênicos , Atividade Motora/fisiologia , RNA Mensageiro/metabolismo , Teste de Desempenho do Rota-Rod , Resultado do TratamentoRESUMO
It has been uncertain whether specific disease-relevant biomarker phenotypes can be found using sporadic Parkinson's disease (PD) patient-derived samples, as it has been proposed that there may be a plethora of underlying causes and pathological mechanisms. Fibroblasts derived from familial PD patients harboring leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and Parkin mutations show clear disease-relevant mitochondrial phenotypes, which are exacerbated under conditions of pharmacological stress. We utilized fibroblasts derived from non-familial sporadic PD patients (without LRRK2 mutations) or LRRK2 mutation carriers to directly compare the cellular phenotypes during and after mitochondrial stress. We then determined the effects of pharmacological LRRK2 kinase inhibition using LRRK2-in-1. We found that there were two distinct populations of sporadic PD patient-derived fibroblast lines. One group of sporadic PD lines was highly susceptible to valinomycin-induced mitochondrial depolarization, emulating the mutant LRRK2 phenotype. These lines showed elevated mitochondrial superoxide/ nitric oxide levels, displayed increased mitochondrial and lysosome co-localization, and an increased rate of mitochondrial collapse, which corresponded with changes in mitochondrial fission and fusion proteins. The application of LRRK2-in-1 reversed decreased levels of mitochondrial and lysosome co-localization and partially restored mitochondrial network associated proteins and the mitochondrial membrane potential in the fibroblasts. This study identifies novel mitochondrial biomarkers in sporadic PD patient-derived fibroblast lines, which could be used as preclinical tools in which to test novel and known neuroprotective compounds.
Assuntos
Biomarcadores/metabolismo , Fibroblastos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Doença de Parkinson/enzimologia , Benzodiazepinonas/farmacologia , Linhagem Celular , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Doença de Parkinson/patologia , Proteínas Quinases/metabolismo , Pirimidinas/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Valinomicina/farmacologiaRESUMO
We have previously shown that local destruction of neural tissue by wild-type herpes simplex virus type 1 (HSV-1) is attenuated by intracerebral infusion of nerve growth factor (NGF). To investigate the effect of NGF on the extent of neurolysis and efficacy of neuronal gene transfer mediated by an HSV-1 amplicon vector system in vivo, rats were stereotaxically injected in the striatum with an amplicon preparation, pHSVlac. This amplicon contains the Escherichia coli lacZ gene under the transcriptional control of the HSV-1 immediate early 4/5 promoter and is packaged by an HSV-1 helper virus carrying a deletion in the immediate early 3 gene. Vector injection was followed by continuous intracerebral infusion of NGF-beta (total dose 5 micrograms) or vehicle solution over 7 days. Animals were sacrificed at the end of the 7-day infusion period for histological analysis of the brains. A distinct zone of inflammation and necrosis surrounded the injection site in all vector-inoculated animals. The volume of striatal tissue destruction was significantly smaller in NGF-treated animals (1.27 +/- 0.19 mm3; mean +/- SEM) than in the vehicle-treated controls (2.16 +/- 0.37 mm3; P < 0.05 by t-test). Immunohistochemical staining for HSV and beta-galactosidase (beta-Gal) in vehicle-treated animals revealed that many striatal cells harbored HSV antigens (3,678 +/- 636), but only a small number expressed the reporter gene at 7 days post-injection (294 +/- 60). NGF infusion did not significantly affect the number of HSV-immunoreactive cells (4,224 +/- 618), or the number of cells expressing beta-Gal (330 +/- 72) at this time.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corpo Estriado/efeitos dos fármacos , Vetores Genéticos/efeitos adversos , Fatores de Crescimento Neural/farmacologia , Simplexvirus/genética , Animais , Corpo Estriado/patologia , Amplificação de Genes , Vetores Genéticos/efeitos dos fármacos , Masculino , Neurônios/virologia , Ratos , Ratos Sprague-Dawley , Simplexvirus/efeitos dos fármacos , Simplexvirus/fisiologiaRESUMO
Gene promoter systems that drive high-level, long-term, and cell-specific transgene expression are of great interest because of their potential utility for gene therapy. To generate an efficient promoter system specific for noradrenergic (NA) neurons, we multimerized an NA-specific cis-regulatory element (PRS2) identified in the human dopamine beta-hydroxylase (hDBH) promoter, and combined it with a minimal promoter (containing a TATA box and transcription start site). Forms of this synthetic promoter that contain 8 or more copies of PRS2 were >50 times more effective than the 1.15-kb hDBH promoter at driving reporter gene expression in cell lines originated from NA neurons. Neither the synthetic promoter nor the 1.15-kb hDBH promoter drove reporter gene expression in nonneuronal cells. Microinjections of an adenoviral vector containing the synthetic promoter directly into rat brain caused more strict NA-specific reporter gene expression than that caused by a vector containing the 1.15-kb hDBH promoter when the targeted region contained large numbers of NA neurons (locus coeruleus). Furthermore, the vector containing the synthetic promoter caused less nonspecific ("leaky") reporter gene expression than that caused by the vector containing the 1.15-kb hDBH promoter when the targeted region was devoid of NA neurons (cerebellum, dentate gyrus). Together, these studies provide in vitro and in vivo evidence that this novel synthetic promoter can target transgene expression to NA neurons even more efficiently and selectively than the naturally occurring, 1.15-kb hDBH promoter.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Neurônios/metabolismo , Regiões Promotoras Genéticas , Receptores Adrenérgicos/metabolismo , Transgenes , Adenoviridae/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Linhagem Celular , Dopamina beta-Hidroxilase/genética , Genes Reporter , Terapia Genética/métodos , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/metabolismo , Ratos , Fatores de Tempo , Transcrição Gênica , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) vectors were combined to construct a hybrid amplicon vector, and then packaged via a helper virus-free system. We tested the HSV/AAV hybrid amplicon vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) vectors at the same multiplicity of infection. The HSA/AAV hybrid vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other vectors tested, only hybrid vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing hybrid vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 x 10(6) transducing units injected), transgene expression was observed within the striatum (ranging from 564 to 8610 cells) and the substantia nigra (via retrograde transport, ranging from 130 to 809 neurons). The HSV/AAV hybrid amplicon vectors transduced predominantly neurons within the striatum, and showed transduction efficacy similar to and in many cases higher than that of HSV amplicon vectors. No immune response was observed in the HSA/AAV hybrid vector-injected brains, as determined by immune markers specific for helper T lymphocytes, cytotoxic T lymphocytes, and microglia. This HSV/AAV hybrid system shows high transduction efficiency and stability in culture. The effective and safe transgene delivery into the nigrostriatal system illustrates its potential for therapeutic application for neurologic disorders, such as Parkinson and Huntington disease.
Assuntos
Corpo Estriado/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Simplexvirus/genética , Substância Negra/metabolismo , Animais , Biomarcadores , Células Cultivadas , Cruzamentos Genéticos , Ratos , Transdução GenéticaRESUMO
One month after an in situ injection of kainic acid into the ventrobasal thalamic complex (VB), the lesioned area is totally depleted of neurons. The present study has been undertaken to determine the cytoarchitecture and connectivity of the nucleus constructed by fetal thalamic neurons implanted into the excitotoxically lesioned area. Adult rats received an injection of kainic acid inducing a total neuronal depletion of the right lateral thalamus (including both the nucleus reticularis thalami and the lateral portion of the ventrobasal complex). One month later, homotypic neurons were taken from the dorsal thalamic primordium of rat embryos (gestational age 15-16 days), dissociated, and injected into the lesioned area as a cell suspension. After 2-4-month survival, the cytoarchitecture of the neonucleus formed by the grafted neurons within the previously neuron-depleted area was analyzed. Additionally, connectivity was analyzed in seven rats in which dorsal column nuclei and/or cortical projections to the area were labeled anterogradely with either 3H-leucine or wheat-germ agglutinin conjugated to HRP, and the animals were perfused and processed following various histological procedures (Nissl staining, autoradiographic processing, and histochemistry for visualization of peroxidase). Fetal neurons grew, differentiated, and progressively occupied the previously neuron-depleted area of the adult host CNS. They organized themselves into a neonucleus with particular cytoarchitectural features including 1) the existence of two concentric zones--a central zone containing neurons and glial cells and a marginal zone only filled with a band of glial cells, 2) an increase in cellular density compared to the intact thalamus, 3) the grouping of neurons in spherical clusters, and 4) apparent polymorphism of neuronal somata. Lemniscal and corticothalamic afferents originating from the host were observed in the neonucleus when the fetal neurons had been implanted correctly into the lesioned area but not when they had been misplaced into either normal thalamic tissue or the internal capsule. The afferents labeled from either the dorsal column nuclei or the somatosensory cortex were, however, less dense in the neonucleus than in the normal thalamus. These results are discussed with regard to the normal cytoarchitecture and connectivity of the ventrobasal complex of the rat thalamus.