Chronic stress accelerates glioblastoma progression via DRD2/ERK/ß-catenin axis and Dopamine/ERK/TH positive feedback loop.

BACKGROUND: After diagnosis, glioblastoma (GBM) patients undertake tremendous psychological problems such as anxiety and depression, which may contribute to GBM progression. However, systematic study about the relationship between depression and GBM progression is still lacking. METHODS: Chronic unpredictable mild stress and chronic restrain stress were used to mimic human depression in mice. Human GBM cells and intracranial GBM model were used to assess the effects of chronic stress on GBM growth. Targeted neurotransmitter sequencing, RNA-seq, immunoblotting and immunohistochemistry were used to detect the related molecular mechanism. RESULTS: Chronic stress promoted GBM progression and up-regulated the level of dopamine (DA) and its receptor type 2 (DRD2) in tumor tissues. Down-regulation or inhibition of DRD2 abolished the promoting effect of chronic stress on GBM progression. Mechanistically, the elevated DA and DRD2 activated ERK1/2 and consequently inhibited GSK3ß activity, leading to ß-catenin activation. Meanwhile, the activated ERK1/2 up-regulated tyrosine hydroxylase (TH) level in GBM cells and then promoted DA secretion, forming an autocrine positive feedback loop. Remarkably, patients with high-depression exhibited high DRD2 and ß-catenin levels, which showed poor prognosis. Additionally, DRD2 specific inhibitor pimozide combined with temozolomide synergistically inhibited GBM growth. CONCLUSIONS: Our study revealed that chronic stress accelerates GBM progression via DRD2/ERK/ß-catenin axis and Dopamine/ERK/TH positive feedback loop. DRD2 together with ß-catenin may serve as a potential predictive biomarker for worse prognosis as well as therapeutic target of GBM patients with depression.

Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Guizhi-Fuling Treatment for Myocardial Ischemia.

Myocardial ischemia is a cardio-physiological condition due to a decrease in blood perfusion to the heart, leading to reduced oxygen supply and abnormal myocardial energy metabolism. Guizhi-Fuling (GZFL) is effective in treating Myocardial ischemia. However, its mechanism of action is unclear and requires further exploration. We attempt to decipher the mechanisms behind GZFL treating Myocardial ischemia by integrating metabolomics and network pharmacology. In this study, myocardial metabolomic analysis was performed using GC/MS to identify the potential mechanism of action of GZFL during myocardial ischemia. Then, network pharmacology was utilized to analyze key pathways and construct a pathway-core target network. Molecular docking was incorporated to validate core targets within network pharmacological signaling pathways. Finally, western blots were utilized to verify core targets of metabolomics, network pharmacology integrated pathways, and key signaling targets. Thus, 22 critical biomarkers of GZFL for treating myocardial ischemia were identified. Most of these metabolites were restored using modulation after GZFL treatment. Based on the network pharmacology, 297 targets of GZFL in treating myocardial ischemia were identified. The further comprehensive analysis focused on three key targets, such as Tyrosine hydroxylase (TH), myeloperoxidase (MPO), and phosphatidylinositol 3-kinases (PIK3CA), and their related metabolites and pathways. Compared with the model group, the protein expression levels of TH, MPO and PIK3CA were reduced in GZFL. Therefore, the mechanism of GZFL for treating myocardial ischemia could inhibit myocardial inflammatory factors, reduce myocardial inflammation, and restore endothelial function while controlling norepinephrine release and uric acid concentration.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats.

BACKGROUND AIMS: Gingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. METHODS: RNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. RESULTS: Intravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium-binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. CONCLUSIONS: The authors' research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

Sapropterina para o tratamento da fenilcetonúria / Sapropterin for treatment of phenylketonuria

INTRODUÇÃO: A FNC por deficiência de fenilalanina hidroxilase (FAH) é uma doença de padrão de herança autossômico recessivo, sendo classificada em FNC clássica, FNC leve ou hiperfenilalaninemia (HFA) não-FNC. A fenilalanina (FAL) é um aminoácido essencial, metabolizado no fígado pela enzima FAH e convertida no aminoácido tirosina (TIR). A não conversão de FAL em TIR leva ao aumento dos níveis plasmáticos de FAL e redução dos níveis de TIR. A doença não tratada caracteriza­se por atraso do desenvolvimento neuropsicomotor, retardo mental e crises convulsivas. O principal tratamento consiste em dieta restrita em FAL e uso de fórmula metabólica rica em aminoácidos, vitaminas e minerais e isenta em FAL. Embora não sejam deficientes em tetrahidrobiopterina (BH4), aproximadamente 30% dos pacientes com deficiência de FAH respondem à administração de BH4 com aumento do metabolismo de FAL para TIR. Dicloridato de sapropterina é a forma sintética do BH4 e administrado por via oral. TECNOLOGIA: Dicloridrato de sapropterina (Kuvan®). PERGUNTA: O uso de dicloridrato de sapropterina, isolado ou associado à dieta e uso de fórmula metabólica é eficaz e seguro quando comparado ao uso de dieta e fórmula metabólica sem BH4 em pacientes com FNC? EVIDÊNCIAS CIENTÍFICAS: Foram incluídos 4 ensaios clínicos randomizados (ECR) e 8 ensaios clínicos abertos prospectivos. Os estudos, na sua grande maioria, incluíram pequeno número de pacientes dada a prevalência da FNC, os quais, possuíam idades (crianças, adolescentes e adultos) e manifestações fenotípicas (FCN clássica e leve) variáveis. Foram identificadas duas revisões sistemáticas. Entretanto, a que não apresentou meta-análise não foi considerada, pois priorizou-se relatar os dados dos estudos primários. Mesmo a segunda revisão sistemática com meta-análise incluiu somente 2 estudos e para a grande maioria dos desfechos considerados não foi possível estimar uma única medida de associação. Os 2 estudos dessa revisão sistemática também foram considerados individualmente no presente parecer. A maioria dos ensaios clínicos investigou prioritariamente o desfecho nível de FAL plasmática, com amostras compostas por adultos, adolescentes ou crianças. Tais estudos demonstraram que, em pacientes com FNC responsivos à sapropterina, o tratamento, na maioria das vezes em conjunto com a dieta, reduziu substancialmente a concentração plasmática de FAL, oferecendo, assim, um melhor controle dos níveis plasmáticos da mesma. Os ensaios clínicos que avaliaram o desfecho tolerância à FAL, em menor número, demonstraram um aumento dessa tolerância o suficiente para permitir uma dieta menos restritiva ou, em alguns casos, substitui-la (descontinuação da dietoterapia). Os principais eventos adversos descritos foram sintomas relacionados ao sistema nervoso (ex.: cefaleia, enxaqueca, dificuldade de concentração) e distúrbios gastrointestinais (vômito, diarreia, gastroenterite). De modo geral, os achados apontam para um perfil de segurança aceitável para doses que não ultrapassam valores de 20 mg/kg/dia, sendo avaliada na grande maioria dos estudos ainda por um curto espaço de tempo. Eventos adversos após mais de um ano de tratamento são menos estudados, ainda que alguns estudos tenham apresentado seguimentos longos, como 2 e 7 anos. Uma vez que a evidência disponível para os desfechos de benefício de maior relevância foi de qualidade moderada, existe recomendação fraca a favor da inclusão da sapropterina no SUS como tratamento complementar na FNC para pacientes responsivos à mesma. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: Foi elaborado um modelo de impacto orçamentário para estimar os possíveis gastos decorrentes da incorporação do dicloridrato de sapropterina para tratamento da FNC no SUS em um horizonte temporal de 5 anos (2019 até 2023). Levando em consideração o custo anual das intervenções e a projeção do número de pacientes por intervenção, a incorporação da sapropterina geraria um impacto orçamentário incremental de aproximadamente R$ 79,3 milhões em 2019 e R$ 299,7 milhões após 5 anos (2019 a 2023). A análise de sensibilidade univariada estimou que o impacto orçamentário incremental da incorporação do dicloridrato de sapropterina pode variar de aproximadamente R$ 66,4 a R$ 102,58 milhões no primeiro ano e de R$ 251 a R$ 388 milhões ao longo dos cinco anos após a incorporação no SUS. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Foi identificado um medicamento de substituição enzimática, Pegvaliase (PAL-PEG), em fase 3 de pesquisa clínica, com registro aprovado no FDA em 2018. RECOMENDAÇÃO INICIAL: Pelo exposto, a CONITEC em sua 69ª reunião ordinária, no dia 02 de agosto de 2018, recomendou a não incorporação no SUS da sapropterina para FNC. Considerou-se que não foram apresentadas novas e robustas evidências ou argumentos que respaldem a tecnologia. Além disso, os membros ressaltaram o alto custo da tecnologia frente à incerteza do benefício que ela representa. A matéria foi disponibilizada em consulta pública. CONSULTA PÚBLICA: A Consulta Pública nº 43/2018 foi realizada entre os dias 21/08/2018 a 10/09/2018. Foram recebidas 919 contribuições, sendo 63 (6,9%) pelo formulário para contribuições técnico-científicas e 856 (93,1%) pelo formulário para contribuições sobre experiência ou opinião de pacientes, familiares, amigos ou cuidadores de pacientes, profissionais de saúde ou pessoas interessadas no tema. Das 63 contribuições recebidas de cunho técnicocientífico, 42 (67%) discordaram totalmente, dezessete (27%) discordaram parcialmente, um (2%) concordou totalmente e três (5%) concordou parcialmente da recomendação preliminar da CONITEC. Das 856 contribuições, 30 (4%) e quatro (1%) concordaram totalmente e parcialmente com a recomendação preliminar da CONITEC, respectivamente. Do restante, 105 (12%) discordaram parcialmente e 717 (84%) discordaram totalmente da recomendação preliminar da CONITEC. Após apreciação das contribuições encaminhadas pela consulta pública o plenário recomendou a incorporação do dicloridrato de sapropterina para o tratamento de mulheres com fenilcetonúria, com teste de responsividade positivo ao medicamento, e que estejam no período pré-concepcional ou em período gestacional, mediante negociação de preço e conforme protocolo clínico e diretrizes terapêuticas do Ministério da Saúde. RECOMENDAÇÃO FINAL DA CONITEC: Os membros da CONITEC presentes na 72ª reunião ordinária, no dia 8 de novembro de 2018, deliberaram, por unanimidade, recomendar a incorporação no SUS do dicloridrato de sapropterina para o tratamento de mulheres com Fenilcetonúria, com teste de responsividade positivo ao medicamento, e que estejam no período pré-concepcional ou em período gestacional. Foi assinado aos 08 (oito) dias do mês de novembro de 2018 registro de deliberação n° 390/2018 pela incorporação do medicamento dicloridrato de sapropterina ao SUS. DECISÃO: Incorporar o dicloridrato de sapropterina no tratamento da fenilcetonúria mediante negociação de preço e conforme Protocolo Clínico e Diretrizes Terapêuticas do Ministério da Saúde, no âmbito do Sistema Único de Saúde - SUS. Dada pela Portaria nº 78, publicada no DOU nº 241, seção 1, página 77, em 17 de dezembro de 2018.

Informe de evaluación científica en la evidencia disponible: fenilcetonuria / Relatório de avaliação científica das evidências disponíveis: fenilcetonúria

INTRODUCCIÓN: La fenilcetonuria es una enfermedad congénita del metabolismo, causada en la mayoría de los casos por el déficit de la enzima hepática fenilalanina hidroxilasa (1,2), responsable de transformar la fenilalanina a tirosina, esto da como resultado concentraciones elevadas de fenilalanina y sus metabolitos, fenilacetato y fenilactato en sangre. Además la fenilalanina tendrá interferida su vía de transformación en tirosina, ya que la fenilalanina hidroxilasa no se habrá formado bien, lo que se traduce en un retardo mental. La incidencia de fenilcetonuria es de 13.500 a 19.000 nacimientos en EEUU. Las manifestaciones clínicas de la fenilcetonuria en los recién nacidos son raras, en general son asintomáticos y no puede causar síntomas hasta la infancia temprana. En los pacientes no tratados la enfermedad se manifiesta con la incapacidad intelectual, un área de cognición que es particularmente afectada es la función ejecutiva. Otros hallazgos incluyen anormalidades de la marcha y postura. TECNOLOGÍAS SANITARIA DE INTERÉS: Sapropterina: es una versión sintética de la 6R-BH4 natural, que es un cofactor de las hidrolasas de fenilalanina, tirosina y triptófano. La justificación de la administración de sapropterina en pacientes con fenilcetonuria que responde a BH4 (tetrahidrobiopterina) es aumentar la actividad de la fenilalanina hidroxilasa defectuosa y así aumentar o restituir el metabolismo oxidativo de la fenilalanina lo suficiente para reducir o mantener los niveles plasmáticos de fenilalanina, evitar o reducir aún más la acumulación de fenilalanina y aumentar la tolerancia a la ingesta de fenilalanina en la dieta. La justificación de la administración de la administración de sapropterina en pacientes con deficiencia de BH4 es restituir los niveles deficitarios, restableciendo la actividad de la fenilalanina hidroxilasa (EMA). EFICACIA DE LOS TRATAMIENTOS: La información sobre los efectos de la Sapropterina está basada en dos estudios. El resumen de los resultados es el siguiente. Los resultados arrojan que existe un beneficio del tratamiento de la Fenilcetonuria con Sapropterina para pacientes que responden a la misma. El tratamiento resultó eficaz en disminuir considerablemente la concentración de Fenililanina en sangre (3,4) y una tolerancia mayor a la misma. A pesar de lo anterior, cabe destacar que es necesario estudios que consideren la efectividad de la Sapropterina en el largo plazo, que estudie desenlaces finales y no solo intermedios. A pesar de que existen estudios que consideran un período relativamente largo de estudio (5), dando indicios de buenos resultados del tratamiento con Sapropterina, se necesitan estudios que aborden la investigación con mayor control sobre la población (ensayos aleatorios controlados) para evaluar la efectividad en el largo plazo con mayor certeza. Es también importante mayor investigación que genere evidencia sobre los efectos del tratamiento según sea la severidad de la enfermedad, ya que los estudios encontrados no permiten tales aseveraciones. ALTERNATIVAS DISPONIBLES: Dieta: Sustituto proteico y Alimentos hipoprotéicos. RESULTADOS DE LA BÚSQUEDA DE EVIDENCIA: La información sobre los efectos de la Sapropterina está basada en dos estudios. El resumen de los resultados es el siguiente. Los resultados arrojan que existe un beneficio del tratamiento de la Fenilcetonuria con Sapropterina para pacientes que responden a la misma. El tratamiento resultó eficaz en disminuir considerablemente la concentración de Fenilalanina en sangre y una tolerancia mayor a la misma. A pesar de lo anterior, cabe destacar que es necesario estudios que consideren la efectividad de la Sapropterina en el largo plazo, que estudie desenlaces finales y no solo intermedios. A pesar de que existen estudios que consideran un período relativamente largo de estudio, dando indicios de buenos resultados del tratamiento con Sapropterina, se necesitan estudios que aborden la investigación con mayor control sobre la población (ensayos aleatorios controlados) para evaluar la efectividad en el largo plazo con mayor certeza. Es también importante mayor investigación que genere evidencia sobre los efectos del tratamiento según sea la severidad de la enfermedad, ya que los estudios encontrados no permiten tales aseveraciones. CONCLUSIÓN: Para dar cumplimiento al artículo 28° del Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con Sistema de Protección Financiera, según lo establecido en los artículos 7°y 8° de la ley N°20.850, aprobado por el decreto N°13 del Ministerio de Salud, se concluye que el presente informe de evaluación se considera no favorable, dado que su impacto presupuestario supera el 80% del fondo disponible, de acuerdo a lo establecido en el Título III. de las Evaluaciones Favorables de la Norma Técnica N° 0192 de este mismo Ministerio.

Recent progress in gene therapy for Parkinson's disease.

Parkinson's disease (PD) is an age-related and the second most common neurodegenerative disorder beyond Alzheimer's disease. A neuropathological hallmark of PD is a prominent loss of dopaminergic neurons in the substantia nigra projecting into the caudate and putamen. Oral administration of L-dopa and/or dopamine agonists ameliorates cardinal motor symptoms of PD. However, an intermittent and long-term treatment with L-dopa frequently induces adverse side effects such as motor fluctuations and dyskinesia. As alternative therapeutic strategies, the following four approaches are currently under evaluation for clinical gene therapy trials in PD; 1) recombinant adeno-associated virus 2 system encoding aromatic L-amino acid decarboxylase (AADC), 2) glutamic acid decarboxylase (GAD) and 3) Neurturin, and 4) equine infectious anemia virus-based lentiviral system encoding AADC, tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GCH) in a single transcriptional unit. GAD and Neurturin have been assessed in double blind placebocontrolled phase II studies; GAD showed a significant improvement in motor function, and Neurturin, although it failed to show significant effects at 12 months post-treatment, exhibited promising outcomes in additional examinations at 18 months. The other two approaches also represented significant effects in phase I or I/II studies. Adverse side effects due to surgery have not been observed. Here, we review preclinical and clinical trials encouraging further investigations of curative treatment for the patients suffering from PD.

BCG vaccine-induced neuroprotection in a mouse model of Parkinson's disease.

There is a growing interest in using vaccination with CNS antigens to induce autoreactive T cell responses that home to damaged areas in the CNS and ameliorate neurodegenerative disease. Neuroprotective vaccine studies have focused on administering oligodendrocyte antigens or Copaxone® in complete Freund's adjuvant (CFA). Theoretical considerations, however, suggest that vaccination with a neuronal antigen may induce more robust neuroprotective immune responses. We assessed the neuroprotective potential of vaccines containing tyrosine hydroxylase (a neuronal protein involved in dopamine synthesis) or Copaxone® in CFA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Surprisingly, we observed that the main beneficial factor in these vaccines was the CFA. Since the major immunogenic component in CFA is Mycobacterium tuberculosis, which closely related to the bacille Calmette-Guérin (BCG) that is used in human vaccines, we tested BCG vaccination in the MPTP mouse model. We observed that BCG vaccination partially preserved markers of striatal dopamine system integrity and prevented an increase in activated microglia in the substantia nigra of MPTP-treated mice. These results support a new neuroprotective vaccine paradigm in which general (nonself-reactive) immune stimulation in the periphery can limit potentially deleterious microglial responses to a neuronal insult and exert a neurorestorative effect in the CNS. Accordingly, BCG vaccination may provide a new strategy to augment current treatments for a wide range of neuropathological conditions.

Gene therapy in hemiparkinsonian rhesus monkeys: long-term survival and behavioral recovery by transplantation of autologous human tyrosine hydroxylase-expressing neural stem cells.

BACKGROUND AIMS: Neural stem cells (NSC) derived from bone marrow stromal cells (BMSC) (BMSC-D-NSC) are remarkably versatile in response to environmental signals, which render them useful in the search for neurodegenerative disease treatments. METHODS: We isolated NSC from rhesus monkey bone marrow (BM), transfected them with the human tyrosine hydroxylase (hTH) gene, and transplanted them into 1-methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned hemiparkinsonian rhesus monkeys to determine changes in neural transmitter production and alterations in behavior. RESULTS: hTH-expressing cells produced monoamine agents in vitro, such as noradrenalin and dopamine. After cell transplantation in the caudate nucleus and substantia nigra of the experimental monkeys, their disease symptoms and dysfunctional glucose metabolism and dopamine transport were ameliorated. CONCLUSIONS: hTH-expressing BMSC-D-NSC survived in transplantation sites and assumed normal dopaminergic neuronal properties, playing an instrumental role in functional restoration.

A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase.

The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 microM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD.

Tyrosine hydroxylase replacement in experimental Parkinson's disease with transvascular gene therapy.

Transvascular gene therapy of Parkinson's disease (PD) is a new approach to the gene therapy of PD and involves the global distribution of a therapeutic gene to brain after an intravenous administration and transport across the blood-brain barrier (BBB). This is enabled with the development of a nonviral gene transfer technology that encapsulates plasmid DNA inside pegylated immunoliposomes or PILs. An 85- to 100-nm liposome carries the DNA inside the nanocontainer, and the liposome surface is conjugated with several thousand strands of 2000-Da polyethyleneglycol (PEG). This PEGylation of the liposome stabilizes the structure in the blood stream. The liposome is targeted across the BBB via attachment to the tips of 1-2% of the PEG strands of a receptor-specific monoclonal antibody (mAb) directed at a BBB receptor, such as the insulin receptor or transferrin receptor (TfR). Owing to the expression of the insulin receptor or the TfR on both the BBB and the neuronal plasma membrane, the PIL is able to reach the neuronal nuclear compartment from the circulation. Brain-specific expression is possible with the combined use of the PIL gene transfer technology and brain-specific gene promoters. In the 6-hydroxydopamine rat model of experimental PD, striatal tyrosine hydroxylase (TH) activity is completely normalized after an intravenous administration of TfRmAb-targeted PILs carrying a TH expression plasmid. A treatment for PD may be possible with dual gene therapy that seeks both to replace striatal TH gene expression with TH gene therapy, and to halt or reverse neurodegeneration of the nigro-striatal tract with neurotrophin gene therapy.

Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes.

One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.

Cultured human foetal cerebral cortex, transfected with tyrosine hydroxylase cDNA, as a source of neural transplant material.

Obtaining an adequate supply of foetal dopaminergic tissue to treat Parkinson's disease by neural transplantation can be difficult. In this study primary cultures of human foetal cerebral cortex cells were transfected, using cationic lipids, with a eukaryotic expression vector (pCIneo-THI) containing the cDNA for human tyrosine hydroxylase isoform I (TH). Cortical cells from human (10-14 week) foetuses were cultured for 11 days in vitro and transfected twice with pCIneo-THI during this time. The double transfection process resulted in 3-4% of the cells becoming TH positive. When grafted into the striatum of 6-OHDA lesioned rats the transfected foetal cerebral cortex cells reduced amphetamine-induced circling behaviour by 75%, while grafts of untransfected cells had no significant effect on turning. TH transfected foetal cerebral cortex cells may therefore be a useful alternative supply of tissue for use in neural transplants to treat Parkinson's disease.

Gene therapy of Parkinson disease model rat by direct injection of plasmid DNA-lipofectin complex.

Lipofectin-mediated gene transfer was used to introduce plasmid harboring the tyrosine hydroxylase (TH) gene into the striatum of rats with lesions of the nigrostriatal pathway. The rotational asymmetry of Parkinson disease model rat was reduced quickly and significantly, suggesting that plasmid-DNA-transfected brain cells can generate L-dopa locally in the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input. Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) also confirm that striatal cells can express exogenous TH gene. Such in vivo plasmid DNA transfer strategy may be useful in other neurologic disease therapy, especially acute brain insults.