RESUMO
The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.
Assuntos
Terapia Genética , Vetores Genéticos/efeitos adversos , Neurônios/efeitos dos fármacos , Parvovirinae/genética , Medula Espinal/efeitos dos fármacos , Animais , Transporte Axonal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Dependovirus , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteoglicanas de Heparan Sulfato/administração & dosagem , Proteoglicanas de Heparan Sulfato/genética , Humanos , Infusões Intraventriculares , Neurônios Motores/efeitos dos fármacos , Neurônios/patologia , Primatas , Medula Espinal/patologia , Tálamo/efeitos dos fármacosRESUMO
Brain-derived neurotrophic factor (BDNF) gene delivery to the entorhinal cortex is a candidate for treatment of Alzheimer's disease (AD) to reduce neurodegeneration that is associated with memory loss. Accurate targeting of the entorhinal cortex in AD is complex due to the deep and atrophic state of this brain region. Using MRI-guided methods with convection-enhanced delivery, we were able to accurately and consistently target AAV2-BDNF delivery to the entorhinal cortex of non-human primates; 86 ± 3% of transduced cells in the targeted regions co-localized with the neuronal marker NeuN. The volume of AAV2-BDNF (3 × 108 vg/µl) infusion linearly correlated with the number of BDNF labeled cells and the volume (mm3) of BDNF immunoreactivity in the entorhinal cortex. BDNF is normally trafficked to the hippocampus from the entorhinal cortex; in these experiments, we also found that BDNF immunoreactivity was elevated in the hippocampus following therapeutic BDNF vector delivery to the entorhinal cortex, achieving growth factor distribution through key memory circuits. These findings indicate that MRI-guided infusion of AAV2-BDNF to the entorhinal cortex of the non-human primate results in safe and accurate targeting and distribution of BDNF to both the entorhinal cortex and the hippocampus. These methods are adaptable to human clinical trials.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Dependovirus/genética , Córtex Entorrinal/metabolismo , Imageamento por Ressonância Magnética/métodos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Meios de Contraste/farmacocinética , Feminino , Gadolínio/farmacocinética , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos/farmacocinética , Hipocampo/metabolismo , Macaca fascicularis , Macaca mulatta , Masculino , Neurônios/virologia , Compostos Organometálicos/farmacocinética , Transporte ProteicoRESUMO
Many studies have demonstrated that adeno-associated virus serotype 9 (AAV9) transduces astrocytes and neurons when infused into rat or nonhuman primate (NHP) brain. We previously showed in rats that transduction of antigen-presenting cells (APC) by AAV9 encoding a foreign protein triggered a full neurotoxic immune response. Accordingly, we asked whether this phenomenon occurred in NHP. We performed parenchymal or intrathecal infusion of AAV9 encoding green fluorescent protein (GFP), a non-self protein derived from jellyfish, or human aromatic L-amino acid decarboxylase (hAADC), a self-protein, in separate NHP. Animals receiving AAV9-GFP into cisterna magna (CM) became ataxic, indicating cerebellar pathology, whereas AAV9-hAADC animals remained healthy. In transduced regions, AAV9-GFP elicited inflammation associated with early activation of astrocytic and microglial cells, along with upregulation of major histocompatibility complex class II (MHC-II) in glia. In addition, we found Purkinje neurons lacking calbindin after AAV9-GFP but not after AAV9-hAADC delivery. Our results demonstrate that AAV9-mediated expression of a foreign-protein, but not self-recognized protein, triggers complete immune responses in NHP regardless of the route of administration. Our results warrant caution when contemplating use of serotypes that can transduce APC if the transgene is not syngeneic with the host. This finding has the potential to complicate preclinical toxicology studies in which such vectors encoding human cDNA's are tested in animals.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Dependovirus , Vetores Genéticos , Inflamação/genética , Inflamação/imunologia , Animais , Sistema Nervoso Central/patologia , Corpo Estriado/imunologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dependovirus/genética , Dependovirus/imunologia , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Proteínas de Fluorescência Verde/genética , Humanos , Inflamação/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Transdução Genética , TransgenesRESUMO
Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence-alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD.
Assuntos
Alcoolismo , Animais , Masculino , Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/metabolismo , Alcoolismo/terapia , Alcoolismo/tratamento farmacológico , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Etanol/metabolismo , Etanol/farmacologia , Etanol/uso terapêutico , Terapia Genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Núcleo Accumbens/metabolismo , Primatas/genética , Área Tegmentar Ventral/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene at chromosome 12q12 are the most common genetic cause of sporadic and familial late-onset Parkinson's disease. Our aim was to identify novel LRRK2 mutations in late-onset Parkinson's disease families. DESIGN: We analyzed chromosome 12p11.2-q13.1 haplotypes in 14 late-onset Parkinson's disease families without known LRRK2 mutations. RESULTS: Haplotype analysis identified 12 families in which the affected subjects shared chromosome 12p11.2-q13.1 haplotypes. LRRK2 sequencing revealed a novel co-segregating missense mutation in exon 36 (c.5281A>C; p.S1761R) located within a highly conserved region of the COR [C-terminal of ROC (Ras of complex proteins)] domain wherein it could deregulate LRRK2 kinase activity by modifying ROC-COR dimer stability. p.S1761R was present in a late-onset Parkinson's disease family and in 2 unrelated Parkinson's disease subjects, but not in 2491 healthy controls. LRRK2 p.S1761R carriers developed levodopa-responsive asymmetrical parkinsonism, with variable age at onset (range: 37-72 years) suggesting age-dependent penetrance. These findings indicate that mutations interfering with LRRK2 ROC-COR domain dimerization lead to typical Parkinson's disease.
Assuntos
Arginina/genética , Mutação/genética , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Adulto , Fatores Etários , Idoso , Cromossomos Humanos Par 12 , Análise Mutacional de DNA , Saúde da Família , Feminino , Ligação Genética , Haplótipos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Parkinson's disease (PD) and essential tremor (ET) may share some etiopathogenic factors. A genome-wide association study has shown that LINGO1 gene variants are associated with increased risk of ET. We hypothesized that LINGO1 variants could increase susceptibility to PD. METHODS: A large series of PD subjects and healthy controls were genotyped for rs9652490 and rs11856808 LINGO1 single nucleotide polymorphisms (SNPs). RESULTS: We found an increased frequency of the rs11856808(T/T) genotype in PD compared with controls (odds ratio = 1.46; corrected P value = 0.02). A recessive genetic model was the best fit for rs11856808 influence on PD (recessive gene action test: corrected P value = 0.01). Stratification analysis showed that rs11856808(T/T) genotype frequency was higher in the tremor-dominant PD and the classical PD (C-PD) subgroups (recessive gene action test for the C-PD subgroup: corrected P value = 0.004). DISCUSSION: Our results indicate that LINGO1 variants could increase risk of PD, specifically those presenting the non-rigid-akinetic phenotypes, which suggests that LINGO1 may have a role in the etiology of tremor in PD at least in the Spanish population.
Assuntos
Predisposição Genética para Doença , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Fenótipo , Espanha , Adulto JovemRESUMO
Recent genome-wide association studies of Parkinson's disease have nominated 3 new susceptibility loci (PARK16-18) and confirmed 2 known risk genes (MAPT and SNCA) in populations of European ancestry. We sought to replicate these findings. We genotyped single-nucleotide polymorphisms in each of these genes/loci in 1445 Parkinson's disease patients and 1161 controls from northern Spain. Logistic regression was used to test for association between genotype and Parkinson's disease under an additive model, adjusting for sex, age, and site. We also performed analyses stratified by age at onset. Single-nucleotide polymorphisms in MAPT (rs1800547; P = 3.1 × 10(-4) ) and SNCA (rs356219; P = 5.5 × 10(-4) ) were significantly associated with Parkinson's disease. However, none of the markers in PARK16-18 associated with Parkinson's disease in the overall sample, or in any age stratum, with P values ranging from .09 to .88. Although our data further validate MAPT and SNCA as Parkinson's disease susceptibility genes, we failed to replicate PARK16, PARK17, and PARK18. Potential reasons for the discordance between our study and previous genome-wide association studies include effects of population structure, power, and population-specific environmental interactions. Our findings suggest that additional studies of PARK16-18 are necessary to establish the role of these loci in modifying risk for Parkinson's disease in European-derived populations. © 2011 Movement Disorder Society.
Assuntos
Predisposição Genética para Doença , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único/genética , alfa-Sinucleína/genética , Proteínas tau/genética , Idoso , Idoso de 80 Anos ou mais , Replicação do DNA/genética , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , EspanhaRESUMO
Phosphatase and tensin homolog-induced putative kinase 1 gene mutations have been associated with autosomal recessive early-onset Parkinson's disease. To date, no neuropathological reports have been published from patients with Parkinson's disease with both phosphatase and tensin homolog-induced putative kinase 1 gene copies mutated. We analysed the coding region of phosphatase and tensin homolog-induced putative kinase 1 gene in a large Spanish family with six members with parkinsonism. The phenotype was characterized by an early-onset (mean: 31.6, standard deviation: 9.6 years, range: 14-45 years), slowly progressive levodopa-responsive parkinsonism, initial gait impairment and psychiatric symptoms. We identified two segregating pathogenic phosphatase and tensin homolog-induced putative kinase 1 mutations that were either in homozygous or heterozygous compound state in all affected family members. We found an exon 7 deletion (g.16089_16383del293; c.1252_1488del) and a novel+1U1-dependent 5' splice-site mutation in exon 7 (g.16378G>A; c.1488+1G>A). Leukocyte-derived messenger RNA analysis showed that both mutations caused exon 7 skipping and c.1488+1G>A also lead to an in-frame transcript with a 33 base-pair deletion (p.L485_R497del) resulting from activation of a 5' cryptic exon 7 splice site. Single photon emission computed tomography quantification of striatal dopamine transporter binding (123I-Ioflupane) revealed a posterior-anterior gradient similar to that of idiopathic Parkinson's disease, but there was no correlation between striatal reduced uptake and disease duration. Post-mortem neuropathological examination of an early-onset Parkinson's disease carrier of two heterozygous compound phosphatase and tensin homolog-induced putative kinase 1 mutations showed neuronal loss in the substantia nigra pars compacta, Lewy bodies and aberrant neurites in the reticular nuclei of the brainstem, substantia nigra pars compacta and Meynert nucleus, but the locus ceruleus and the amygdala were spared. This is the first neuropathological report of the brain from an early-onset phosphatase and tensin homolog-induced putative kinase 1-linked parkinsonism showing that mutated phosphatase and tensin homolog-induced putative kinase 1 protein induces Lewy body pathology. Unbalanced preservation of the locus ceruleus may well play a role in the slow evolution of motor symptoms and, probably, in the psychiatric symptoms often encountered in Parkinson's disease associated with phosphatase and tensin homolog-induced putative kinase 1 mutation.
Assuntos
Corpos de Lewy/genética , Corpos de Lewy/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Proteínas Quinases/genética , Adolescente , Adulto , Idoso , Feminino , Ligação Genética , Humanos , Masculino , Mutação/genética , LinhagemRESUMO
Progressive nonfluent aphasia (PNFA) is an early stage of frontotemporal degeneration. We identified a novel Cys521Tyr progranulin gene variant in a PNFA family that potentially disrupts disulphide bridging causing protein misfolding. To identify early neurodegeneration changes, we performed neuropsychological and neuroimaging studies in 6 family members (MRI [magnetic resonance imaging], fMRI [functional MRI], and 18f-fluorodeoxygenlucose positron emission tomography, including 4 mutation carriers, and in 9 unrelated controls. Voxel-based morphometry (VBM) of the carriers compared with controls showed significant cortical atrophy in language areas. Grey matter loss was distributed mainly in frontal lobes, being more prominent on the left. Clusters were located in the superior frontal gyri, left inferior frontal gyrus, left middle frontal gyrus, left middle temporal gyri and left posterior parietal areas, concordant with (18)FDG-PET hypometabolic areas. fMRI during semantic and phonemic covert word generation (CWGTs) and word listening tasks (WLTs) showed recruitment of attentional and working memory networks in the carriers indicative of functional reorganization. During CWGTs, activation in left prefrontal cortex and bilateral anterior insulae was present whereas WLT recruited mesial prefrontal and anterior temporal cortex. These findings suggest that Cys521Tyr could be associated with early brain impairment not limited to language areas and compensated by recruitment of bilateral auxiliary cortical areas.
Assuntos
Afasia Primária Progressiva/genética , Afasia Primária Progressiva/patologia , Lobo Frontal/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação , Lobo Temporal/patologia , Afasia Primária Progressiva/diagnóstico por imagem , Demência/diagnóstico por imagem , Demência/genética , Demência/patologia , Diagnóstico Precoce , Lobo Frontal/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador , Testes de Linguagem , Imageamento por Ressonância Magnética , Testes Neuropsicológicos , Tomografia por Emissão de Pósitrons , Progranulinas , Espanha , Lobo Temporal/diagnóstico por imagemRESUMO
MicroRNAs are small RNA sequences that negatively regulate gene expression by binding to the 3' untranslated regions of mRNAs. MiR-133b has been implicated in Parkinson's disease (PD) by a mechanism that involves the regulation of the transcription factor PITX3. The variation in these genes could contribute to the risk of developing PD. We searched for DNA variants in miR-133 and PITX3 genes in PD patients and healthy controls from Spain. We found common DNA variants in the three miR-133 genes. Genotyping of a first set of patients (n = 777) and controls (n = 650) showed a higher frequency of homozygous for a miR-133b variant (-90 del A) in PD-patients (6/575; 1%) than in healthy controls (0/650) (P = 0.03). However, this association was not confirmed in a second set of patients (1/250; 0.4%) and controls (2/210; 1%). No common PITX3 variants were associated with PD, although a rare missense change (G32S) was found in only one patient and none of the controls. In conclusion, we report the variation in genes of a pathway that has been involved in dopaminergic neuron differentiation and survival. Our work suggests that miR-133 and PITX3 gene variants did not contribute to the risk for PD.
Assuntos
Variação Genética , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Doença de Parkinson/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Espanha , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
The role of the plasma level of homocysteine (Hcy), as a primary outcome, and the effect of silent cerebrovascular lesions and genetic variants related to Hcy metabolism, as secondary outcomes, in the cognitive decline and dementia in Parkinson's disease (PD) were studied. This case-control study focused on 89 PD patients of minimum 10 years of evolution and older than 60 years, who were neuropsychologically classified either as cognitively normal (n = 37), having mild cognitive impairment (Petersen criteria) (n = 22), or suffering from dementia (DSM-IV) (n = 30), compared with cognitively normal age-matched control subjects (n = 30). Plasma levels of Hcy, vitamins B12 and B6, folic acid, polymorphisms in genes related to Hcy metabolism (MTHFR, MTR, MTRR, and CBS) and silent cerebrovascular events were analyzed. Plasma levels of Hcy were increased in PD patients (P = 0.0001). There were no differences between the groups of patients. The brain vascular burden was similar among PD groups. There was no association between polymorphisms in the studied genes and the Hcy plasma levels or cognitive status in PD patients. We found no evidence for a direct relationship between Hcy plasma levels and cognitive impairment and dementia in PD. No indirect effect through cerebrovascular disease or genetic background was found either.
Assuntos
Transtornos Cognitivos/sangue , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Homocisteína/sangue , Doença de Parkinson/sangue , Doença de Parkinson/genética , Doença de Parkinson/patologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Carbono-Nitrogênio Ligases/genética , Estudos de Casos e Controles , Transtornos Cognitivos/etiologia , Cistationina beta-Sintase/genética , Depressão/etiologia , Feminino , Ácido Fólico/sangue , Humanos , Imunoensaio/métodos , Modelos Logísticos , Imageamento por Ressonância Magnética/métodos , Masculino , Entrevista Psiquiátrica Padronizada , Pessoa de Meia-Idade , Testes Neuropsicológicos , Doença de Parkinson/complicações , Polimorfismo Genético , Índice de Gravidade de Doença , Vitamina B 12/sangue , Vitamina B 6/sangueRESUMO
Gene therapy shows great promise for the treatment of neurological disorders, and accessing cerebrospinal fluid (CSF) from the cerebellomedullary cistern through the posterior atlanto-occipital membrane has become a common route of delivery in preclinical studies. Unlike direct brain parenchymal infusions, CSF delivery offers broader coverage to the central and peripheral nervous system. This prospectively increases its translational value, more specially to treat global brain dysfunctions in which the pathology is disseminated throughout the brain and not focalized in one specific brain structure. Also, from the practical point of view, this approach offers a more reliable method for neurological gene replacement in infants, whose immature cranial suture preclude the use of skull-mounted devices. Here we describe a consistent, precise, and safe method for CSF injection.
Assuntos
Cisterna Magna/metabolismo , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Animais , Líquido Cefalorraquidiano/metabolismo , Terapia Genética , Injeções Epidurais , Macaca fascicularis , Macaca mulattaRESUMO
OBJECTIVE: Successful convection-enhanced delivery of therapeutic agents to subcortical brain structures requires accurate cannula placement. Stereotactic guiding devices have been developed to accurately target brain nuclei. However, technologies remain limited by a lack of MRI compatibility, or by devices' size, making them suboptimal for direct gene delivery to brain parenchyma. The goal of this study was to validate the accuracy of a novel frameless skull-mounted ball-joint guide array (BJGA) in targeting the nonhuman primate (NHP) brain. METHODS: Fifteen MRI-guided cannula insertions were performed on 9 NHPs, each targeting the putamen. Optimal trajectories were planned on a standard MRI console using 3D multiplanar baseline images. After cannula insertion, the intended trajectory was compared to the final trajectory to assess deviation (euclidean error) of the cannula tip. RESULTS: The average cannula tip deviation was 1.18 ± 0.60 mm (mean ± SD) as measured by 2 independent reviewers. Topological analysis showed a superior, posterior, and rightward directional bias, and the intra- and interclass correlation coefficients were > 0.85, indicating valid and reliable intra- and interobserver evaluation. CONCLUSIONS: The data demonstrate that the BJGA can be used to reliably target subcortical brain structures by using MRI guidance, with accuracy comparable to current frameless stereotactic systems. The size and versatility of the BJGA, combined with a streamlined workflow, allows for its potential applicability to a variety of intracranial neurosurgical procedures, and for greater flexibility in executing MRI-guided experiments within the NHP brain.
Assuntos
Imageamento por Ressonância Magnética/métodos , Neuronavegação/métodos , Procedimentos Neurocirúrgicos/métodos , Crânio/diagnóstico por imagem , Crânio/cirurgia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/cirurgia , Macaca mulatta , Imageamento por Ressonância Magnética/instrumentação , Masculino , Neuronavegação/instrumentação , Procedimentos Neurocirúrgicos/instrumentação , Técnicas Estereotáxicas/instrumentaçãoRESUMO
OBJECTIVE: To develop and assess a convective delivery technique that enhances the effectiveness of drug delivery to nonspherical brain nuclei, the authors developed an occipital "infuse-as-you-go" approach to the putamen and compared it to the currently used transfrontal approach. METHODS: Eleven nonhuman primates received a bilateral putamen injection of adeno-associated virus with 2 mM gadolinium-DTPA by real-time MR-guided convective perfusion via either a transfrontal (n = 5) or occipital infuse-as-you-go (n = 6) approach. RESULTS: MRI provided contemporaneous assessment and monitoring of putaminal infusions for transfrontal (2 to 3 infusion deposits) and occipital infuse-as-you-go (stepwise infusions) putaminal approaches. The infuse-as-you-go technique was more efficient than the transfrontal approach (mean 35 ± 1.1 vs 88 ± 8.3 minutes [SEM; p < 0.001]). More effective perfusion of the postcommissural and total putamen was achieved with the infuse-as-you-go versus transfronatal approaches (100-µl infusion volumes; mean posterior commissural coverage 76.2% ± 5.0% vs 32.8% ± 2.9% [p < 0.001]; and mean total coverage 53.5% ± 3.0% vs 38.9% ± 2.3% [p < 0.01]). CONCLUSIONS: The infuse-as-you-go approach, paralleling the longitudinal axis of the target structure, provides a more effective and efficient method for convective infusate coverage of elongated, irregularly shaped subcortical brain nuclei.
RESUMO
Here we evaluated the utility of MRI to monitor intrathecal infusions in nonhuman primates. Adeno-associated virus (AAV) spiked with gadoteridol, a gadolinium-based MRI contrast agent, enabled real-time visualization of infusions delivered either via cerebromedullary cistern, lumbar, cerebromedullary and lumbar, or intracerebroventricular infusion. The kinetics of vector clearance from the cerebrospinal fluid (CSF) were analyzed. Our results highlight the value of MRI in optimizing the delivery of infusate into CSF. In particular, MRI revealed differential patterns of infusate distribution depending on the route of delivery. Gadoteridol coverage analysis showed that cerebellomedullary cistern delivery was a reliable and effective route of injection, achieving broad infusate distribution in the brain and spinal cord, and was even greater when combined with lumbar injection. In contrast, intracerebroventricular injection resulted in strong cortical coverage but little spinal distribution. Lumbar injection alone led to the distribution of MRI contrast agent mainly in the spinal cord with little cortical coverage, but this delivery route was unreliable. Similarly, vector clearance analysis showed differences between different routes of delivery. Overall, our data support the value of monitoring CSF injections to dissect different patterns of gadoteridol distribution based on the route of intrathecal administration.
RESUMO
Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.
Assuntos
Dependovirus/metabolismo , Terapia Genética , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo A/terapia , Sorogrupo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Inflamação/patologia , Injeções , Fígado/patologia , Camundongos Knockout , Atividade Motora , Primatas , Esfingomielina Fosfodiesterase/administração & dosagem , Esfingomielina Fosfodiesterase/sangue , Esfingomielina Fosfodiesterase/genética , TransgenesRESUMO
BACKGROUND: In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Previous work has shown that parenchymal delivery of adeno-associated virus (AAV) in the thalamus by convection-enhanced delivery (CED) can lead to large-scale transduction via axonal transport in distal areas including cortex. We used this approach to obtain widespread cortical expression of light-sensitive ion channels. NEW METHOD: AAV vectors co-expressing channelrhodopsin-2 (ChR2) and yellow fluorescent protein (YFP) genes were infused into thalamus of three rhesus macaques under MR-guided CED. After six to twelve weeks recovery, in vivo optical stimulation and single cell recording in the cortex was carried out using an optrode in anesthetized animals. Post-mortem immunostaining against YFP was used to estimate the distribution and level of expression of ChR2 in thalamus and cortex. RESULTS: Histological analysis revealed high levels of transduction in cortical layers. The patterns of expression were consistent with known thalamo-cortico-thalamic circuits. Dense expression was seen in thalamocortiocal axonal fibers in layers III, IV and VI and in pyramidal neurons in layers V and VI, presumably corticothalamic neurons. In addition we obtained reliable in vivo light-evoked responses in cortical areas with high levels of expression. COMPARISON WITH EXISTING METHODS: Thalamic CED is very efficient in achieving large expressing areas in comparison to convectional techniques both in minimizing infusion time and in minimizing damage to the brain. CONCLUSION: MR-guided CED infusion into thalamus provides a simplified approach to transduce large cortical areas by thalamo-cortico-thalamic projections in primate brain.
Assuntos
Dependovirus/genética , Vetores Genéticos/administração & dosagem , Macaca mulatta , Optogenética/métodos , Tálamo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/fisiologia , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Convecção , Dermoscopia , Feminino , Imageamento Tridimensional , Imuno-Histoquímica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imageamento por Ressonância Magnética , Masculino , Modelos Animais , Vias Neurais/citologia , Vias Neurais/fisiologia , Estimulação Luminosa , Tálamo/citologia , Tálamo/diagnóstico por imagem , Tálamo/fisiologiaRESUMO
Eighteen loci and several susceptibility genes have been related to Parkinson's disease (PD). However, most studies focus on single genes in small PD series. Our aim was to establish the genetic background of a large Spanish PD sample. Pooled-DNA target sequencing of 7 major PD genes (SNCA, PARK2, PINK1, DJ-1, LRRK2, GBA, and MAPT) was performed in 562 PD cases. Forty-four variants were found among 114 individuals (20.28%, p<0.05). Among these variants, 30 were found in Mendelian genes (68.18%) and 14 in PD susceptibility genes (31.82%). Seven novel variants were identified. Interestingly, most variants were found in PARK2 and PINK1 genes, whereas SNCA and DJ-1 variants were rare. Validated variants were also genotyped in Spanish healthy controls (n = 597). Carriers of heterozygous PARK2 variants presented earlier disease onset and showed dementia more frequently. PD subjects carrying 2 variants at different genes (1.42%) had an earlier age of onset and a predominantly akinetic-rigid PD phenotype (55.6%, p < 0.05), suggesting that the accumulation of genetic risk variants could modify PD phenotype.
Assuntos
Predisposição Genética para Doença , Doença de Parkinson/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Proteínas Quinases/genética , Análise de Sequência de DNA , Espanha , Ubiquitina-Proteína Ligases/genética , População Branca/genética , Adulto JovemRESUMO
In Parkinson's disease (PD), aromatic L-amino acid decarboxylase (AADC) is the rate-limiting enzyme in the conversion of L-DOPA (Sinemet) to dopamine (DA). Previous studies in PD animal models demonstrated that lesion of dopaminergic neurons is associated with profound loss of AADC activity in the striatum, blocking efficient conversion of L-DOPA to DA. Relatively few studies have directly analyzed AADC in PD brains. Thus, the aim of this study was to gain a better understanding of regional changes in AADC activity, DA, serotonin and their monoamine metabolites in the striatum of PD patients and experimentally lesioned animals (rat and MPTP-treated nonhuman primate, NHP). Striatal AADC activity was determined post mortem in neuropathologically confirmed PD subjects, animal models and controls. A regional analysis was performed for striatal AADC activity and monoamine levels in NHP tissue. Interestingly, analysis of putaminal AADC activity revealed that control human striatum contained much less AADC activity than rat and NHP striata. Moreover, a dramatic loss of AADC activity in PD striatum compared to controls was detected. In MPTP-treated NHP, caudate nucleus was almost as greatly affected as putamen, although mean DA turnover was higher in caudate nucleus. Similarly, DA and DA metabolites were dramatically reduced in different regions of PD brains, including caudate nucleus, whereas serotonin was relatively spared. After L-DOPA administration in MPTP-treated NHP, very poor conversion to DA was detected, suggesting that AADC in NHP nigrostriatal fibers is mainly responsible for L-DOPA to DA conversion. These data support further the rationale behind viral gene therapy with AAV2-hAADC to restore AADC levels in putamen and suggest further the advisability of expanding vector delivery to include coverage of anterior putamen and the caudate nucleus.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Núcleo Caudado/metabolismo , Doença de Parkinson/metabolismo , Putamen/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Descarboxilases de Aminoácido-L-Aromático/genética , Corpo Estriado/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Dopamina/metabolismo , Ativação Enzimática , Feminino , Terapia Genética , Vetores Genéticos/genética , Humanos , Levodopa/metabolismo , Levodopa/uso terapêutico , Macaca mulatta , Masculino , Doença de Parkinson/genética , Doença de Parkinson/terapia , RatosRESUMO
Gene transfer technology offers great promise as a potential therapeutic approach to the brain but has to be viewed as a very complex technology. Success of ongoing clinical gene therapy trials depends on many factors such as selection of the correct genetic and anatomical target in the brain. In addition, selection of the viral vector capable of transfer of therapeutic gene into target cells, along with long-term expression that avoids immunotoxicity has to be established. As with any drug development strategy, delivery of gene therapy has to be consistent and predictable in each study subject. Failed drug and vector delivery will lead to failed clinical trials. In this article, we describe our experience with AAV viral vector delivery system, that allows us to optimize and monitor in real time viral vector administration into affected regions of the brain. In addition to discussing MRI-guided technology for administration of AAV vectors we have developed and now employ in current clinical trials, we also describe ways in which infusion cannula design and stereotactic trajectory may be used to maximize the anatomical coverage by using fluid backflow. This innovative approach enables more precise coverage by fitting the shape of the infusion to the shape of the anatomical target.