RESUMO
Shati/Nat8l was identified as an upregulated molecule in the nucleus accumbens (NAc) of mice following repeated methamphetamine administration. Region-specific roles of this molecule are associated with psychiatric disorders. In the present study, we examined the importance of Shati/Nat8l in the hippocampus because of its high expression in this region. Mice with a hippocampus-specific knockdown of Shati/Nat8l (hippocampal Shati-cKD) were prepared by the microinjection of adeno-associated virus (AAV) vectors carrying Cre into the hippocampus of Shati/Nat8lflox/flox mice, and their phenotypes were investigated. Drastic reduction in the expression and function of Shati/Nat8l in the hippocampus was observed in Shati-cKD mice. These mice exhibited cognitive dysfunction in behavioral experiments and impaired the electrophysiological response to the stimuli, which elicits long-term potentiation. Shati/Nat8l in the hippocampus is suggested to possibly play an important role in synaptic plasticity to maintain cognitive function. This molecule could be a therapeutic target for hippocampus-related disorders such as dementia.
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Diabetic retinopathy (DR) can cause visual impairment and blindness, and the increasing global prevalence of diabetes underscores the need for effective therapies to prevent and treat DR. Therefore, this study aimed to evaluate the protective effect of pemafibrate treatment against DR, using a Spontaneously Diabetic Torii (SDT) fatty rat model of obese type 2 diabetes. SDT fatty rats were fed either a diet supplemented with pemafibrate (0.3 mg/kg/d) for 16 weeks, starting at 8 weeks of age (Pf SDT fatty: study group), or normal chow (SDT fatty: controls). Normal chow was provided to Sprague-Dawley (SD) rats (SD: normal controls). Electroretinography (ERG) was performed at 8 and 24 weeks of age to evaluate the retinal neural function. After sacrifice, retinal thickness, number of retinal folds, and choroidal thickness were evaluated, and immunostaining was performed for aquaporin-4 (AQP4). No significant differences were noted in food consumption, body weight, or blood glucose level after pemafibrate administration. Triglyceride levels were reduced, and high-density lipoprotein cholesterol levels were increased. Extension of oscillatory potential (OP)1 and OP3 waves on ERG was suppressed in the Pf SDT fatty group. Retinal thickness at 1500 microns from the optic disc improved in the Pf SDT fatty group. No significant improvements were noted in choroidal thickness or number of retinal folds. Quantitative analyses showed that AQP4-positive regions in the retinas were significantly larger in the Pf SDT fatty group than in the SDT fatty group. The findings suggest that pemafibrate treatment can exert protective effects against DR.
Assuntos
Benzoxazóis , Butiratos , Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Ratos , Animais , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/prevenção & controle , Ratos Sprague-Dawley , Modelos Animais de DoençasRESUMO
BACKGROUND: The delivery of adeno-associated virus (AAV) vectors via the cerebrospinal fluid (CSF) has emerged as a valuable method for widespread transduction in the central nervous system. Although infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, the cisterna magna has been recognized as an alternative target for clinical studies because it can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. METHODS: We evaluated the early distribution of fluorine-18-labeled AAV9 vectors infused into the lateral ventricle or cisterna magna of four non-human primates using positron emission tomography. The expression of the green fluorescent protein was immunohistochemically determined. RESULTS: In both approaches, the labeled vectors diffused into the broad arachnoid space around the brain stem and cervical spinal cord within 30 min. Both infusion routes efficiently transduced neurons in the cervical spinal cord. CONCLUSIONS: For gene therapy that primarily targets the cervical spinal cord and brainstem, such as amyotrophic lateral sclerosis, cisterna magna infusion would be a feasible and effective administration method.
Assuntos
Terapia Genética , Medula Espinal , Animais , Transdução Genética , Medula Espinal/metabolismo , Terapia Genética/métodos , Primatas/genética , Vetores Genéticos/genética , Dependovirus/genéticaRESUMO
Aromatic L-amino acid decarboxylase deficiency results in decreased neurotransmitter levels and severe motor dysfunction. Twenty-six patients without head control received bilateral intraputaminal infusions of a recombinant adeno-associated virus type 2 vector containing the human aromatic L-amino acid decarboxylase gene (eladocagene exuparvovec) and have completed 1-year evaluations. Rapid improvements in motor and cognitive function occurred within 12 months after gene therapy and were sustained during follow-up for >5 years. An increase in dopamine production was demonstrated by positron emission tomography and neurotransmitter analysis. Patient symptoms (mood, sweating, temperature, and oculogyric crises), patient growth, and patient caretaker quality of life improved. Although improvements were observed in all treated participants, younger age was associated with greater improvement. There were no treatment-associated brain injuries, and most adverse events were related to underlying disease. Post-surgery complications such as cerebrospinal fluid leakage were managed with standard of care. Most patients experienced mild to moderate dyskinesia that resolved in a few months. These observations suggest that eladocagene exuparvovec treatment for aromatic L-amino acid decarboxylase deficiency provides durable and meaningful benefits with a favorable safety profile.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Qualidade de Vida , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Descarboxilases de Aminoácido-L-Aromático/líquido cefalorraquidiano , Descarboxilases de Aminoácido-L-Aromático/deficiência , Descarboxilases de Aminoácido-L-Aromático/genética , Dopamina , Terapia Genética/efeitos adversos , HumanosRESUMO
BACKGROUND: Despite the increasing availability of effective drugs, around one-third of patients with epilepsy are still resistant to pharmacotherapy. Gene therapy has been suggested as a plausible approach to achieve seizure control, in particular for patients with focal epilepsy. Because seizures develop across wide spans of the brain in many forms of epilepsy, global delivery of the vectors is necessary to tackle such generalized seizures. Neuroligin 2 (NL2) is a postsynaptic cell adhesion molecule that induces or strengthens inhibitory synaptic function by specifically combining with neurexin 1. METHODS: In the present study, we applied an adeno-associated virus (AAV) type 9 vector expressing NL2 to modulate neuronal excitability in broad areas of the brain in epileptic (EL) mice, a model of polygene epilepsy. We administered the AAV vector expressing Flag-tagged NL2 under the synapsin I promoter (AAV-NL2) via cardiac injection 6 weeks after birth. RESULTS: Significant reductions in the duration, strength and frequency of seizure were observed during a 14-week observation period in NL2-treated EL mice compared to untreated or AAV-green fluorescent protein-treated EL mice. No behavioral abnormality was observed in NL2-treated EL mice in an open-field test. Immunohistochemical examination at 14 weeks after AAV-NL2 injection revealed the expression of exogenous NL2 in broad areas of the brain, including the hippocampus and, in these areas, NL2 co-localized with postsynaptic inhibitory molecule gephyrin. CONCLUSIONS: Global brain delivery of NL2 by systemic administration of AAV vector may provide a non-invasive therapeutic approach for generalized epilepsy.
Assuntos
Epilepsia , Sinapses , Animais , Encéfalo , Moléculas de Adesão Celular Neuronais , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/terapia , Humanos , Camundongos , Proteínas do Tecido Nervoso , Convulsões/genética , Convulsões/metabolismo , Convulsões/terapia , Sinapses/metabolismoRESUMO
Methamphetamine (METH), the most widely distributed psychostimulant, aberrantly activates the reward system in the brain to induce addictive behaviors. The presynaptic protein "Piccolo", encoded by Pclo, was identified as a METH-responsive protein with enhanced expression in the nucleus accumbens (NAc) in mice. Although the physiological and pathological significance of Piccolo has been identified in dopaminergic signaling, its role in METH-induced behavioral abnormalities and the underlying mechanisms remain unclear. To clarify such functions, mice with Piccolo knockdown in the NAc (NAc-miPiccolo mice) by local injection of an adeno-associated virus vector carrying miRNA targeting Pclo were generated and investigated. NAc-miPiccolo mice exhibited suppressed hyperlocomotion, sensitization, and conditioned place preference behavior induced by systemic administration of METH. The excessive release of dopamine in the NAc was reduced in NAc-miPiccolo mice at baseline and in response to METH. These results suggest that Piccolo in the NAc is involved in METH-induced behavioral alterations and is a candidate therapeutic target for the treatment of drug addiction.
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Estimulantes do Sistema Nervoso Central , Metanfetamina , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Metanfetamina/farmacologia , Camundongos , Núcleo Accumbens/metabolismo , RecompensaRESUMO
As the elderly population rapidly increases worldwide, the onset of cognitive dysfunction is expected to increase. Although neuronal plasticity, neurogenesis, and mitochondrial dysfunction have been reported to be involved in cognitive function, the detailed mechanism of cognitive impairment accompanied by aging is poorly understood as there are many confounding factors associated with aging. Therefore, effective treatments for aging have not yet been developed, and the establishment of therapeutic strategies has not progressed accordingly. We have previously found a decline of cognitive function in the developmental stage in mice who lack the expression of Shati/Nat8l, an N-acetyl transferase However, the contribution of Shati/Nat8l to cognitive impairment in aged mice has not yet been investigated. In this study, we aimed to investigate the role of Shati/Nat8l in cognitive function during aging. We observed a reduction in Shati/Nat8l mRNA expression in the dorsal hippocampus of mice as a result of their aging. Moreover, the cognitive dysfunction observed in aged mice was reversed by Shati/Nat8l overexpression in the dorsal hippocampus. Shati/Nat8l overexpression in the dorsal hippocampus of mice did not alter the expression of neurotrophic factors or mitochondrial function-related genes, including Bdnf or Pgc-1α, which are suggested to be downstream genes of Shati/Nat8l. Decreased N-acetyl aspartate (NAA) in aged mice was upregulated by Shati/Nat8l overexpression, suggesting that the Shati/Nat8l-NAA pathway determines cognitive function with aging. Taken together, Shati/Nat8l and NAA in the dorsal hippocampus may be novel targets for the treatment of cognitive impairment.
Assuntos
Acetiltransferases , Disfunção Cognitiva , Acetiltransferases/genética , Envelhecimento , Animais , Ácido Aspártico/metabolismo , Cognição , Disfunção Cognitiva/metabolismo , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Alzheimer's disease (AD) is a type of dementia characterized by the deposition of amyloid ß, a causative protein of AD, in the brain. Shati/Nat8l, identified as a psychiatric disease related molecule, is a responsive enzyme of N-acetylaspartate (NAA) synthesis. In the hippocampi of AD patients and model mice, the NAA content and Shati/Nat8l expression were reported to be reduced. Having recently clarified the involvement of Shati/Nat8l in cognitive function, we examined the recovery effect of the hippocampal overexpression of Shati/Nat8l in AD model mice (5XFAD). Shati/Nat8l overexpression suppressed cognitive dysfunction without affecting the Aß burden or number of NeuN-positive neurons. In addition, brain-derived neurotrophic factor mRNA was upregulated by Shati/Nat8l overexpression in 5XFAD mice. These results suggest that Shati/Nat8l overexpression prevents cognitive dysfunction in 5XFAD mice, indicating that Shati/Nat8l could be a therapeutic target for AD.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Acetiltransferases/genética , Peptídeos beta-Amiloides , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios/metabolismoRESUMO
Glucose transporter 1 deficiency syndrome (GLUT1DS) is caused by haplo-insufficiency of SLC2A1, which encodes GLUT1, resulting in impaired hexose transport into the brain. Previously, we generated a tyrosine-mutant AAV9/3 vector in which SLC2A1 was expressed under the control of the endogenous GLUT1 promoter (AAV-GLUT1), and confirmed the improved motor function and cerebrospinal fluid glucose levels of Glut1-deficient mice after cerebroventricular injection of AAV-GLUT1. In preparation for clinical application, we examined the expression of transgenes after intra-cisterna magna injection of AAV-GFP (tyrosine-mutant AAV9/3-GFP with the CMV promoter) and AAV-GLUT1. We injected AAV-GFP or AAV-GLUT1 (1.63 × 1012 vector genomes/kg) into the cisterna magna of pigs to compare differential promoter activity. After AAV-GFP injection, exogenous GFP was expressed in broad areas of the brain and peripheral organs. After AAV-GLUT1 injection, exogenous GLUT1 was expressed predominantly in the brain. At the cellular level, exogenous GLUT1 was mainly expressed in the endothelium, followed by glia and neurons, which was contrasted with the neuronal-predominant expression of GFP by the CMV promotor. We consider intra-cisterna magna injection of AAV-GLUT1 to be a feasible approach for gene therapy of GLUT1DS.
Assuntos
Cisterna Magna , Dependovirus , Animais , Dependovirus/genética , Vetores Genéticos/genética , Transportador de Glucose Tipo 1/genética , Camundongos , Suínos , TransgenesRESUMO
ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4+/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4+/-/ARF5-/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4+/-/ARF5-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4+/-/ARF5-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4+/-/ARF5-/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4+/-/ARF5-/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4+/-/ARF5-/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Axônios/metabolismo , Transtornos dos Movimentos/etiologia , Canal de Sódio Disparado por Voltagem NAV1.6/fisiologia , Células de Purkinje/metabolismo , Tremor/etiologia , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Potenciais de Ação , Animais , Dependovirus/genética , Eletroencefalografia , Eletromiografia , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Genótipo , Movimentos da Cabeça , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.6/deficiência , Técnicas de Patch-Clamp , Transporte Proteico , Células de Purkinje/fisiologia , Teste de Desempenho do Rota-Rod , Método Simples-Cego , Tremor/metabolismo , Tremor/fisiopatologiaRESUMO
In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-[18F]fluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.10.1093/brain/awy331_video1awy331media15991361892001.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Descarboxilases de Aminoácido-L-Aromático/deficiência , Terapia Genética/métodos , Processos Mentais/fisiologia , Destreza Motora/fisiologia , Adolescente , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico por imagem , Descarboxilases de Aminoácido-L-Aromático/genética , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.
Assuntos
Axônios/fisiologia , Regeneração Nervosa/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Traumatismos da Medula Espinal/terapia , Proteínas Quinases Ativadas por AMP , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Neurogênese/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/metabolismoRESUMO
Shati/Nat8l is a novel N-acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Shati/Nat8l mRNA is expressed in various brain areas, including the prefrontal cortex (PFC), where the expression level is higher than that in other brain regions. Shati/Nat8l overexpression in the nucleus accumbens (NAc) attenuates the pharmacological response to METH via mGluR3. Meanwhile, dopamine (DA) and glutamate dysregulations have been reported in the medial prefrontal cortex (mPFC) and NAc after METH self-administration and during reinstatement. However, the mechanism, the reward system, and function of Shati/Nat8l in the mPFC is unclear. Here, we injected an adeno-associated virus (AAV) vector containing Shati/Nat8l into the mPFC of mice, to overexpress Shati/Nat8l in the mPFC (mPFC-Shati/Nat8l). Interestingly, the METH-induced conditioned place preference (CPP) was attenuated in the mPFC-Shati/Nat8l mice, but locomotor activity was not. Additionally, immunohistochemical results from mice that were injected with AAV-GFP showed fluorescence in the mPFC and other brain regions, mainly the NAc, indicating an mPFC-NAc top-down connection. Finally, in vivo microdialysis experiments revealed that Shati/Nat8l overexpression in the mPFC reduced extracellular DA levels and suppressed the METH-induced DA increase in the NAc. Moreover, decreased extracellular glutamate levels were observed in the NAc. These results indicate that Shati/Nat8l overexpression in the mPFC attenuates METH-induced CPP by decreasing extracellular DA in the NAc. In contrast, Shati/Nat8l-mPFC overexpression did not alter METH-induced hyperlocomotion. This study demonstrates that Shati/Nat8l in the mPFC attenuates METH reward-seeking behaviour but not the psychomotor activity of METH.
Assuntos
Acetiltransferases/genética , Condicionamento Clássico , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Locomoção/genética , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Técnicas de Introdução de Genes , Locomoção/efeitos dos fármacos , Masculino , Metanfetamina/farmacologia , Camundongos , MicrodiáliseRESUMO
Early-phase pathologies of Alzheimer's disease (AD) are attracting much attention after clinical trials of drugs designed to remove beta-amyloid (Aß) aggregates failed to recover memory and cognitive function in symptomatic AD patients. Here, we show that phosphorylation of serine/arginine repetitive matrix 2 (SRRM2) at Ser1068, which is observed in the brains of early phase AD mouse models and postmortem end-stage AD patients, prevents its nuclear translocation by inhibiting interaction with T-complex protein subunit α. SRRM2 deficiency in neurons destabilized polyglutamine binding protein 1 (PQBP1), a causative gene for intellectual disability (ID), greatly affecting the splicing patterns of synapse-related genes, as demonstrated in a newly generated PQBP1-conditional knockout model. PQBP1 and SRRM2 were downregulated in cortical neurons of human AD patients and mouse AD models, and the AAV-PQBP1 vector recovered RNA splicing, the synapse phenotype, and the cognitive decline in the two mouse models. Finally, the kinases responsible for the phosphorylation of SRRM2 at Ser1068 were identified as ERK1/2 (MAPK3/1). These results collectively reveal a new aspect of AD pathology in which a phosphorylation signal affecting RNA splicing and synapse integrity precedes the formation of extracellular Aß aggregates and may progress in parallel with tau phosphorylation.
Assuntos
Doença de Alzheimer/genética , Proteínas de Transporte/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Transporte Ativo do Núcleo Celular , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Cognição , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Cultura Primária de Células , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas tau/metabolismoRESUMO
DNA damage and repair is a critical domain of many neurodegenerative diseases. In this study, we focused on RpA1, a candidate key molecule in polyQ disease pathologies, and tested the therapeutic effect of adeno-associated virus (AAV) vector expressing RpA1 on mutant Ataxin-1 knock-in (Atxn1-KI) mice. We found significant effects on motor functions, normalized DNA damage markers (γH2AX and 53BP1), and improved Purkinje cell morphology; effects that lasted for 50 weeks following AAV-RpA1 infection. In addition, we confirmed that AAV-RpA1 indirectly recovered multiple cellular functions such as RNA splicing, transcription and cell cycle as well as abnormal morphology of dendrite and dendritic spine of Purkinje cells in Atxn1-KI mice. All these results suggested a possibility of gene therapy with RpA1 for SCA1.
Assuntos
Ataxina-1/genética , Reparo do DNA , Mutação , Proteína de Replicação A/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Ciclo Celular , DNA/metabolismo , Dano ao DNA , Dependovirus , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Terapia Genética , Camundongos , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Células de Purkinje/fisiologia , RNA/metabolismo , Splicing de RNA , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Ataxias Espinocerebelares/fisiopatologia , Transcrição GênicaRESUMO
BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). RESULTS: Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. CONCLUSIONS: Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.
Assuntos
Dependovirus/genética , Terapia Genética , Transportador de Glucose Tipo 1/genética , Animais , Encéfalo/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Glucose/líquido cefalorraquidiano , Transportador de Glucose Tipo 1/líquido cefalorraquidiano , Humanos , Fígado/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ratos , TransgenesRESUMO
Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Imagem Molecular , Dados de Sequência Molecular , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/metabolismo , Agregados Proteicos , Ligação Proteica/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
Background: Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3. Methods: Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests. Results: The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes N-acetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor. Conclusions: Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3-mediated functional control of the serotonergic neuronal system.
Assuntos
Corpo Estriado/metabolismo , Depressão/genética , Depressão/patologia , Regulação da Expressão Gênica/genética , Receptores de Glutamato Metabotrópico/metabolismo , Serotonina/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Depressão/metabolismo , Dipeptídeos/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Elevação dos Membros Posteriores , Humanos , Relações Interpessoais , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise , Microinjeções , Natação/psicologia , Transdução GenéticaRESUMO
Clinical studies of gene therapy for Parkinson's disease are based on three kinds of strategies. 1. Restoration of dopamine production in the putamen by introducing genes of dopamine-synthesizing enzymes. 2. Protection of nigrostriatal projections by gene transfer of neurotrophic factors into the putamen and substantia nigra. 3. Modulation of subthalamic nucleus neural activity by gene delivery of an enzyme to synthesize inhibitory transmitter y-aminobutyric acid. In all studies, procedures were well tolerated and no adverse effects attributed to viral vectors were reported. The beneficial effects of putaminal gene transfer on motor symptoms have also been confirmed in children with aromatic L-amino acid decarboxylase deficiency. For a neuroprotective strategy, early intervention is necessary before degeneration has proceeded too far.