RESUMO
Precise genome manipulation in specific cell types and subtypes in vivo is crucial for neurobiological research because of the cellular heterogeneity of the brain. Site-specific recombinase systems in the mouse, such as Cre-loxP, improve cell type-specific genome manipulation; however, undesirable expression of cell type-specific Cre can occur. This could be due to transient expression during early development, natural expression in more than one cell type, kinetics of recombinases, sensitivity of the Cre reporter, and disruption in cis-regulatory elements by transgene insertion. Moreover, cell subtypes cannot be distinguished in cell type-specific Cre mice. To address these issues, we applied an intersectional genetic approach in mouse using triple recombination systems (Cre-loxP, Flp-FRT and Dre-rox). As a proof of principle, we labelled heterogeneous cell subtypes and deleted target genes within given cell subtypes by labelling neuropeptide Y (NPY)-, calretinin (calbindin 2) (CR)- and cholecystokinin (CCK)-expressing GABAergic neurons in the brain followed by deletion of RNA-binding Fox-1 homolog 3 (Rbfox3) in our engineered mice. Together, our study applies an intersectional genetic approach in vivo to generate engineered mice serving dual purposes of simultaneous cell subtype-specific labelling and gene knockout.
Assuntos
Integrases , Recombinases , Camundongos , Animais , Técnicas de Inativação de Genes , Integrases/metabolismo , Recombinases/genética , Recombinases/metabolismo , Transgenes , Encéfalo/metabolismo , Camundongos TransgênicosRESUMO
Epilepsy is a common neurological disorder, which has been linked to mutations or deletions of RNA binding protein, fox-1 homolog (Caenorhabditis elegans) 3 (RBFOX3)/NeuN, a neuronal splicing regulator. However, the mechanism of seizure mediation by RBFOX3 remains unknown. Here, we show that mice with deletion of Rbfox3 in gamma-aminobutyric acid (GABA) ergic neurons exhibit spontaneous seizures and high premature mortality due to increased presynaptic release, postsynaptic potential, neuronal excitability, and synaptic transmission in hippocampal dentate gyrus granule cells (DGGCs). Attenuating early excitatory gamma-aminobutyric acid (GABA) action by administering bumetanide, an inhibitor of early GABA depolarization, rescued premature mortality. Rbfox3 deletion reduced hippocampal expression of vesicle-associated membrane protein 1 (VAMP1), a GABAergic neuron-specific presynaptic protein. Postnatal restoration of VAMP1 rescued premature mortality and neuronal excitability in DGGCs. Furthermore, Rbfox3 deletion in GABAergic neurons showed fewer neuropeptide Y (NPY)-expressing GABAergic neurons. In addition, deletion of Rbfox3 in NPY-expressing GABAergic neurons lowered intrinsic excitability and increased seizure susceptibility. Our results establish RBFOX3 as a critical regulator and possible treatment path for epilepsy.
Assuntos
Proteínas de Ligação a DNA , Neurônios GABAérgicos , Proteínas do Tecido Nervoso , Neuropeptídeo Y , Convulsões , Proteína 1 Associada à Membrana da Vesícula , Animais , Bumetanida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Giro Denteado/metabolismo , Antagonistas GABAérgicos/farmacologia , Neurônios GABAérgicos/metabolismo , Deleção de Genes , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeo Y/metabolismo , Convulsões/genética , Convulsões/metabolismo , Proteína 1 Associada à Membrana da Vesícula/genética , Proteína 1 Associada à Membrana da Vesícula/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/ß-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3ß inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.
Assuntos
Glicogênio Sintase Quinase 3 beta , Células-Tronco Pluripotentes Induzidas , Mucopolissacaridose II , Neurônios , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Mucopolissacaridose II/patologia , Mucopolissacaridose II/metabolismo , Mucopolissacaridose II/genética , Diferenciação Celular/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Degeneração Neural/patologia , Cálcio/metabolismoRESUMO
Dysfunction of RBFOX3 has been identified in neurodevelopmental disorders such as autism spectrum disorder, cognitive impairments and epilepsy and a causal relationship with these diseases has been previously demonstrated with Rbfox3 homozygous knockout mice. Despite the importance of RBFOX3 during neurodevelopment, the function of RBFOX3 regarding neurogenesis and synaptogenesis remains unclear. To address this critical question, we profiled the developmental expression pattern of Rbfox3 in the brain of wild-type mice and analyzed brain volume, disease-relevant behaviors, neurogenesis, synaptic plasticity, and synaptogenesis in Rbfox3 homozygous knockout mice and their corresponding wild-type counterparts. Here we report that expression of Rbfox3 differs developmentally for distinct brain regions. Moreover, Rbfox3 homozygous knockout mice exhibited cold hyperalgesia and impaired cognitive abilities. Focusing on hippocampal phenotypes, we found Rbfox3 homozygous knockout mice displayed deficits in neurogenesis, which was correlated with cognitive impairments. Furthermore, RBFOX3 regulates the exons of genes with synapse-related function. Synaptic plasticity and density, which are related to cognitive behaviors, were altered in the hippocampal dentate gyrus of Rbfox3 homozygous knockout mice; synaptic plasticity decreased and the density of synapses increased. Taken together, our results demonstrate the important role of RBFOX3 during neural development and maturation. In addition, abnormalities in synaptic structure and function occur in Rbfox3 homozygous knockout mice. Our findings may offer mechanistic explanations for human brain diseases associated with dysfunctional RBFOX3.
Assuntos
Hipocampo/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Neurogênese , Proteínas Nucleares/genética , Sinapses/metabolismo , Animais , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Plasticidade Neuronal , Proteínas Nucleares/metabolismo , Sinapses/patologiaRESUMO
RBFOX3 mutations are linked to epilepsy and cognitive impairments, but the underlying pathophysiology of these disorders is poorly understood. Here we report replication of human symptoms in a mouse model with disrupted Rbfox3. Rbfox3 knockout mice displayed increased seizure susceptibility and decreased anxiety-related behaviors. Focusing on hippocampal phenotypes, we found Rbfox3 knockout mice showed increased expression of plasticity genes Egr4 and Arc, and the synaptic transmission and plasticity were defective in the mutant perforant pathway. The mutant dentate granules cells exhibited an increased frequency, but normal amplitude, of excitatory synaptic events, and this change was associated with an increase in the neurotransmitter release probability and dendritic spine density. Together, our results demonstrate anatomical and functional abnormality in Rbfox3 knockout mice, and may provide mechanistic insights for RBFOX3-related human brain disorders.