RESUMO
The single nucleotide polymorphism rs13166360, causing a substitution of valine (Val) 147 to leucine (Leu) in the adenylyl cyclase 2 (ADCY2), has previously been associated with bipolar disorder (BD). Here we show that the disease-associated ADCY2 missense mutation diminishes the enzyme´s capacity to generate the second messenger 3',5'-cylic adenosine monophosphate (cAMP) by altering its subcellular localization. We established mice specifically carrying the Val to Leu substitution using CRISPR/Cas9-based gene editing. Mice homozygous for the Leu variant display symptoms of a mania-like state accompanied by cognitive impairments. Mutant animals show additional characteristic signs of rodent mania models, i.e., they are hypersensitive to amphetamine, the observed mania-like behaviors are responsive to lithium treatment and the Val to Leu substitution results in a shifted excitatory/inhibitory synaptic balance towards more excitation. Exposure to chronic social defeat stress switches homozygous Leu variant carriers from a mania- to a depressive-like state, a transition which is reminiscent of the alternations characterizing the symptomatology in BD patients. Single-cell RNA-seq (scRNA-seq) revealed widespread Adcy2 mRNA expression in numerous hippocampal cell types. Differentially expressed genes particularly identified from glutamatergic CA1 neurons point towards ADCY2 variant-dependent alterations in multiple biological processes including cAMP-related signaling pathways. These results validate ADCY2 as a BD risk gene, provide insights into underlying disease mechanisms, and potentially open novel avenues for therapeutic intervention strategies.
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Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.
Assuntos
Encéfalo/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Proteômica , Células de Purkinje/metabolismoRESUMO
Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.
Assuntos
Animais Geneticamente Modificados/genética , Endonucleases/genética , Técnicas de Inativação de Genes/métodos , Edição de RNA/genética , Animais , Quebras de DNA de Cadeia Dupla , Genoma , Camundongos , Microinjeções , Mutação , RNA Mensageiro/genéticaRESUMO
Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Desoxirribonucleases/genética , Terapia Genética/métodos , Inteínas , Linhagem Celular , Dependovirus/genética , Marcação de Genes , Humanos , Plasmídeos/genética , Streptococcus pyogenes/enzimologia , TransfecçãoRESUMO
The use of TALEN and CRISPR/CAS nucleases is becoming increasingly popular as a means to edit single target sites in one-cell mouse embryos. Nevertheless, an area that has received less attention concerns the engineering of structural genome variants and the necessary religation of two distant double-strand breaks. Herein, we applied pairs of TALEN or sgRNAs and Cas9 to create deletions in the Rab38 gene. We found that the deletion of 3.2 or 9.3 kb, but not of 30 kb, occurs at a frequency of 6-37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to â¼10 kb can be readily achieved for modeling human disease alleles. This work represents an important step towards the establishment of new protocols that support the ligation of remote DSB ends to achieve even larger rearrangements.
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Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.
Assuntos
Mutagênese Sítio-Dirigida/métodos , Animais , Desoxirribonucleases/química , Desoxirribonucleases/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Camundongos , MicroinjeçõesRESUMO
Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator-like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2-3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.
Assuntos
Marcação de Genes/métodos , Camundongos Knockout/genética , Mutagênese Sítio-Dirigida/métodos , RNA Mensageiro/química , Animais , Sítios de Ligação , Desoxirribonucleases/química , Desoxirribonucleases/genética , Embrião de Mamíferos , Engenharia Genética/métodos , Técnicas de Genotipagem , Camundongos , Microinjeções/métodos , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms.
Assuntos
Engenharia Genética/métodos , Mutagênese , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Animais , Sequência de Bases , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Camundongos Mutantes/genética , Camundongos Transgênicos/genética , Dados de Sequência Molecular , Gravidez , RNA Mensageiro/genética , Proteína FUS de Ligação a RNA/genética , Ativação TranscricionalRESUMO
Deregulated MAP kinase (MAPK) signaling plays key roles in developmental and adult disease processes, but the experimental activation of MAPK is a currently unresolved task. For the reversible induction of MAPK signaling, we generated transgenic mice harboring a tamoxifen inducible BRAF(V637E)ER(T2) fusion protein. The expression of the inducible BRAF kinase can be directed by Cre/loxP-mediated recombination to selected cell types and enables the highly specific activation of MAPK signalling in vivo. We show that MAPK signaling can be transiently activated in the brain, liver, or kidney of Braf(V637E)ER(T2) mice by a single injection of tamoxifen. Braf(V637E)ER(T2) mice provide a new versatile tool to study disease mechanisms elicited by MAPK activation, complementing gene knockout technology that is restricted to the analysis of loss-of-function phenotypes.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas B-raf/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Recombinação Genética/efeitos dos fármacos , Medula Espinal/embriologia , Medula Espinal/metabolismoRESUMO
The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.
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Modelos Animais de Doenças , Doenças Genéticas Inatas/genética , Mutação em Linhagem Germinativa , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fase de Clivagem do Zigoto , Desoxirribonucleases/administração & dosagem , Desoxirribonucleases/genética , Feminino , Técnicas Genéticas , Vetores Genéticos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microinjeções , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto , Gravidez , Homologia de Sequência do Ácido Nucleico , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Tyrosine hydroxylase (TH)-immunoreactive (ir) neurons have been found in the striatum after dopamine depletion; however, little is known about the mechanism underlying their appearance or their functional significance. We previously showed an increase in striatal TH-ir neurons after L-DOPA treatment in mice with unilateral 6-OHDA lesions in the striatum. In the present study, we further examined the time-course and persistence of the effects of chronic L-DOPA treatment on the appearance and regulation of TH-ir neurons as well as their possible function. We found that the L-DOPA-induced increase in striatal TH-ir neurons is dose-dependent and persists for days after L-DOPA withdrawal, decreasing significantly 10 days after L-DOPA treatment ends. Using hemiparkinsonian D1 receptor knock-out (D1R-/-) and D2 receptor knock-out (D2R-/-) mice, we found that the D1R, but not the D2R, is required for the L-DOPA-induced appearance of TH-ir neurons in the dopamine-depleted striatum. Interestingly, our experiments in aphakia mice, which lack Pitx3 expression in the brain, indicate that the L-DOPA-dependent increase in the number of TH-ir neurons is independent of Pitx3, a transcription factor necessary for the development of mesencephalic dopaminergic neurons. To explore the possible function of L-DOPA-induced TH-ir neurons in the striatum, we examined dopamine overflow and forelimb use in L-DOPA-treated parkinsonian mice. These studies revealed a tight spatio-temporal correlation between the presence of striatal TH-ir neurons, the recovery of electrically stimulated dopamine overflow in the lesioned striatum, and the recovery of contralateral forelimb use with chronic L-DOPA treatment. Our results suggest that the presence of TH-ir neurons in the striatum may underlie the long-duration response to L-DOPA following withdrawal. Promotion of these neurons in the early stages of Parkinson's disease, when dopamine denervation is incomplete, may be beneficial for maintaining motor function.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopaminérgicos/farmacologia , Levodopa/farmacologia , Neurônios/efeitos dos fármacos , Transtornos Parkinsonianos/metabolismo , Animais , Corpo Estriado/metabolismo , Dopamina/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool.
Assuntos
Embrião de Mamíferos , Marcação de Genes , Mutação , Animais , Sequência de Bases , Predisposição Genética para Doença , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Gene targeting by sequence-specific nucleases in one-cell embryos provides an expedited mutagenesis approach in rodents. This technology has been recently established to create knockout and knockin mutants through sequence deletion or sequence insertion. This article provides protocols for the preparation and microinjection of nuclease mRNA and targeting vector DNA into fertilized mouse eggs. Furthermore, we provide guidelines for genotyping the desired mouse mutants. Curr. Protoc. Mouse Biol. 2:347-364 © 2012 by John Wiley & Sons, Inc.
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Associative learning depends on multiple cortical and subcortical structures, including striatum, hippocampus, and amygdala. Both glutamatergic and dopaminergic neurotransmitter systems have been implicated in learning and memory consolidation. While the role of glutamate is well established, the role of dopamine and its receptors in these processes is less clear. In this study, we used two models of dopamine D(1) receptor (D(1)R, Drd1a) loss, D(1)R knock-out mice (Drd1a(-/-)) and mice with intrahippocampal injections of Drd1a-siRNA (small interfering RNA), to study the role of D(1)R in different models of learning, hippocampal long-term potentiation (LTP) and associated gene expression. D(1)R loss markedly reduced spatial learning, fear learning, and classical conditioning of the eyelid response, as well as the associated activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. These results provide the first experimental demonstration that D(1)R is required for trace eyeblink conditioning and associated changes in synaptic strength in hippocampus of behaving mice. Drd1a-siRNA mice were indistinguishable from Drd1a(-/-) mice in all experiments, indicating that hippocampal knockdown was as effective as global inactivation and that the observed effects are caused by loss of D(1)R and not by indirect developmental effects of Drd1a(-/-). Finally, in vivo LTP and LTP-induced expression of Egr1 in the hippocampus were significantly reduced in Drd1a(-/-) and Drd1a-siRNA, indicating an important role for D(1)R in these processes. Our data reveal a functional relationship between acquisition of associative learning, increase in synaptic strength at the CA3-CA1 synapse, and Egr1 induction in the hippocampus by demonstrating that all three are dramatically impaired when D(1)R is eliminated or reduced.
Assuntos
Aprendizagem por Associação/fisiologia , Hipocampo/metabolismo , Plasticidade Neuronal/genética , Receptores de Dopamina D1/deficiência , Receptores de Dopamina D1/genética , Transmissão Sináptica/genética , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/fisiopatologia , Região CA3 Hipocampal/metabolismo , Região CA3 Hipocampal/fisiopatologia , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica/genética , Hipocampo/fisiopatologia , Potenciação de Longa Duração/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/genética , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologiaRESUMO
Recent evidence suggests that glutamatergic and dopaminergic afferents must be activated to induce persistent long-term potentiation (LTP) in the hippocampus. Whereas extensive evidence supports the role of glutamate receptors in long-lasting synaptic plasticity and spatial learning and memory, there is less evidence regarding the role of dopamine receptors in these processes. Here, we used dopamine D(1) receptor knockout (D(1)R(-/-)) mice to explore the role of D(1)R in hippocampal LTP and its associated gene expression. We show that the magnitude of early and late phases of LTP (E-LTP and L-LTP) was markedly reduced in hippocampal slices from D(1)R(-/-) mice compared with wild-type mice. SCH23390, a D(1)/D(5)R antagonist, did not further reduce L-LTP in D(1)R(-/-) mice, suggesting that D(5)Rs are not involved. D(1)R(-/-) mice also showed a significant reduction of D(1)R-induced potentiation of N-Methyl-D-aspartic acid-mediated currents, via protein kinase activated by cyclic adenosine 3',5'-monophosphate activation. Finally, LTP-induced expression of the immediate early genes zif268 and arc in the hippocampal CA1 area was abolished in D(1)R(-/-) mice, and these mice showed impaired learning. These results indicate that D(1)R but not D(5)R are critical for hippocampal LTP and for the induction of Zif268 and Arc, proteins required for the transition from E-LTP to L-LTP and for memory consolidation in mammals.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Animais , Aprendizagem/fisiologia , Camundongos , Percepção Espacial/fisiologiaRESUMO
Interactions between the dopaminergic and glutamatergic neurotransmission systems were investigated in the adult brain of wild-type (WT) and transgenic mice lacking the dopamine D(1) or D(2) receptor subtypes. Activity of the glutamine cycle was evaluated by using (13)C NMR spectroscopy, and striatal activity was assessed by c-Fos expression and motor coordination. Brain extracts from (1,2-(13)C(2)) acetate-infused mice were prepared and analyzed by (13)C NMR to determine the incorporation of the label into the C4 and C5 carbons of glutamate and glutamine. D(1)R(-/-) mice showed a significantly higher concentration of cerebral (4,5-(13)C(2)) glutamine, consistent with an increased activity of the glutamate-glutamine cycle and of glutamatergic neurotransmission. Conversely, D(2)R(-/-) mice did not show any significant changes in (4,5-(13)C(2)) glutamate or (4,5-(13)C(2)) glutamine, suggesting that alterations in glutamine metabolism are mediated through D(1) receptors. This was confirmed with D(1)R(-/-) and WT mice treated with reserpine, a dopamine-depleting drug, or with reserpine followed by L-DOPA, a dopamine precursor. Exposure to reserpine increased (4,5-(13)C(2)) glutamine in WT to levels similar to those found in untreated D(1)R(-/-) mice. These values were the same as those reached in the reserpine-treated D(1)R(-/-) mice. Treatment of WT animals with L-DOPA returned (4,5-(13)C(2)) glutamine levels to normal, but this was not verified in D(1)R(-/-) animals. Reserpine impaired motor coordination and decreased c-Fos expression, whereas L-DOPA restored both variables to normal values in WT but not in D(1)R(-/-). Together, our results reveal novel neurometabolic interactions between glutamatergic and dopaminergic systems that are mediated through the D(1), but not the D(2), dopamine receptor subtype.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Neurotransmissores/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Imuno-Histoquímica , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Transgênicos , Atividade Motora/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.