RESUMO
NMDA receptors in mice were mutated by gene targeting to substitute asparagine (N) in position 598 of the NR1 subunit to glutamine (Q) or arginine (R). Animals expressing exclusively the mutated NR1 alleles, NR1(Q/Q) and NR1(-/R) mice, developed a perinatally lethal phenotype mainly characterized by respiratory failure. The dysfunctions were partially rescued in heterozygous mice by the presence of pure wild-type receptors. Thus, NR1(+/Q) mice exhibited reduced life expectancy, with females being impaired in nurturing; NR1(+/R) mice displayed signs of underdevelopment such as growth retardation and impaired righting reflex, and died before weaning. We analyzed the key properties of NMDA receptors, high Ca(2+) permeability, and voltage-dependent Mg(2+) block, in the mutant mice. Comparison of the complex physiological and phenotypical changes observed in the different mutants indicates that properties controlled by NR1 subunit residue N598 are important for autonomic brain functions at birth and during postnatal development. We conclude that disturbed NMDA receptor signaling mediates a variety of neurological phenotypes.
Assuntos
Mutação Puntual , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologia , Alelos , Animais , Cálcio/metabolismo , Homozigoto , Potenciação de Longa Duração , Magnésio/metabolismo , Camundongos , Neocórtex/crescimento & desenvolvimento , Fenótipo , Insuficiência Respiratória/genéticaRESUMO
This manuscript summarizes mouse mutants for ionotropic glutamate receptors that were generated by different laboratories to analyze the function of the NMDA and AMPA receptors in the mouse. Thus, NMDA receptor mutant mice that were generated by the "knock-in" technology demonstrate that the NR1 and the NR2B subunits participate in the formation of NMDA receptors that are involved in vital functions like breathing and suckling of a newborn mouse. Mice that lack NR2A, -2C, and -2D subunits were described to be viable and have been used to study the role of NMDA receptors in adult mice. The depletion of the GluR-B subunit revealed an NMDA receptor-independent form of long-term potentiation (LTP). This AMPA receptor-mediated LTP at CA3/CA1 synapses was also observed in mice that carry an editing-deficient GluR-B allele even though these mice die prematurely after heavy epileptic seizures. In other mutants, the intracellular COOH-terminal domain of the NMDA receptor was truncated; and when compared to NMDA receptor "knock-out" mice, a functional knock-out of the NMDA receptor was observed. However, in the synapses of NR2AC/AC mutants, gatable NMDA receptors were synaptically activated, indicating that the knock-out phenotypes mediated by the COOH-terminally truncated NMDA receptors appear to reflect defective intracellular signaling.
Assuntos
Receptores de AMPA/genética , Receptores de Glutamato/genética , Receptores de N-Metil-D-Aspartato/genética , Animais , Deleção de Genes , Regulação da Expressão Gênica , Potenciação de Longa Duração/genética , Camundongos , Camundongos Knockout , Mutação , Edição de RNA , Sinapses/metabolismoRESUMO
This manuscript summarizes our recent attempts to regulate in vitro and in vivo the expression of genes encoding components and regulators of the postsynaptic machinery along with marker genes such as lacZ and GFP. In particular, we studied tTA-dependent regulation and utilized Cre in combination with reversible silencing by intron engineering of dominant negative alleles. We further present a "knockin" approach for on-site artificial regulation of chromosomal genes.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Virais , Animais , Encéfalo/citologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Genes Reporter/genética , Integrases/genética , Camundongos , Camundongos Transgênicos , Recombinação Genética , Transmissão Sináptica/genética , Tetraciclina/farmacologia , Transativadores/genéticaRESUMO
Glutamate receptor channels are built around an ancient pore loop structure which defines the inner channel environment and which is connected to structures for channel gating. This pore loop, which corresponds to the M2 region of the receptor subunits, enters the lipid bilayer from the intracellular side in an alpha-helical configuration, then kinks to form a random coil and exits the lipid bilayer at the intracellular side. The narrow constriction of the channel is formed by amino acid residues that occupy a position shortly after the end of the alpha-helical part of M2. These residues determine ion selectivity and conductance properties of the glutamate-gated channel. The critical residues are asparagines for NMDA receptor subunits and glutamine or arginine for AMPA and kainate receptor subunits. Presence of arginine in the critical channel position of AMPA and kainate receptors is controlled by site-selective RNA editing. To study the importance of these critical channel residues in the mouse, we introduced codon changes in the endogenous genes for NMDA and AMPA receptor subunits. Our results show that changes in the critical channel position are not tolerated, but lead to early death. Therefore, the impact on adult synaptic function and plasticity by glutamate receptor channels with changed ion selectivity and conductance needs to be addressed by conditional expression of the mutant receptors.
Assuntos
Química Encefálica , Canais de Cálcio/genética , Ativação do Canal Iônico/fisiologia , Transporte de Íons/fisiologia , Proteínas do Tecido Nervoso/genética , Receptores de Glutamato/genética , Adenosina/química , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Códon/genética , Ácido Glutâmico/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Conformação Proteica/efeitos dos fármacos , Subunidades Proteicas , Edição de RNA , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Receptores de Ácido Caínico/efeitos dos fármacos , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-AtividadeRESUMO
Novel spatially restricted genetic manipulations can be used to assess contributions made by synaptic plasticity to learning and memory, not just selectively within the hippocampus, but even within specific hippocampal subfields. Here we generated genetically modified mice (NR1(deltaDG) mice) exhibiting complete loss of the NR1 subunit of the N-methyl-D-aspartate receptor specifically in the granule cells of the dentate gyrus. There was no evidence of any reduction in NR1 subunit levels in any of the other hippocampal subfields, or elsewhere in the brain. NR1(deltaDG) mice displayed severely impaired long-term potentiation (LTP) in both medial and lateral perforant path inputs to the dentate gyrus, whereas LTP was unchanged in CA3-to-CA1 cell synapses in hippocampal slices. Behavioural assessment of NR1(deltaDG) mice revealed a spatial working memory impairment on a three-from-six radial arm maze task despite normal hippocampus-dependent spatial reference memory acquisition and performance of the same task. This behavioural phenotype resembles that of NR1(deltaCA3) mice but differs from that of NR1(deltaCA1) mice which do show a spatial reference memory deficit, consistent with the idea of subfield-specific contributions to hippocampal information processing. Furthermore, this pattern of selective functional loss and sparing is the same as previously observed with the global GluR-A L-alpha-amino-3-hydroxy-5-methyl-4-isoxazelopropionate receptor subunit knockout, a mutation which blocks the expression of hippocampal LTP. The present results show that dissociations between spatial working memory and spatial reference memory can be induced by disrupting synaptic plasticity specifically and exclusively within the dentate gyrus subfield of the hippocampal formation.
Assuntos
Giro Denteado/fisiologia , Memória de Curto Prazo/fisiologia , Rememoração Mental/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Percepção Espacial/fisiologia , Animais , Comportamento Animal/fisiologia , Eletrofisiologia , Imuno-Histoquímica , Hibridização In Situ , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
A functionally critical position (Q/R site) of the AMPA receptor subunit GluR-B is controlled by RNA editing that operates in the nucleus, since in brain and clonal cell lines of neural origin, unspliced GluR-B transcripts occur edited in the Q/R site CAG codon and, additionally, in intronic adenosines. Transfection of GluR-B gene constructs into PC12 cells revealed that the proximal part of the intron downstream of the unedited exonic site is required for Q/R site editing. This intron portion contains an imperfect inverted repeat preceding a 10 nt sequence with exact complementarity to the exon centered on the unedited codon. Single nucleotide substitutions in this short intronic sequence or its exonic complement curtailed Q/R site editing, which was recovered by restoring complementarity in the respective partner strand. Base conversion in the channel-coding region of GluR-B directed by base paired sequences may be executed by a ubiquitous nuclear adenosine deaminase specific for double-stranded RNA.
Assuntos
Edição de RNA , Receptores de AMPA/genética , Adenosina Desaminase/metabolismo , Animais , Sequência de Bases , Expressão Gênica , Genes , Íntrons , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Células PC12 , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 (refs 7, 8) is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. Here we have studied ADAR2-mediated RNA editing by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice was substantially reduced at most of 25 positions in diverse transcripts; the mutant mice became prone to seizures and died young. The impaired phenotype appeared to result entirely from a single underedited position, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically. The critical position specifies an ion channel determinant, the Q/R site, in AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor GluR-B pre-messenger RNA. We conclude that this transcript is the physiologically most important substrate of ADAR2.