Hippocampal CA1-region-restricted knockout of NMDAR1 gene disrupts synaptic plasticity, place fields, and spatial learning.
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Animals↗
Biomedical subjects
S Tonegawa
Publications and source records attributed to S Tonegawa.
Impaired synapse elimination during cerebellar development in PKC gamma mutant mice.
PKC gamma is highly expressed in Purkinje cells (PCs) but not in other types of neurons in the cerebellum. The expression of PKC gamma changes markedly during cerebellar development, being very low at birth and reaching a peak around the third postnatal week. This temporal pattern of PKC gamma expression coincides with the developmental transition from multiple to single climbing fiber innervation onto each PC. In adult mutant mice deficient in PKC gamma, we found that 41% of PCs are still innervated by multiple climbing fibers, while other aspects of the cerebellum including the morphology and excitatory synaptic transmission of PCs appear normal. Thus, elimination of multiple climbing fiber innervation appears to be specifically impaired in the mutant cerebellum. We suggest that the developmental role of PKC gamma may be to act as a downstream element in the signal cascade necessary for the elimination of surplus climbing fiber synapses.
Age Factors↗
Impaired motor coordination correlates with persistent multiple climbing fiber innervation in PKC gamma mutant mice.
It is generally believed that a smooth execution of a compound movement, or motor coordination, requires learning of component movements as well as experience-based refinement of the motor program as a whole. PKC gamma mutant mice display impaired motor coordination but intact eyeblink conditioning, a form of component movement learning. Cerebellar long-term depression, a putative cellular mechanism for component motor learning, is also unimpaired. Thus, PKC gamma mutant mice are defective in refinement of the motor program. In the accompanying paper, we demonstrate that innervation of multiple climbing fibers onto Purkinje cells persists in adulthood in these mutant mice. We propose that this defective elimination of surplus climbing fibers underlies motor discoordination.
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Ontogenic development and tissue distribution of V gamma 1-expressing gamma/delta T lymphocytes in normal mice.
A hamster monoclonal antibody (mAb) recognizing an epitope in the V gamma 1-J gamma 4-C gamma 4 chain of the gamma/delta T cell receptor has been generated. Using this mAb, we have quantitated the occurrence of V gamma 1-bearing gamma/delta T cells in the developing thymus and in the lymphoid organs and several epithelia of adult mice. The V gamma 1-expressing cells constitute a minor gamma/delta T cell subpopulation during fetal and early postnatal life, but they constitute a major population of gamma/delta T cells in the thymus and in the peripheral lymphoid organs in adult mice. In addition, we found that V gamma 1-bearing cells comprise a large proportion (15-60%) of the gamma/delta T cells present in the intestinal epithelium (i-IEL) in all strains of mice tested. V gamma 1+ i-IEL are present in athymic (nude) mice and in antigen-free mice, demonstrating that they can develop extrathymically and that their presence in the intestinal epithelium is independent of the antigenic load of the gut. Our results show that V gamma 1-bearing lymphocytes account for the largest population of gamma/delta T cells in the mouse. This population includes a thymus-dependent component that homes to the secondary lymphoid organs and a thymus-independent component that constitutes a major fraction of the gamma/delta i-IELs.
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Strictly transporter of antigen presentation (TAP)-dependent presentation of an immunodominant cytotoxic T lymphocyte epitope in the signal sequence of a virus protein.
Peptides presented by major histocompatibility complex (MHC) class I molecules are derived from intracellularly synthesized proteins. Cytosolic proteins are fragmented into peptides, which are subsequently transported via the transporter of antigen presentation (TAP) into the endoplasmic reticulum (ER), where they bind to MHC class I molecules. We have investigated the requirements for MHC class I presentation of the immunodominant gp33 cytotoxic T lymphocyte epitope of the lymphocytic choriomeningitis virus. This epitope is located within the leader peptide of the virus glycoprotein. Such an epitope is expected to be presented in a TAP-independent manner, since it is released into the ER by signal peptidase. Taking advantage of TAP1-/- mice, however, we show both in vitro and in vivo that, after virus infection, the presentation of the gp33 epitope is strictly dependent on a functional TAP heterodimer. The results are discussed with respect to peptide trimming processes in the ER.
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Characterization of immature thymocyte lines derived from T-cell receptor or recombination activating gene 1 and p53 double mutant mice.
The T-cell receptor (TCR) beta chain is instrumental in the progression of thymocyte differentiation from the CD4-CD8- to the CD4+CD8+ stage. This differentiation step may involve cell surface expression of novel CD3-TCR complexes. To facilitate biochemical characterization of these complexes, we established cell lines from thymic lymphomas originating from mice carrying a mutation in the p53 gene on the one hand and a mutation in TCR-alpha, TCR-beta, or the recombination activating gene 1 (RAG-1) on the other hand. The cell lines were CD4+CD8+ and appeared to be monoclonal. A cell line derived from a RAG-1 x p53 double mutant thymic lymphoma expressed low levels of CD3-epsilon, -gamma, and -delta on the surface. TCR-alpha x p53 double mutant cell lines were found to express complexes consisting of TCR-beta chains associated with CD3-epsilon, -gamma, and -delta chains and CD3-zeta zeta dimers. These lines will be useful tools to study the molecular structure and signal transducing properties of partial CD3-TCR complexes expressed on the surface of immature thymocytes.
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Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.
Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory.
Animals↗
Mammalian learning and memory studied by gene targeting.
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Animals↗
Homeostatic regulation of intestinal epithelia by intraepithelial gamma delta T cells.
Although T cells bearing gamma delta T-cell receptors have long been known to be present in the epithelial lining of many organs, their specificity and function remain elusive. In the present study, we examined the intestinal epithelia of T-cell-receptor mutant mice, which were deficient in either gamma delta T cells or alpha beta T cells, and of normal littermates. The absence of gamma delta T cells was associated with a reduction in epithelial cell turnover and a downregulation of the expression of major histocompatibility complex class II molecules. No such effects were observed in alpha beta T-cell-deficient mice. These findings indicate that intraepithelial gamma delta T cells regulate the generation and differentiation of intestinal epithelial cells.
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Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.
Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.
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Functional analysis of the TCR alpha- beta+ cells that accumulate in the pneumonic lung of influenza virus-infected TCR-alpha-/- mice.
In mice homozygous (-/-) for a targeted TCR-alpha gene disruption, some thymocytes express a cell-surface TCR-beta chain on the cell surface in the absence of a TCR-alpha chain, and a few CD4+CD8- TCR-alpha-beta+ cells accumulate in the peripheral lymphoid organs. We have infected these mutant mice with an influenza A virus to show that large numbers of TCR-beta+ cells (most of which are CD4+) can be retrieved from the pneumonic lung. Both freshly isolated TCR-alpha-beta+ cells and TCR-alpha-beta+ hybridoma cell lines derived from influenza virus-infected mutant mice respond appropriately to stimulation with anti-CD3 epsilon or the Mls-1 superantigen. It thus seems that CD4+ TCR-alpha-beta+ cells in the peripheral lymphoid organs of TCR-alpha mutant mice can signal through their TCR surface complex. However, there are no indications that CD4+ TCR-alpha-beta+ lymphocytes can either recognize a complex between MHC and influenza virus peptide or act as effector or Th cells. The existence and function of such cells in wild-type mice remains to be established.
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Increase in positive selection of CD8+ T cells in TAP1-mutant mice by human beta 2-microglobulin transgene.
Mice harboring a deletion of the gene encoding the transporter associated with antigen presentation-1 (TAP1) are impaired in providing major histocompatibility complex (MHC) class I molecules with peptides of cytosolic origin and lack stable MHC class I cell surface expression. They consequently have a strongly reduced number of CD8+ T cells. To examine whether selection of CD8+ T cells is dependent on TAP-dependent peptides, we partially restored MHC class I cell surface expression in TAP1-deficient mice by introduction of human beta 2-microglobulin. We show that selection of functional CD8+ T cells can be augmented in vivo in the absence of TAP1-dependent peptides.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Differential reactivity of residual CD8+ T lymphocytes in TAP1 and beta 2-microglobulin mutant mice.
TAP1 -/- and beta 2-microglobulin (beta 2m) -/- mice (H-2b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8+ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8+ T cells than beta 2m -/- mice. Alloreactive CD8+ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in beta 2m -/- mice. Alloreactive CTL generated in TAP1 -/- and beta 2m -/- mice cross-react with H-2b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from beta 2m -/- mice than with similar cells from TAP1 -/- mice. The beta 2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2b MHC class I molecules. Such H-2b-reactive CD8+ CTL from beta 2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2b-reactive CD8+ CTL from neither beta 2m -/- mice nor TAP1 -/- mice killed beta 2m -/- cells. In line with these results, beta 2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8+ T cells differs between TAP1 -/- and beta 2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and beta 2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8+ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Contribution of alpha/beta and gamma/delta T lymphocytes to immunity against Mycobacterium bovis bacillus Calmette Guérin: studies with T cell receptor-deficient mutant mice.
Mutant mice with defined T cell deficiencies were infected with Mycobacterium bovis bacillus Calmette Guérin (BCG) and the relative contribution of alpha/beta T cells and gamma/delta T cells to the host immune response was assessed. Recombinase activating gene (RAG-1)-/- mutants as well as T cell receptor (TcR) beta-/-, but not TcR-delta-/-, mutants succumbed to M. bovis BCG infection and failed to develop granulomatous lesions. Antigen-induced IFN-gamma production by spleen cells in vitro was abrogated in RAG-1-/- mutants and markedly diminished in TcR-beta-/- and TcR-delta-/- mice. Reconstitution experiments suggest that both alpha/beta and gamma/delta T cells are essential for antigen-specific IFN-gamma secretion. Our data formally prove the crucial role of alpha/beta T cells and reveal accessory functions of gamma/delta T cells in optimum immunity against M. bovis BCG.
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TAP1-independent loading of class I molecules by exogenous viral proteins.
Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1-/-) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glyco- and nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1-/- cells as by control cells. Peptide regurgitation was not involved. Since particulate, non-replicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.
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Presentation of endogenous viral proteins in association with major histocompatibility complex class II: on the role of intracellular compartmentalization, invariant chain and the TAP transporter system.
Major histocompatibility complex (MHC) class II-associated antigen presentation is mainly linked to processing of exogenous antigens upon cellular uptake by endocytosis, but has also been observed for endogenously synthesized antigens. We have studied the MHC class II-associated presentation of the endogenously synthesized membrane associated glycoprotein (GP) and the cytosolic nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in professional antigen presenting cells (APC) of mice. Since LCMV is a noncytopathic virus and minimally affects cellular protein synthesis, it is a convenient virus for the study of antigen presentation. In contrast, most other studies assessing class II-associated presentation of endogeneously synthesized viral antigens used cytolytic viruses such as vaccinia, measles and influenza virus, which drastically interfere with host cell functions. In addition, most studies were performed using non-professional APC. We found that class II-associated presentation of endogenously synthesized membrane associated LCMV-GP was efficient and could not be inhibited by chloroquine or leupeptin. Neither the transporter associated with processing (TAP) system nor the invariant chain (Ii) were significantly involved in this process. In contrast, MHC class II-associated presentation of endogenously synthesized cytosolic LCMV-NP was not observed even in Ii-deficient APC. Thus, MHC class II loading of endogenously synthesized LCMV-GP apparently does not require processing in acidic endosomal compartments as defined by chloroquine and leupeptin insensitivity. Furthermore, although the TAP molecules transport peptides of up to 15 amino acids in length, which potentially could bind to MHC class II molecules in the endoplasmic reticulum, such a process apparently does not occur for either the glycoprotein or the nucleoprotein. Therefore, the subcellular localization of an endogenously synthesized protein influences crucially whether or not MHC class II loading can occur independently of the acidic compartments usually involved in MHC class II loading.
ATP-Binding Cassette Transporters↗
Evidence against a role for metabotropic glutamate receptors in mossy fiber LTP: the use of mutant mice and pharmacological antagonists.
We have used a number of approaches to address a possible role of metabotropic glutamate receptors (mGluRs) in mossy fiber long-term potentiation (LTP) in the hippocampus. We have used two types of mutant mice--one lacking the mGluR1 subtype of receptor and one lacking the gamma isoform of protein kinase C. In neither type of mouse did we find any alteration in the magnitude of mossy fiber LTP. We next examined whether mGluRs might modulate the magnitude and/or threshold for the induction of mossy fiber LTP. In these experiments we used tetani that were either just subthreshold or just suprathreshold for generating LTP. The mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] did not convert a subthreshold tetanus into a suprathreshold tetanus, nor did (+)MCPG have any effect on the small amount of LTP that was generated by a just suprathreshold tetanus. Based on our studies, we have been unable to identify a role for mGluRs in mossy fiber LTP.
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Hippocampal long-term potentiation is normal in heme oxygenase-2 mutant mice.
We have generated mice deficient in HO-2, the major cerebral isoform of heme oxygenase, in order to assess the potential role of carbon monoxide as a retrograde messenger in hippocampal LTP. Cerebral HO catalytic activity was markedly reduced in the HO-2 mutant mice, yet no differences were found between wild types and mutants in gross neuroanatomical structure, in basal hippocampal synaptic transmission, or in the amount of potentiation produced by various LTP induction protocols. Furthermore, zinc protoporphyrin IX, an inhibitor of HO, had nearly identical inhibitory effects on LTP in wild-type and HO-2 mutant hippocampal slices. Our data indicate that carbon monoxide produced endogenously by HO is unlikely to be a neuromodulator required for LTP in the hippocampus.
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