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Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.

Yamasaki N, Maekawa M, Kobayashi K, Kajii Y, Maeda J, Soma M, Takao K, Tanda K, Ohira K, Toyama K, Kanzaki K, Fukunaga K, Sudo Y, Ichinose H, Ikeda M, Iwata N, Ozaki N, Suzuki H, Higuchi M, Suhara T, Yuasa S, Miyakawa T - Mol Brain (2008)

Bottom Line: Transcriptome analysis of the hippocampus of these mutants revealed that the expression levels of more than 2000 genes were significantly changed.Moreover, c-Fos expression in the DG after electric footshock was almost completely and selectively abolished in the mutants.Nearly half of the differentially-expressed probes in the schizophrenia-enriched cluster encoded genes that are involved in neurogenesis or in neuronal migration/maturation, including calbindin, a marker for mature DG neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Frontier Technology Center, Kyoto University Graduate School of Medicine, Japan. yamasaki@kuhp.kyoto-u.ac.jp

ABSTRACT
Elucidating the neural and genetic factors underlying psychiatric illness is hampered by current methods of clinical diagnosis. The identification and investigation of clinical endophenotypes may be one solution, but represents a considerable challenge in human subjects. Here we report that mice heterozygous for a mutation of the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII+/-) have profoundly dysregulated behaviours and impaired neuronal development in the dentate gyrus (DG). The behavioral abnormalities include a severe working memory deficit and an exaggerated infradian rhythm, which are similar to symptoms seen in schizophrenia, bipolar mood disorder and other psychiatric disorders. Transcriptome analysis of the hippocampus of these mutants revealed that the expression levels of more than 2000 genes were significantly changed. Strikingly, among the 20 most downregulated genes, 5 had highly selective expression in the DG. Whereas BrdU incorporated cells in the mutant mouse DG was increased by more than 50 percent, the number of mature neurons in the DG was dramatically decreased. Morphological and physiological features of the DG neurons in the mutants were strikingly similar to those of immature DG neurons in normal rodents. Moreover, c-Fos expression in the DG after electric footshock was almost completely and selectively abolished in the mutants. Statistical clustering of human post-mortem brains using 10 genes differentially-expressed in the mutant mice were used to classify individuals into two clusters, one of which contained 16 of 18 schizophrenic patients. Nearly half of the differentially-expressed probes in the schizophrenia-enriched cluster encoded genes that are involved in neurogenesis or in neuronal migration/maturation, including calbindin, a marker for mature DG neurons. Based on these results, we propose that an "immature DG" in adulthood might induce alterations in behavior and serve as a promising candidate endophenotype of schizophrenia and other human psychiatric disorders.

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Immature DG of Alpha-CaMKII+/- Mice. (A) The number of BrdU-positive cells in the subgranular zone of the DG is increased in the alpha-CaMKII +/- mice at 7 weeks old, as observed 1 d after BrdU administration. The number of BrdU-positive cells within the DG was 52.8% higher (p = 0.00001) in the alpha-CaMKII +/- mice than in the wild-type mice. (B) In alpha-CaMKII+/- mice, the number of cells expressing calbindin (a mature-neuron marker) was decreased. (C) In alpha-CaMKII+/- mice, the number of cells expressing PSA-NCAM (a late-progenitor and immature-neuron marker) and calretinin (an immature-neuron marker) was markedly increased. CaMKII was co-expressed in a subset of PSA-NCAM-, calretinin-, and calbindin-expressing cells, indicated by the white arrowheads. For the analysis of expression of markers related to neurogenesis and differentiation, at least five mice were examined in each group. (D) Rapid Golgi staining showed that the number of Golgi-impregnated cells was decreased in the DG of alpha-CaMKII+/- mice (see Additional file 1, Figure S9) and that Golgi-impregnated cells in the DG of alpha-CaMKII+/- mice had less branching and shorter dendrites. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars indicate s.e.m.
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Figure 3: Immature DG of Alpha-CaMKII+/- Mice. (A) The number of BrdU-positive cells in the subgranular zone of the DG is increased in the alpha-CaMKII +/- mice at 7 weeks old, as observed 1 d after BrdU administration. The number of BrdU-positive cells within the DG was 52.8% higher (p = 0.00001) in the alpha-CaMKII +/- mice than in the wild-type mice. (B) In alpha-CaMKII+/- mice, the number of cells expressing calbindin (a mature-neuron marker) was decreased. (C) In alpha-CaMKII+/- mice, the number of cells expressing PSA-NCAM (a late-progenitor and immature-neuron marker) and calretinin (an immature-neuron marker) was markedly increased. CaMKII was co-expressed in a subset of PSA-NCAM-, calretinin-, and calbindin-expressing cells, indicated by the white arrowheads. For the analysis of expression of markers related to neurogenesis and differentiation, at least five mice were examined in each group. (D) Rapid Golgi staining showed that the number of Golgi-impregnated cells was decreased in the DG of alpha-CaMKII+/- mice (see Additional file 1, Figure S9) and that Golgi-impregnated cells in the DG of alpha-CaMKII+/- mice had less branching and shorter dendrites. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars indicate s.e.m.

Mentions: These marked changes in behavior led us to examine the brains of the alpha-CaMKII mutants. In the mutants, the amount of alpha CaMKII was decreased by 20% to 60% in the cingulate cortex, amygdala, and hippocampus, and the amount of phosphorylated beta-CaMKII was increased in the hippocampus, which is probably a compensatory effect. The amounts of proteins, such as CN and phosphorylated extracellular signal-regulated kinase, were also altered (see Additional file 1, Figure S2). There were no dramatic differences in monoamine content in any region investigated, except for an increase in striatal dopamine turnover in the mutant mice (see Additional file 1, Figure S3). Next, we analyzed the transcriptome of the hippocampus, a region that has an essential role in working memory and locomotor activity in rodents, of alpha-CaMKII+/- mice, by using Affymetrix GeneChips. There were more than 2000 genes that were significantly up- or downregulated (see Additional file 2, Table S1 and S2). There was a more than two-fold increase in the gene expression levels of dopamine D1A receptors, therefore we performed autoradiography studies for five major neurotransmitter receptors and two transporters (see Additional file 1, Figure S4 and S5). Consistent with the microarray results, dopamine D1-like receptor binding was dramatically and selectively increased in the DG of alpha-CaMKII+/- mice (Figure 2A and 2B). N-methyl-D-aspartate (NMDA) receptor binding was downregulated in the hippocampus, especially in the DG (Figure 2C and 2D). Based on the highly selective changes in receptor binding, we examined the location of genes whose expression levels were changed in the mutant brain. A database search using Allen Brain Atlas [23] revealed that, among the 20 most downregulated genes in the alpha-CaMKII +/- hippocampus, 5 genes (DSP, TDO2, NPNT, IL1R1, and PNCK) had highly selective expression in the DG (Figure 2Eā€“2J, and Additional file 1, Figure S6). Moreover, c-Fos expression in the DG 2 h after electric shock was almost completely and selectively abolished in alpha-CaMKII+/- mice, which suggests that their DG was functionally downregulated (Figure 2K, 2L and Additional file 1, Figure S7). These findings in the DG, a region in which 3000 to 4000 neurons are born each day in rodents [24], led us to examine the cell proliferation in the mutant mouse DG. BrdU-labeled cells were dramatically increased by 53% in the mutants (Figure 3A). Consistent with this finding, many of the genes involved in the brain derived neurotrophic factor ā€“ mitogen-activated protein kinase (BDNF-MAPK) pathway, which has an important role in neurogenesis [25], were significantly upregulated or downregulated in the hippocampus of the alpha-CaMKII+/- mice, including a 32 to 35% up-regulation of BDNF probes (see Additional file 1, Figure S8). Dysregulation of the expression of BDNF-MAPK pathway genes could be related to the increased cell proliferation in the mutants.


Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.

Yamasaki N, Maekawa M, Kobayashi K, Kajii Y, Maeda J, Soma M, Takao K, Tanda K, Ohira K, Toyama K, Kanzaki K, Fukunaga K, Sudo Y, Ichinose H, Ikeda M, Iwata N, Ozaki N, Suzuki H, Higuchi M, Suhara T, Yuasa S, Miyakawa T - Mol Brain (2008)

Immature DG of Alpha-CaMKII+/- Mice. (A) The number of BrdU-positive cells in the subgranular zone of the DG is increased in the alpha-CaMKII +/- mice at 7 weeks old, as observed 1 d after BrdU administration. The number of BrdU-positive cells within the DG was 52.8% higher (p = 0.00001) in the alpha-CaMKII +/- mice than in the wild-type mice. (B) In alpha-CaMKII+/- mice, the number of cells expressing calbindin (a mature-neuron marker) was decreased. (C) In alpha-CaMKII+/- mice, the number of cells expressing PSA-NCAM (a late-progenitor and immature-neuron marker) and calretinin (an immature-neuron marker) was markedly increased. CaMKII was co-expressed in a subset of PSA-NCAM-, calretinin-, and calbindin-expressing cells, indicated by the white arrowheads. For the analysis of expression of markers related to neurogenesis and differentiation, at least five mice were examined in each group. (D) Rapid Golgi staining showed that the number of Golgi-impregnated cells was decreased in the DG of alpha-CaMKII+/- mice (see Additional file 1, Figure S9) and that Golgi-impregnated cells in the DG of alpha-CaMKII+/- mice had less branching and shorter dendrites. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars indicate s.e.m.
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Figure 3: Immature DG of Alpha-CaMKII+/- Mice. (A) The number of BrdU-positive cells in the subgranular zone of the DG is increased in the alpha-CaMKII +/- mice at 7 weeks old, as observed 1 d after BrdU administration. The number of BrdU-positive cells within the DG was 52.8% higher (p = 0.00001) in the alpha-CaMKII +/- mice than in the wild-type mice. (B) In alpha-CaMKII+/- mice, the number of cells expressing calbindin (a mature-neuron marker) was decreased. (C) In alpha-CaMKII+/- mice, the number of cells expressing PSA-NCAM (a late-progenitor and immature-neuron marker) and calretinin (an immature-neuron marker) was markedly increased. CaMKII was co-expressed in a subset of PSA-NCAM-, calretinin-, and calbindin-expressing cells, indicated by the white arrowheads. For the analysis of expression of markers related to neurogenesis and differentiation, at least five mice were examined in each group. (D) Rapid Golgi staining showed that the number of Golgi-impregnated cells was decreased in the DG of alpha-CaMKII+/- mice (see Additional file 1, Figure S9) and that Golgi-impregnated cells in the DG of alpha-CaMKII+/- mice had less branching and shorter dendrites. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars indicate s.e.m.
Mentions: These marked changes in behavior led us to examine the brains of the alpha-CaMKII mutants. In the mutants, the amount of alpha CaMKII was decreased by 20% to 60% in the cingulate cortex, amygdala, and hippocampus, and the amount of phosphorylated beta-CaMKII was increased in the hippocampus, which is probably a compensatory effect. The amounts of proteins, such as CN and phosphorylated extracellular signal-regulated kinase, were also altered (see Additional file 1, Figure S2). There were no dramatic differences in monoamine content in any region investigated, except for an increase in striatal dopamine turnover in the mutant mice (see Additional file 1, Figure S3). Next, we analyzed the transcriptome of the hippocampus, a region that has an essential role in working memory and locomotor activity in rodents, of alpha-CaMKII+/- mice, by using Affymetrix GeneChips. There were more than 2000 genes that were significantly up- or downregulated (see Additional file 2, Table S1 and S2). There was a more than two-fold increase in the gene expression levels of dopamine D1A receptors, therefore we performed autoradiography studies for five major neurotransmitter receptors and two transporters (see Additional file 1, Figure S4 and S5). Consistent with the microarray results, dopamine D1-like receptor binding was dramatically and selectively increased in the DG of alpha-CaMKII+/- mice (Figure 2A and 2B). N-methyl-D-aspartate (NMDA) receptor binding was downregulated in the hippocampus, especially in the DG (Figure 2C and 2D). Based on the highly selective changes in receptor binding, we examined the location of genes whose expression levels were changed in the mutant brain. A database search using Allen Brain Atlas [23] revealed that, among the 20 most downregulated genes in the alpha-CaMKII +/- hippocampus, 5 genes (DSP, TDO2, NPNT, IL1R1, and PNCK) had highly selective expression in the DG (Figure 2Eā€“2J, and Additional file 1, Figure S6). Moreover, c-Fos expression in the DG 2 h after electric shock was almost completely and selectively abolished in alpha-CaMKII+/- mice, which suggests that their DG was functionally downregulated (Figure 2K, 2L and Additional file 1, Figure S7). These findings in the DG, a region in which 3000 to 4000 neurons are born each day in rodents [24], led us to examine the cell proliferation in the mutant mouse DG. BrdU-labeled cells were dramatically increased by 53% in the mutants (Figure 3A). Consistent with this finding, many of the genes involved in the brain derived neurotrophic factor ā€“ mitogen-activated protein kinase (BDNF-MAPK) pathway, which has an important role in neurogenesis [25], were significantly upregulated or downregulated in the hippocampus of the alpha-CaMKII+/- mice, including a 32 to 35% up-regulation of BDNF probes (see Additional file 1, Figure S8). Dysregulation of the expression of BDNF-MAPK pathway genes could be related to the increased cell proliferation in the mutants.

Bottom Line: Transcriptome analysis of the hippocampus of these mutants revealed that the expression levels of more than 2000 genes were significantly changed.Moreover, c-Fos expression in the DG after electric footshock was almost completely and selectively abolished in the mutants.Nearly half of the differentially-expressed probes in the schizophrenia-enriched cluster encoded genes that are involved in neurogenesis or in neuronal migration/maturation, including calbindin, a marker for mature DG neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Frontier Technology Center, Kyoto University Graduate School of Medicine, Japan. yamasaki@kuhp.kyoto-u.ac.jp

ABSTRACT
Elucidating the neural and genetic factors underlying psychiatric illness is hampered by current methods of clinical diagnosis. The identification and investigation of clinical endophenotypes may be one solution, but represents a considerable challenge in human subjects. Here we report that mice heterozygous for a mutation of the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII+/-) have profoundly dysregulated behaviours and impaired neuronal development in the dentate gyrus (DG). The behavioral abnormalities include a severe working memory deficit and an exaggerated infradian rhythm, which are similar to symptoms seen in schizophrenia, bipolar mood disorder and other psychiatric disorders. Transcriptome analysis of the hippocampus of these mutants revealed that the expression levels of more than 2000 genes were significantly changed. Strikingly, among the 20 most downregulated genes, 5 had highly selective expression in the DG. Whereas BrdU incorporated cells in the mutant mouse DG was increased by more than 50 percent, the number of mature neurons in the DG was dramatically decreased. Morphological and physiological features of the DG neurons in the mutants were strikingly similar to those of immature DG neurons in normal rodents. Moreover, c-Fos expression in the DG after electric footshock was almost completely and selectively abolished in the mutants. Statistical clustering of human post-mortem brains using 10 genes differentially-expressed in the mutant mice were used to classify individuals into two clusters, one of which contained 16 of 18 schizophrenic patients. Nearly half of the differentially-expressed probes in the schizophrenia-enriched cluster encoded genes that are involved in neurogenesis or in neuronal migration/maturation, including calbindin, a marker for mature DG neurons. Based on these results, we propose that an "immature DG" in adulthood might induce alterations in behavior and serve as a promising candidate endophenotype of schizophrenia and other human psychiatric disorders.

Show MeSH
Related in: MedlinePlus