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

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 null 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.

Figure 1

Dysregulated Behaviors of Alpha-CaMKII+/- Mice. (A, B) In the spatial working memory version of the eight-arm radial maze, the alpha-CaMKII+/- mice performed significantly worse than control mice with respect to the number of different arm choices in the first eight entries (P = 0.0022) and made significantly more revisiting errors than controls (P = 0.0026). (C) Mutant mice had normal performance in reference memory tasks in the eight-arm radial maze (P = 0.6394). Activity level of control mice (D-F) and alpha-CaMKII+/- mice (G-I) in their home cage. Mutant mice were hyperactive and showed a periodic mood-change-like activity pattern. (J) The variability in the activity in the home cage during the dark period was greater in mutant mice (coefficient of variation, P = 0.0088, Mann-Whitney U test). (K) The activity of mutant mice increased throughout the dark period (genotype effect, P < 0.0001, interaction between genotype and time, P < 0.0001, repeated measures ANOVA). Error bars indicate s.e.m.

Figure 2

Specific Abnormalities in the DG of Alpha-CaMKII+/- Mice. (A, B) Dopamine D1 receptor binding was highly increased in the DG of alpha-CaMKII+/- mice. (C, D) NMDA receptor binding was similarly downregulated in the DG and CA1 of alpha-CaMKII+/- mice. (E-J) Among the downregulated genes in the hippocampus of alpha-CaMKII+/- mice, several genes were expressed selectively in the DG (Allen Brain Atlas [23] [Internet]. Seattle, WA: Allen Institute for Brain Science. © 2004–2008. Available from: .). Graphs indicate the relative expression levels of each gene in the microarray experiment, which are confirmed by quantitative RT-PCR (see Additional file 1, Figure S6). (K, L) c-Fos expression following electric foot shock was selectively decreased in the DG of mutant mice at the age of 8 weeks. BLA, basolateral amygdaloid nucleus; MeA, medial amygdaloid nucleus; LimCX, limbic cortex; Ent, entorhinal cortex; CPU, caudate nucleus/putamen; NAC, nucleus accumbens; SNr, substantia nigra pars reticulata; CA1rad, stratum radiatum of CA1; CA1or, stratum oriens of CA1; MDT, mediodorsal thalamic nucleus; CTX, cingulated cortex; SEP, septum; OrCX, orbital cortex; TGN, tegmental nuclei; CB, cerebellum; Ncx, neocortex; LH, lateral hypothalamus; CeA, central amygdaloid nucleus. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars indicate s.e.m.

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.

Figure 4

Altered Granule Cell Excitability and Mossy Fiber Synaptic Transmission in Alpha-CaMKII+/- Mice. (A) Examples of action potential firing of granule cells in wild-type and mutant mice evoked by steady depolarizing currents (320 pA, 400 ms). (B) Increased input resistance in mutant mice (P = 0.0029). (C) Reduced spike latency in mutant mice (P < 0.0001). Depolarizing currents (320 pA) were injected into granule cells and the latency of the first action potential was measured. (D) The maximal number of spikes evoked by 400 ms depolarizing currents was decreased in mutant mice (P < 0.0001). (E) The efficacy of basal transmission at the mossy fiber synapse was increased in mutant mice (P < 0.0001). The ratio of the peak EPSP amplitude to fiber volley amplitude is shown. (F) Reduced paired-pulse facilitation ratios of EPSPs at intervals ranging from 50 to 5000 ms in mutant mice. (G) Mutant mice had greatly reduced frequency facilitation at 1 Hz. Sample EPSPs were recorded at the time indicated by the numbers in the graph. Error bars indicate s.e.m.

Figure 5

Clustering of Human Post-Mortem Brains by Biomarkers Derived from Alpha-CaMKII+/- Mice. (A) We selected 10 genes (ADCY8, CCND1, LOC151835, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and TDO2), which were differentially expressed in the mutant hippocampus, as a set of biomarkers to characterize the mutants, and performed the statistical clustering of the expression data of these 10 genes in 166 hippocampi of human post-mortem brains. The cluster analysis classified the subjects into two clusters (Cluster A and Cluster B), and Cluster B contained significantly more schizophrenic, schizoaffective and bipolar patients than Cluster A (P = 0.0041, χ² test). (B) We compared the gene expression profile between the schizophrenic patients in Cluster B (= schizophrenia-enriched cluster) (n = 19) and the controls with no major psychiatric diagnosis and no CNS-related illness in Cluster A (= control-enriched cluster) (n = 30), using the following criteria; (i) gene expression level was significantly different between two groups (p < 0.01, fold change > 2.0), (ii) present flags were marked in over 75% of hippocampi in at least one of the two groups, (iii) gene expression was not affected by age and gender. We found that 26 probes met these criteria and nearly half of them encoded genes which are known to be involved in neurogenesis or cell-migration/maturation.

Figure 6

Immature DG of Alpha-CaMKII+/- Mice. (A) Cells proliferate in the subgranular layer (SGL), and migrate and differentiate into mature neurons in the granular layer (GL) in the adult hippocampus. Each stage of the neuronal development has cell markers and specific morphological and physiological properties. In the DG of alpha-CaMKII+/- mice, the number of cells expressing Polysialic acid-NCAM (PSA-NCAM), a marker for late-stage progenitor cells and immature neurons, and calretinin, a marker for immature neurons, was dramatically increased, whereas the amount of calbindin, a marker for mature neurons in the DG, -positive cells was markedly reduced. Electrophysiological study of the DG neurons showed that input resistance was high and the number of spikes during sustained depolarization was decreased in mutant mice. Furthermore, morphological analysis revealed that dendritic branching and length were decreased in the mutant DG. Collectively, the mutant DG granule cells had many features that are characteristic of immature DG neurons. Red arrows represent the specific changes in the DG of alpha-CaMKII+/- mice. (B) A schematic model of gene-to-behavior pathways in alpha-CaMKII+/- mice. Alpha-CaMKII deficiency leads multiple abnormal gene expression and signal transduction, which causes "immature DG" and impaired function of hippocampus and other brain regions, resulting in abnormal behaviors of alpha-CaMKII+/- mice. Based on the finding that expression changes of genes related to neurogenesis and neural maturation/migration, including calbindin, in hippocampus is associated with higher incidence of schizophrenic patients, "Immature DG" and its equivalent hippocampal functional abnormalities may serve as a promising candidate endophenotype of psychiatric disorders, such as schizophrenia and bipolar disorders.