MeCP2 Deficiency Disrupts Kainate-Induced Presynaptic Plasticity in the Mossy Fiber Projections in the Hippocampus

Abstract

Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events in vivo in the hippocampus of MeCP2 mutant mice. While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.

Keywords: MeCP2; activity-dependent gene expression; autism; neurogenesis; presynaptic plasticity.

FIGURE 1

KA-induced seizures failed to increase IPT size in a mouse model of MeCP2-308 mutation. (A) Hippocampal MF-CA3 circuit. The MF consist of the axons of the granule cells of the DG that project through the main tract (MT) in the lucid stratum or through the IPT located mainly in the stratum oriens. (B) Seizures activity over time after KA administration in MeCP2-308 MUT model. No differences in the development or severity of seizures were observed between WT and MUT mice in response to KA (n = 5 mice per experimental group, two-way ANOVA p = 0.7023, followed by Tukey’s HSD test). (C–F) Representative images of IPT stained with anti-Synaptoporin in coronal hippocampal sections; IPT is indicated by black arrows. Two weeks after KA injection, WT mice (D) but not MUT mice (F) showed increased IPT size, in comparison with WT and MUT controls (C,E). Scale bar: 200 μm. (G) Quantitative analysis of IPT volume in WT and MeCP2-308 MUT mice. KA-treated WT mice showed an increase in the IPT related to the controls; however, KA-treated MUT mice showed similar IPT size as MUT control. N = 4 mice per experimental group. (H,I) KA-induced gene expression: BDNF and Arc expression were determined by real-time RT-PCR from total hippocampus. Six hours after KA injection, a significant increase in the expression of the early activation genes Arc (H) and BDNF (I) was observed. Fold change was calculated in reference to the WT control group, which was normalized to 1. Results are represented as the ratio between the relative amount of the gene of interest and GAPDH. N = 4 mice per group. cDNA was prepared individually for each mouse and real-time RT-PCR reactions were run in duplicates for each mouse. Data analysis: two-way ANOVA followed by Tukey’s HSD test. ^*p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 2

KA-induced seizures failed to increase IPT size in a mouse model of MeCP2 deficiency. (A) Seizures activity over time after KA administration. Six-week-old MeCP2-Bird KO mice elicit significantly higher seizures scores than WT littermates, indicative of greater sensitivity to KA. N = 4–5 mice per experimental group, two-way ANOVA, followed by Tukey’s HSD test, ^*p < 0.05. (B) Quantitative analysis of IPT volume in WT and MeCP2-Bird KO mice. KA-treated WT mice showed an increase in the IPT volume with respect to controls; however, KA-treated KO mice showed similar IPT size as KO control. N = 4 mice per experimental group. (C,D) KA-induced neuronal gene expression: BDNF and Arc expression were quantified by real-time RT-PCR from total hippocampus. Six hours after KA injection, a significant increase in the expression of the early activation genes Arc (C) and BDNF (D) was observed. (E,F) Sema3F and Plexin A3 expression was assessed by real-time RT-PCR from total hippocampus. Two weeks after KA-seizures induction, a significant decrease in Sema3F was observed in KA-treated WT mice related to WT controls, whereas an opposite response was registered between KA-injected KO mice and KO controls. Basal Sema3F levels in KO mice were significantly lower than in the WT littermates (E). Regarding PlxnA3, no changes of expression were observed after KA treatment in WT or KO mice; however, PlxnA3 expression was significantly decreased in KO mice (F). (C–F) Fold change was calculated in reference to the WT control group, which was normalized to 1. Results are represented as the ratio between the relative amount of the gene of interest and GAPDH. N = 5 mice per experimental group. cDNA was prepared individually for each mouse and real-time PCR reactions were run in duplicates for each mouse. Data analysis: two-way ANOVA followed by Tukey’s HSD test, ^*p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (E′,F′) In order to better evidence KA effect on each genotype, the expression levels generated by KA were re-calculated in relation to their respective control groups, WT or MUT. The dotted line on the Y-axis (Y = 1) represents the control values for each genotype. Mann–Whitney t-test with 95% confidence intervals, ^*p < 0.05.

FIGURE 3

Seizures induce similar rate of adult neurogenesis in WT and MeCP2-308 MUT mice. (A,B) IHC for BrDU labeling. Eight days after KA injection, a strong increase in the number of BrDU-positive cells was observed in DG from KA-treated mice (B) compared with control littermates (A). Scale bar: 50 μm. GLC, granule cell layer; HL, hilus. (C) BrDU-positive cells per DG in KA-treated and controls WT and MUT mice. A single seizure episode induces an increase in the number of BrDU-positive cells that was detected in both WT and MUT mice, respective to their controls. (D) Quantification of BrdU-positive cells by DG in WT and MUT mice injected with KA. A similar decrease in the number of BrdU-positive cells was observed in the WT and MUT mice between days 8 and 35 post KA-induced activity. (E,F) Double labeling IHC for BrdU and neuronal or astroglial markers in hippocampus 8 days after KA administration. (E) Double labeling of newborn DG cells (BrdU in red, white arrows) and Tuj1 (green) a neuronal marker. (F) Double labeling of newborn DG cells (BrdU in red, white arrows) and GFAP (in green) labeling astroglial cells in hippocampus. Scale bar: 50 μm. n = 3 mice per each experimental group. Data analysis: two-way ANOVA followed by Tukey’s HSD test, ^*p < 0.05; ^∗∗p < 0.01.

FIGURE 4

Defective maturation is recapitulated in MeCP2 mutant adult-born granule neurons. (A) Triple IHC labeling of BrdU-positive cells and neuronal markers NeuN and DCX. Examples of BrDU-positive neurons in intermediate state of maturation that co-express both NeuN and DCX (right panel). Example of a mature neuron that only expresses NeuN (left panel) and an immature neuron that only expresses DCX (middle panel). The vertical panels are unique optical planes showing the three fluorescence channels separately and their overlapping in the upper panel. Scale bar: 15 μm. (B) Percentages of BrdU-positive cells in different stages of maturation in WT and MUT mice exposed to KA. 5 weeks after seizures, WT mice showed a high percentage of mature new granule cells (90%), while MUT mice presented significantly less mature cells and more immature cells. Each bar represents the mean ± standard error (SEM) of BrdU-positive cells co-localizing with the corresponding markers, normalized to the total number of BrdU-positive cells in each mouse. Each bar is subdivided to indicate the mean percentage of BrdU-positive cells at different states of maturation. n = 4–5 mice per experimental group. Data analysis: two-way ANOVA followed by Sidak’s test, ^*p < 0.05; ^∗∗p < 0.01.

FIGURE 5

Kainic-induced Sema3F and receptors levels in WT and MeCP2-308 MUT mice. (A–C) Sema3F, Plexin A3, and Npn-2 expression were assessed by real-time RT-PCR from total hippocampus. Two weeks after KA-seizures induction, a significant decrease in Sema3F was observed in KA-treated WT mice related to WT controls, whereas no differences were registered between KA-injected MUT and MUT control mice (A,A′). Regarding PlxnA3, no changes of expression were observed after KA treatment in WT or MUT mice (B,B′); however, Npn-2 expression in MUT mice was significantly lower than WT mice, independently of treatment (C,C′). (A–C) Fold change was calculated in reference to the WT control group, which was normalized to 1. Results are represented as the ratio between the relative amount of the gene of interest and GAPDH. N = 5 mice per experimental group. cDNA was prepared individually for each mouse and real-time PCR reactions were run in duplicates for each mouse. Data analysis: two-way ANOVA followed by Tukey’s HSD test, ^*p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (A′–C′) In order to better evidence KA effect on each genotype, the expression levels generated by KA were re-calculated in relation to their respective control groups, WT or MUT. The dotted line on the Y-axis (Y = 1) represents the control values for each genotype. A′–C′: Mann–Whitney t-test with 95% confidence intervals.

FIGURE 6

Kainic-induced BDNF expression is deficient in MeCP2-308 MUT mice. (A,A′) BDNF expression was assessed by real-time RT-PCR from total hippocampus. (A) Two weeks after KA-seizures induction, a significant increase in BDNF was observed in KA-treated WT mice related to WT controls, whereas no differences were registered between KA-injected MUT and MUT control mice. Fold change was calculated in reference to the WT control group, which was normalized to 1. Results are represented as the ratio between the relative amount of the gene of interest and GAPDH. N = 5 mice per experimental group. cDNA was prepared individually for each mouse and real-time PCR reactions were run in duplicates for each mouse. (A′) In order to better evidence KA effect on each genotype, the expression levels generated by KA were re-calculated in relation to their respective control groups, WT or MUT. The dotted line on the Y-axis (Y = 1) represents the control values for each genotype. Data analysis: (A) two-way ANOVA followed by Tukey’s HSD test, ^*p < 0.05; ^∗∗p < 0.01. (A′) Mann–Whitney t-test with 95% confidence intervals, ^∗∗p < 0,01. (B–E) IHC- and pTrkB-positive cells quantification in DG. Two weeks after KA treatment, an increase in pTrkB-positive cells (white arrows) was observed in WT mice DG injected with KA (C) in comparison to KA-injected MUT mice (E) or control mice (B,D). IHC labeling pTrkB (green) and Dapi (blue). Scale bar: 50 μm. (F) Quantification of pTrkB-positive cells per DG in WT and MUT mice injected with KA and control. A single seizure episode leads to an increase in the number of pTrkB-positive cells, detected 2 weeks after seizure activity only in WT mice. n = 3 mice per experimental group. Data analysis: two-way ANOVA followed by Tukey’s HSD test, ^*p < 0.05.

FIGURE 7

Mecp2-mutant mice show defective spatial memory evaluated through the Barnes maze. (A) Learning: the acquisition was reflected by lower escape latencies along the trainings. For both experimental groups, a similar decrease in the escape latency time (measured in seconds) throughout the successive trainings was observed. n = 16-19 mice per experimental group. Data analysis: two-way ANOVA followed by Tukey’s HSD test. (B–D) Memory: relative percentage of time spent and number of snouts made by the animals in the target quadrant and in the escape hole during the 2 min of the final memory test. (B) Percentage of time spent in target quadrant for WT and MUT mice. The analysis shows that WT mice spent significantly more time in the target quadrant than MUT mice. Percentage of snouts in the target quadrant holes (C) and in the escape hole (D) in relation to the total snouts performed in the holes of the platform in the 2 min of the final memory test. Both the percentage of snouts in the “target” quadrant (C) and in the escape hole (D) was significantly higher in WT mice when compared with the MUT. n = 16–19 mice per experimental group. Data analysis: Mann–Whitney t-test with 95% confidence interval. ^*p < 0.05; ^∗∗∗p < 0.001; ^****p < 0.0001.

FIGURE 8

Schematic summary of the activity-induced presynaptic plasticity regulated by MeCP2. In WT mice, seizures activity increases the level of neurogenesis, accompanied with higher levels of BDNF and lower of Sema3F, allowing IPT to grow (A,B). Conversely, MUT animals show abnormal activity-dependent presynaptic plasticity characterized by the lack of IPT growth which correlates with imbalanced trophic and guidance support (BDNF and Sema3F abnormal levels). Although activity do increases the neurogenesis in MUT mice, adult born granule cells show developmental restraining and recapitulate maturational defects (C,D). These results are in line with deficits in spatial memory observed in MUT mice. This study set the basis to establish a model to evaluate the effect of manipulating specific pathways involved in axonal guidance, synaptogenesis, and neuronal maturation to correlate with changes in behavior.