Modulation of cognition and neuronal plasticity in gain- and loss-of-function mouse models of the schizophrenia risk gene Tcf4

Abstract

The transcription factor TCF4 was confirmed in several large genome-wide association studies as one of the most significant schizophrenia (SZ) susceptibility genes. Transgenic mice moderately overexpressing Tcf4 in forebrain (Tcf4tg) display deficits in fear memory and sensorimotor gating. As second hit, we exposed Tcf4tg animals to isolation rearing (IR), chronic social defeat (SD), enriched environment (EE), or handling control (HC) conditions and examined mice with heterozygous deletion of the exon 4 (Tcf4Ex4δ^+/-) to unravel gene-dosage effects. We applied multivariate statistics for behavioral profiling and demonstrate that IR and SD cause strong cognitive deficits of Tcf4tg mice, whereas EE masked the genetic vulnerability. We observed enhanced long-term depression in Tcf4tg mice and enhanced long-term potentiation in Tcf4Ex4δ^+/- mice indicating specific gene-dosage effects. Tcf4tg mice showed higher density of immature spines during development as assessed by STED nanoscopy and proteomic analyses of synaptosomes revealed concurrently increased levels of proteins involved in synaptic function and metabolic pathways. We conclude that environmental stress and Tcf4 misexpression precipitate cognitive deficits in 2-hit mouse models of relevance for schizophrenia.

Fig. 1. Experimental design and Tcf4Ex4δ^+/− mouse model.

a, b Housing conditions (a) and experimental timeline (b). Tcf4tg and wt mice were housed in enriched environment (EE) or isolation rearing (IR) from age 4 weeks onwards (cohort 1) or were kept individually and subjected to 3 weeks of social defeat (SD) or handling control conditions (HC) from age 3 weeks onwards (cohort 2). Tcf4Ex4δ^+/− and wt animals (cohort 3) were housed in IR from age 4 weeks onwards. All mice were subjected to behavioral phenotyping from the age of 3 months. w, weeks; m, month(s). c Schematic representation of the human TCF4 and mouse Tcf4 gene. Black rectangles, exons; gray arrows, enrichment of SZ-associated risk single nucleotide polymorphisms (SNPs); gray line and gray arrow heads, 5′ region covering partial deletions associated with intellectual disability; dashed line, 3′ region with high occurrence of mutations causing mental retardation of the Pitt-Hopkins syndrome type; white rectangle, exon 4 excised in Tcf4Ex4δ^+/− mice (see main text for citations). d The Tcf4Ex4 construct containing two marker genes (LacZ and neo) flanked by FRT sites and loxP sites flanking Tcf4 exon 4, excision of which leads to Tcf4Ex4δ^+/− genotype. loxP, lox recognition sites, FRT, flippase recognition target. e, f Relative expression levels of indicated Tcf4 exons in prefrontal cortex (PFC; e) and hippocampus (Hi; f) of Tcf4Ex4δ^+/− mice, normalized to wt levels (dashed line) and housekeeping genes Actb and Rpl13. Tcf4Ex4δ^+/−, n = 5; wt, n = 6. Data represent mean ± SEM. ***, p < 0.001; *, p < 0.05.

Fig. 2. Tcf4 gene-dosage modulates cognition in environment-dependent manner.

Tcf4tg mice (cohort 1, a, b; cohort 2, d, e) and Tcf4Ex4δ^+/− mice (cohort 3, g, h) were submitted to spatial learning in the water maze task. Cumulated results from water maze and other behavioral profiling (Table 1) are depicted as radar charts for Tcf4tg (c, f) and Tcf4Ex4δ^+/− (i) mice. a In the initial learning task in Morris Water Maze, Tcf4tg and wt mice in isolation rearing (IR) learned slower than mice in enriched environment (EE) (p < 0.001, environmental effect), as measured by the latency to reach a hidden platform. The effect was independent of genotype (p = 0.210, G×E interaction test). b During reversal learning, Tcf4tg mice subjected to IR needed more time to reach the platform than IR subjected wt littermates (p = 0.005), indicating an impairment of cognitive flexibility. c Behavioral profiles show impaired spatial learning upon IR (blue) and no cognitive deficits upon EE (green) in Tcf4tg mice compared to wt mice from the corresponding environment (black). Green and blue stars indicate significant differences (see Table 1 and Dataset Supplementary Fig. 1 for details) between Tcf4tg EE (green) vs. reference (wt EE, black) and between Tcf4tg IR (blue) and reference (wt IR, black), respectively. IR significantly impaired cognition in Tcf4tg mice (cognitive symptom class: p = 0.005, cognitive domain flexibility learning: p = 0.005, cognitive trait cue memory: p = 0.001; all passing multiple-tesing correction). However, at the superdomain level displayed here, only nominal significance was reached (spatial learning p = 0.019, fear memory p = 0.044, blue stars in brackets). d During initial learning, socially defeated (SD) Tcf4tg mice displayed longer platform latencies than mutants from the handling control (HC) group or wt animals (p ≤ 0.001). Tcf4tg HC mice showed slightly delayed platform latencies than wt HC animals (p = 0.007, not reaching significance after Bonferroni correction). e In reversal learning (i.e. flexibility learning), Tcf4tg mice needed significantly more time to reach the platform than wt animals in both SD (p < 0.001) and HC groups (p = 0.002). f Behavioral profiles of Tcf4tg mice from SD and HC. Spatial learning in Tcf4tg mice is significantly impaired upon SD (red) and mildly in HC (gray) compared to wt mice in the corresponding conditions (black) as indicated by stars of corresponding colors (multiple-testing adjusted significance, see Table 1 and Dataset Supplementary Fig. 1 for details). Pain sensitivity was not assessed in this cohort (as indicated by white circle). g, h Tcf4Ex4δ^+/− mice housed in IR displayed higher platform latencies than wt controls in initial learning (p < 0.001)(g) and flexibility learning (p < 0.001)(h). i Behavioral profiles of Tcf4Ex4δ^+/− mice (blue) show that spatial learning and working memory are impaired compared to wt mice (black), as indicated by blue stars (multiple-testing adjusted significance, see Table 1 and Supplementary Table 1 for details). a, b, d, e, g, h Data represent mean ± SEM. n = 12–16 mice per genotype and housing conditions. See Table 1 and Dataset Supplementary Fig. 1 for detailed statistics.

Fig. 3. The expression level of Tcf4 regulates long-term synaptic plasticity without change in basal synaptic transmission.

Electrophysiological characterization of Tcf4tg (a, c, e) and Tcf4Ex4δ^+/− mice (b,d,f). Values from Tcf4tg and Tcf4Ex4δ^+/− are plotted in gray and those from littermate wt controls in black, respectively. a, b) Input/output relationship of excitatory synapses examined using extracellular field potential recording in the stratum radiatum layer CA1 regions of acute hippocampal slices from Tcf4tg and wt (a) and Tcf4Ex4δ^+/− and wt mice (b). A summary plot of the slope of the field excitatory postsynaptic potential (fEPSP) in mV/ms versus the fiber volley (FV) indicates no differences between the genotypes. Tcf4tg n = 28, wt n = 32; (a) and Tcf4Ex4δ^+/− n = 24, wt n = 15; (b). c Superimposed pooled LTP data showing the normalized changes in fEPSP slope. LTP induced by strong stimulation (100 Hz x 1) for 1 s in CA1. The fEPSP slope was measured and expressed as a mean percentage against time. The levels of LTP are unchanged in Tcf4tg mice (effect of genotype, p = 0.186). Summary graphs of LTP obtained by extracellular field recordings in CA1 stratum radiatum of acute hippocampal slices from Tcf4tg and wt mice (n = 28 and 32 slices, respectively). d Superimposed pooled LTP data showing the normalized changes in fEPSP slope. LTP induced by strong stimulation (100 Hz x 1) for 1 s in CA1. Summary graphs of LTP obtained by extracellular field recordings in CA1 stratum radiatum of acute hippocampal slices from Tcf4Ex4δ^+/− mice and wt controls (n = 22 and 34 slices, respectively) showing significantly increased LTP in Tcf4Ex4δ^+/− mice (effect of genotype, p < 0.001). e Superimposed pooled LTD data showing the normalized changes in fEPSP slope. LTD triggered by low stimulation (1 Hz) for 15 min. Summary graphs of LTD in the acute hippocampal slices from Tcf4tg and wt mice (n = 24 and 15 slices, respectively). LTD is significantly increased in Tcf4tg mice (effect of genotype, p < 0.001). f Superimposed pooled LTD data showing the normalized changes in fEPSP slope. LTD triggered by low stimulation (1 Hz) for 15 min. Summary graphs of LTD in the acute hippocampal slices from Tcf4Ex4δ^+/− and wt mice (n = 29 and 20 slices, respectively). LTD is unchanged in Tcf4Ex4δ^+/− mice (effect of genotype, p = 0.143). Data represent mean ± SEM. LTP, long-term potentiation; LTD, long-term depression; fEPSP, field excitatory postsynaptic potentials. wt, filled black circles; open gray circles represent Tcf4tg, (c, e) or Tcf4Ex4δ^+/− (d, f) vs littermate wt animals 4–5 weeks of age, respectively.

Fig. 4. Tcf4 overexpression leads to an increase of premature spine numbers and proteome alterations in peripubertal mice.

a Experimental design. Group-housed Tcf4tg and wt mice were analyzed (*) at the age of 4 weeks (upper bar) (n = 4, each). Independent cohorts of Tcf4tg and wt mice were group-housed (middle bar) or exposed to chronic social defeat (SD, bottom bar) and analyzed at the age of 12 weeks (*). b Total spine density in the frontal cortex of Tcf4tg and wt mice was analyzed with STED nanoscopy. Spine density was significantly increased in 4-week-old Tcf4tg mice (p = 0.031). There was no difference in total spine density between the genotypes upon group housing (p = 0.686) or SD (p = 0.786) in 12-week-old mice. Subjecting mice to SD during puberty reduced total spine densities significantly (p = 0.003) without genotype differences. Data represent mean ± SEM. See Supplementary Fig. 6 for detailed analysis of spine types. c The analysis of electron-microscopic pictures for asymmetric/glutamatergic synapses reveals no differences between Tcf4tg and wt mice (n = 5, each). in the anterior cingulate cortex (ACC) nor the orbitofrontal cortex (OFC) at 4 weeks of age (p > 0.400). d Visualization of proteomic data from cytoplasmic and synaptosomal fractions isolated from frontal cortex of 4 weeks old Tcf4tg mice and wt controls (n = 4, each). Network graph of differentially regulated proteins in cytoplasmic (circular nodes), synaptosomal (diamond-shape nodes) and in both (hexagonal nodes) fractions. KEGG networks and overrepresented pathways visualized with ClueGo and CluePedia in Cytoscape are depicted as filled hexagons (blue = synapse associated pathways; grey = metabolic pathways). Primary data are from Supplementary Table 2.