Tcf4 Regulates Synaptic Plasticity, DNA Methylation, and Memory Function

Abstract

Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/-) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/-) mice.

Keywords: DNA methylation; E2-2; HDAC inhibitor; Pitt-Hopkins syndrome; autism; autism spectrum disorder; epigenetics; language; learning and memory; neuroepigenetics; next-generation sequencing; schizophrenia; social interactions; transcription; transcription factor 4.

Figure 1. Tcf4(+/−) Mice Are Hyperactive, Display Motor Incoordination, Have Reduced Prepulse Inhibition, and Show Autistic-like Behavior

(A) Homology model of Tcf4 protein (teal) with NeuroD2 (tan) bound to DNA. Mutations in Tcf4 protein that cause PTHS (yellow) are predominately basic residues necessary for DNA backbone association. (B and C) Open field. Distance traveled per minute during a 30 m trial. Tcf4(+/−) mice show hyperactivity and a reduced ability to habituate to an open field. (D) Thigmotaxis, a behavior associated with anxiety, during the open field task. (E and F) Elevated plus maze. Arm entries during a 5 m trial. Tcf4(+/−) mice cross more frequently between arms but do not elicit significantly altered anxiety toward open arms. (G) Dynamic weight bearing. Tcf4(+/−) mice place more body weight on the front left paw than on the front right. (H) Representative images of Tcf4(+/−) mice placing more pressure on the front left paw than on the front right while ambulating across a catwalk. (I) Three-chamber social interaction task. Mice are placed into the center (C) of a three-chamber apparatus, where the distal chambers contained either an empty cylindrical cage (E) or a cylindrical cage holding a novel mouse (M) of equivalent age, gender, and strain. Tcf4(+/−) mice show social aversion, preferring the chamber with an empty cage to that with the novel mouse. (J) Start reflex. Tcf4(+/−) mice have an enhance startle reflex to unexpected 120 dB sound bursts. (K) Tcf4(+/−) mice have deficits in prepulse inhibition of the startle reflex when the 120 dB sound burst is preempted by 69, 73, and 81 dB pulses over 65 dB background white noise. (L) USVs in Tcf4(+/−) pups P3 and P5 were significantly infrequent when removed from the nest. (M and N) Occurrence (M) and traces (N) across ultrasonic frequencies elicited by P3 pups. All data shown are means ± SEM from 12 mice per genotype at 2–3 months of age, with the exception of the USV experiments, which were performed with pups (P3–P11) and 6 mice per genotype. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. Tcf4(+/−) Mice Have Deficits in Associative and Spatial Learning and Memory, Prepulse Inhibition, and Enhanced LTP across Schaffer Collaterals

(A–C) MWM hidden platform task. Tcf4(+/−) mice show reduced spatial learning in locating the hidden platform. (D) 24 hr OLM. Mice interacted for 10 min with 50 ml beakers, and 24 hr memory was tested by moving one beaker to a novel location and measuring the amount of time interacting with the novel location (t_N) versus the familiar location (t_F). Discrimination index = (t_N − t_F)/(t_N × t_F). (E) Trace threat recognition training. Mice were presented with a 30 s white noise tone (CS, line), followed by a 15 s delay, and then a 1 s, 0.5 mA foot shock (US, arrow) three times over the course of a 7.5 m trial. Tcf4(+/−) mice do not show deficits in freezing during training. (F) 24 hr contextual test. Tcf4(+/−) mice show reduced freezing during a 5 m test in the training context. (G) 24 hr cued test. Tcf4(+/−) mice show reduced freezing when placed in a novel context and presented with a 3 m white noise tone. (H) Tcf4(+/−) mice have enhanced LTP across Schaffer collaterals when activated with (1×) theta burst stimulation. All data shown are means ± SEM from 6–12 mice per genotype at 2–3 months of age. LTP from 55–58 hippocampal slices per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Gene Expression Patterns and Altered CpG Methylation Caused by Tcf4(+/−) in the Hippocampus

(A) Heatmap of poly(A) RNA-seq DEGs in the Tcf4(+/−) hippocampus showing individual replicates (FDR < 0.05). (B) Gene expression grouped by their relation to PTHS, neuroactive receptors, and myelination (blue, downregulated; orange, upregulated; white, not significant). (C) Relative global methylation across gene bodies. Curve thickness is ±SEM. (D) DEG gene body and TSS methylation changes. (E) Top ten represented upregulated KEGG pathways in Tcf4(+/−) mice as ranked by Benjamini and Hochberg (B–H) adjusted p value. (F) Downregulated KEGG pathways in Tcf4(+/−) mice. (G) In Tcf4(+/−) hippocampal tissue, 97 deregulated genes are affected by the administration of the class I Hdac inhibitor CI-994 and have a strong negative correlation (R² = 0.72). All data shown are from four mice per genotype at 2 months of age.

Figure 4. HDAC Inhibition Rescues LTP and Memory in Tcf4(+/−) Mice

(A) TSA reduces LTP in the CA1 area of Tcf4(+/−) mice. (B) TSA enhances LTP in the CA1 area of WT controls. (C) TSA normalizes LTP in Tcf4(+/−) mice relative to untreated WT controls. (D and E) SAHA has no significant effect on Tcf4(+/−) hyperactivity or thigmotaxis in an open field. (F and G) SAHA administered 25 mg/kg i.p. for 10 days rescues Tcf4(+/−) deficits in 24 hr contextual and trace-cued threat recognition memory. (H) Schematic of the experimental paradigm to test SAHA-mediated memory retrieval post-training. Tcf4(+/−) mice were trained using the trace threat recognition protocol, tested for memory at 24 hr, and then treated with SAHA for 10 days before being test again for remote memory recall. (I) Tcf4(+/−) mice have 24 hr memory deficits. Bars represent freezing during the first 2 minutes of the cue presentation. (J) SAHA treatment after training improves remote memory recall in Tcf4(+/−) mice. All data shown are means ± SEM from six mice per group at 2 months of age. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Effects of SAHA on Tcf4(+/−) and Memory-Associated Gene Expression

(A) Tcf4(+/−) and WT mice were treated with SAHA or vehicle control for 10 days and either kept naive or trained in threat recognition. RNA and methylated DNA were then isolated from the CA1 region of the hippocampus 1 hr after training. (B) Correlation between the original Tcf4(+/−) RNA-seq and the Tcf4(+/−) naive vehicle group DEG lists relative to WT controls. (C) Expression of all genes in the WT naive vehicle group, and significant changes in transcription associated with training. (D) Chord diagram showing the number of overlapping DEGs between each of the seven most relevant pairwise comparisons among the eight datasets. The most significant overlaps in gene identity between comparisons are filled with color, and the significance of the overlap is listed on the perimeter. (E) Expression profiles of Tcf4(+/−) DEGs for select datasets. (F) RNA-seq read density and normalized expression for the dysregulated IEGs Npas4 and Dusp1. (G) SAHA elicits an expression profile similar to that of training in WT mice. Scatterplot of WT naive SAHA versus WT naive vehicle and WT trained vehicle versus WT naive vehicle. Only genes significant in both comparisons are shown. (H) SAHA normalizes the expression of Tcf4(+/−) DEGs post-training. Scatterplot of Tcf4(+/−) naive vehicle versus WT naive vehicle and Tcf4(+/−) trained SAHA versus Tcf4(+/−) trained vehicle. Only genes significant in both comparisons are shown. All data shown are means ± SEM from three mice per group at 2 months of age. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. Tcf4(+/−) DMRs Are Associated with Trained DEGs

(A) Chord diagram showing the number of overlapping genes containing DMRs between each of the seven most relevant pairwise comparisons among the eight datasets. The most significant overlaps in gene identity between comparisons are filled with color, and the significance of the overlap is listed on the perimeter. (B) Cross-comparison of DEGs and genes containing DMRs for each pairwise comparison, in which significant overlaps (p < 0.05) between datasets are shown as circles in the matrix. (C) Differential CpG methylation profile of expressed and silenced genes in the hippocampus of WT mice. Curve thickness is ±SEM. (D) Differential CpG methylation profile of genes upregulated and downregulated after training. Genes upregulated after training in WT mice are significantly hypomethylated in Tcf4(+/−) mice. (E) Genes with significantly upregulated expression after training in WT mice have significantly increased baseline expression in Tcf4(+/−) naive mice compared to WT naive controls. (F) Interactome network derived from the original Tcf4(+/−), Tcf4(+/−) naive vehicle, and Tcf4(+/−) trained vehicle DEGs, highlighting gene products with a high degree of interaction among these Tcf4(+/−) DEGs. (G) Ubc and Hdac2 are identified as the most interconnected gene products in the Tcf4(+/−) DEG datasets.

Figure 7. HDAC2 Knockdown Enhances Tcf4 Expression and Rescues Spatial and Threat Recognition Memory in Tcf4(+/−) Mice

(A) Schematic of surgical, behavioral, and expression analysis. Mice were injection with HDAC2-ASO1 or control at 2 months of age followed by a behavioral battery including open field (OF), object location memory (OLM), Morris water maze (MWM), and contextual threat recognition (TR). At 5 months, RNA was extracted from the hippocampus for analysis. (B and C) HDAC2-ASO1 has no significant effect on Tcf4(+/−) hyperactivity or thigmotaxis in an open field. (D) HDAC2-ASO1 significantly improves 24 hr object location memory in Tcf4(+/−) and wild-type mice. (E) HDAC2-ASO1 rescues 24 hr contextual threat recognition memory in Tcf4(+/−) treated mice. (F–H) HDAC2-ASO1 rescues spatial memory in the MWM task by increasing the time spent in the target quadrant and the number of platform crossing in Tcf4(+/−) treated mice. (I) PCR expression analysis. HDAC2 knockdown 3 months after ICV injection boosts Tcf4 transcription and normalizes the expression of Lefty1, Grin2a, and Tet2. All data shown are means ± SEM from seven to ten mice per group at 2–5 months of age. #p = 0.052, *p < 0.05, **p < 0.01, ***p < 0.001.