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. 2022 May 10:11:e72290.
doi: 10.7554/eLife.72290.

Rescue of behavioral and electrophysiological phenotypes in a Pitt-Hopkins syndrome mouse model by genetic restoration of Tcf4 expression

Hyojin Kim  1   2 , Eric B Gao  1   2 , Adam Draper  1   2 , Noah C Berens  3 , Hanna Vihma  1   2 , Xinyuan Zhang  4 , Alexandra Higashi-Howard  4 , Kimberly D Ritola  5 , Jeremy M Simon  2   6   7 , Andrew J Kennedy  4 , Benjamin D Philpot  1   2   7
Affiliations

Rescue of behavioral and electrophysiological phenotypes in a Pitt-Hopkins syndrome mouse model by genetic restoration of Tcf4 expression

Hyojin Kim et al. Elife. .

Abstract

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by monoallelic mutation or deletion in the transcription factor 4 (TCF4) gene. Individuals with PTHS typically present in the first year of life with developmental delay and exhibit intellectual disability, lack of speech, and motor incoordination. There are no effective treatments available for PTHS, but the root cause of the disorder, TCF4 haploinsufficiency, suggests that it could be treated by normalizing TCF4 gene expression. Here, we performed proof-of-concept viral gene therapy experiments using a conditional Tcf4 mouse model of PTHS and found that postnatally reinstating Tcf4 expression in neurons improved anxiety-like behavior, activity levels, innate behaviors, and memory. Postnatal reinstatement also partially corrected EEG abnormalities, which we characterized here for the first time, and the expression of key TCF4-regulated genes. Our results support a genetic normalization approach as a treatment strategy for PTHS, and possibly other TCF4-linked disorders.

Keywords: Pitt-Hopkins syndrome; gene therapy; genetics; genomics; mouse; neurodevelopmental disorder; neuroscience.

PubMed Disclaimer

Conflict of interest statement

HK, EG, AD, NB, HV, XZ, AH, KR, JS, AK, BP No competing interests declared

Figures

Figure 1.
Figure 1.. Embryonic, pan-cellular reinstatement of Tcf4 fully rescues behavioral deficits in a mouse model of Pitt-Hopkins syndrome.
(A) Schematic depicting a conditional Pitt-Hopkins syndrome mouse model in which expression of the bHLH region of Tcf4 is prevented by the insertion of a loxP-P2A-GFP-STOP-loxP cassette into intron 17 of Tcf4 (Tcf4STOP/+). Adenovirus splicing acceptor is shown by the blue box. Crossing Tcf4STOP/+ mice with Actb-Cre+/– transgenic mice can produce mice with embryonic pan-cellular reinstatement of Tcf4 expression (Tcf4STOP/+::Actb-Cre). (B) Relative Tcf4 mRNA expression in embryonic brain lysates from Tcf4+/+, Tcf4STOP/+, and Tcf4STOP/+::Actb-Cre. The primers were designed to detect Tcf4 exon 18. (C) Representative Western blot for TCF4 and β-tubulin loading control protein and quantification of relative intensity of TCF4 protein in embryonic brain lysates from Tcf4+/+, Tcf4STOP/+, and Tcf4STOP/+::Actb-Cre mice. The data were analyzed by one-way ANOVA followed by Bonferroni’s post hoc. (D) Left panel: Heatmaps indicate time spent in proximity to one object located in the familiar position and the other object relocated to the novel position. Right panel: Percent time interacting with the novel location object (Tcf4+/+: n = 36, Tcf4STOP/+: n = 22, Tcf4STOP/+::Actb-Cre: n = 15). (E) Open field data. Left panel: Distance traveled per 5 min. Center panel: Total distance traveled for the 30 min testing period. Right panel: Percent time spent in the center zone (Tcf4+/+: n = 30, Tcf4STOP/+: n = 23, Tcf4STOP/+::Actb-Cre: n = 10). (F) Nest building data. Left panel: Representative images of nests built by Tcf4STOP/+ and Tcf4STOP/+::Actb-Cre mice. Right panel: Percentage of nest material used during the 4 day nest building period (Tcf4+/+: n = 13, Tcf4STOP/+: n = 10, Tcf4STOP/+::Actb-Cre: n = 5). (G) Elevated plus maze data. Left panel: Heatmaps reveal relative time spent on the elevated plus maze. Right panels: Percent time spent in the closed and open arms, percent of entries made into the closed and open arms, and percent time spent in the center zone (Tcf4+/+: n = 30, Tcf4STOP/+: n = 23, Tcf4STOP/+::Actb-Cre: n = 10). Values are means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Body and brain weight analysis.
(A) DAB immunostaining of GFP (indicating presence of the STOP cassette) in sagittal brain sections of adult Tcf4+/+, Tcf4STOP/+, and Tcf4STOP/+::Actb-Cre mice. Scale bar = 1 mm. (B) Adult male (Tcf4+/+: n = 13, Tcf4STOP/+: n = 11, Tcf4STOP/+::Actb-Cre: n = 5) and female (Tcf4+/+: n = 22, Tcf4STOP/+: n = 21, Tcf4STOP/+::Actb-Cre: n = 5) body weights of each genotypic group. (C) Adult brain weight measured from dissected brains (Tcf4+/+: n = 4; 1 male; 3 females, Tcf4STOP/+: n = 7; 4 males; 3 females, Tcf4STOP/+::Actb-Cre: n = 4; 1 male; 3 females). Values are means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001.
Figure 2.
Figure 2.. Embryonic reinstatement of Tcf4 expression in glutamatergic or GABAergic neurons rescues selective behavioral deficits in a mouse model of PTHS.
(A) Schematic representation of cell type-specific Tcf4 reinstatement strategy. Tcf4STOP/+::Neurod6-Cre or Tcf4STOP/+::Gad2-Cre mice normalize Tcf4 expression in glutamatergic or GABAergic neurons, respectively, while controls (Tcf4+/+, Neurod6-Cre+/–, or Gad2-Cre+/– mice) have normal Tcf4 expression. (B) Total distance traveled for the 30 min testing period in the open field (Left panel: Control: n = 14, Tcf4STOP/+: n = 7, Tcf4STOP/+::Neurod6-Cre: n = 7, and right panel: Control: n = 19, Tcf4STOP/+: n = 7, Tcf4STOP/+::Gad2-Cre: n = 11). (C) Percent time interacting with the novel location object (Left panel: Control: n = 14, Tcf4STOP/+: n = 8, Tcf4STOP/+::Neurod6-Cre: n = 7, and right panel: Control: n = 18, Tcf4STOP/+: n = 7, Tcf4STOP/+::Gad2-Cre: n = 9). (D) Percent time spent in closed and open arms and percent entries made into the closed and open arms (Left panel: Control: n = 15, Tcf4STOP/+: n = 8, Tcf4STOP/+::Neurod6-Cre: n = 7, and right panel: Control: n = 19, Tcf4STOP/+: n = 7, Tcf4STOP/+::Gad2-Cre: n = 11). (E) Representative images of nests built by mice and percentage of nest material used during the 7 day nest building period (Left panel: Control: n = 15, Tcf4STOP/+: n = 8, Tcf4STOP/+::Neurod6-Cre: n = 7, and right panel: Control: n = 19, Tcf4STOP/+: n = 7, Tcf4STOP/+::Gad2-Cre: n = 11). Values are means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Single-cell RNA sequencing reveals cell type-specific Tcf4 expression in the neonatal and adult mouse brain.
(A) Average expression of Tcf4 in 19 cell types identified in the P0 cortex data analyzed from reference (Loo et al., 2019). Cell type names are colored by principal cell classifications. (B) Average expression of Tcf4 in cell types identified in the adult mouse brain data analyzed from reference (Zeisel et al., 2015). Bars are colored by brain region, and clusters were aggregated into principal cell types. Values are means ± SEM.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Behavioral phenotypes are not affected in Neurod6-Cre +/- and Gad2-Cre +/- mice.
(A–B) Behavioral performance of Cre transgenic mice (Tcf4+/+::Neurod6-Cre and Tcf4+/+::Gad2-Cre) was statistically indistinguishable from wildtype mice (Tcf4+/+) in open field (A: Tcf4+/+: n = 7, Tcf4+/+::Neurod6-Cre: n = 7, B: Tcf4+/+: n = 9, Tcf4+/+::Gad2-Cre: n = 10), object location memory (A: Tcf4+/+: n = 6, Tcf4+/+::Neurod6-Cre: n = 8, B: Tcf4+/+: n = 8, Tcf4+/+::Gad2-Cre: n = 10), elevated plus maze (A: Tcf4+/+: n = 6, Tcf4+/+::Neurod6-Cre: n = 9, B: Tcf4+/+: n = 9, Tcf4+/+::Gad2-Cre: n = 10), and nest building assay (A: Tcf4+/+: n = 6, Tcf4+/+::Neurod6-Cre: n = 9, B: Tcf4+/+: n = 9, Tcf4+/+::Gad2-Cre: n = 10). Values are means ± SEM.
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. Behavioral outcomes of Tcf4STOP/+::Gad2-Cre and Tcf4STOP/+::Olig2-Cre mice.
(A–C) Total distance traveled for the 30-min testing period (A: Tcf4+/+: n = 14, Tcf4STOP/+: n = 17, B: Tcf4+/+::Gad2-Cre: n = 12, Tcf4STOP/+::Gad2-Cre: n = 8, C: Tcf4+/+::Olig2-Cre: n = 13, Tcf4STOP/+::Olig2-Cre: n = 9) and percent time interacting with the novel location object (A: Tcf4+/+: n = 13, Tcf4STOP/+: n = 14, B: Tcf4+/+::Gad2-Cre: n = 8, Tcf4STOP/+::Gad2-Cre: n = 8, C: Tcf4+/+::Olig2-Cre: n = 7, Tcf4STOP/+::Olig2-Cre: n = 8). Behavioral outcomes of Tcf4STOP/+ and Tcf4STOP/+::Gad2-Cre mice were independently replicated. Reinstating Tcf4 from GABAergic neurons and oligodendrocytes did not rescue hyperactivity and memory function deficit. Values are means ± SEM. *p < 0.05, **p < 0.005, ****p < 0.0001. These experiments were performed at Bates College in the lab of Dr. Andrew Kennedy.
Figure 3.
Figure 3.. Neonatal ICV delivery of PHP.eB/Cre yields Cre expression by approximately P10-P17.
(A) A timeline of experiment to evaluate timing of Cre biodistribution following intracerebroventricular (ICV) injection of 1 µl of 3.2 × 1013 vg/ml AAV9/PHP.eB-hSyn-Cre to P1 mice. (B) In situ hybridization for Cre mRNA, and (C) immunofluorescence staining for CRE protein in the cortex and hippocampus of P4, P7, P10, and P17 wildtype mice neonatally treated with PHP.eB/Cre. Py. = Stratum pyramidale. Scale bars = 100 µm (B) and 250 µm (C). (D–F) CRE immunofluorescence staining in sagittal section of P60 wildtype mouse brain. Scale bars = 1 mm (D), 500 µm (E), and 100 µm (F). (G) Relative Tcf4 transcript levels detected in the brains of P10 Tcf4STOP/+ mice treated with vehicle or PHP.eB/Cre and P17 Tcf4STOP/+ mice treated with vehicle or PHP.eB/Cre.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Cre immunofluorescence staining in sagittal sections of P4, P7, P10, and P17 mice.
(A) Dual immunostaining of neuronal marker (NeuN) and Cre in P17 brain of a mouse treated with PHP.eB/Cre. CRE protein was detected in NeuN-positive cells (green circle), but absent in NeuN-negative cells (yellow circle). Scale bars = 100 µm. (B–E) CRE protein spatial pattern in lateral (top row) and medial (bottom row) sagittal sections at different postnatal time points. Arrows indicate the brain area close to the injection site. Scale bars = 1 mm.
Figure 4.
Figure 4.. Neonatal ICV injection of PHP.eB/Cre improves behavioral phenotypes in Tcf4STOP/+ mice.
(A) Experimental timeline for evaluation of behavioral phenotypes in Tcf4+/+ and Tcf4STOP/+ mice treated with vehicle or PHP.eB/Cre. (B) Left panel: Distance traveled per 5 min. Right panel: Total distance traveled for the 30-min testing period. (C) Left panel: Heatmaps indicate time spent in proximity to one object located in the familiar position and the other object relocated to a novel position. Right panel: Percent time interacting with the novel location object. (D) Left panel: Heatmaps reveal time spent in elevated plus maze. Right panels: Percent time spent in the closed and open arms and percent entries made into the closed and open arms. (E) Left panel: Percentage of nest material used during the 7-day nest building period. Right panel: Percentage of nest material used per day. (F) Body weight analysis of P65-69 male and female mice. (G) Adult brain weight analysis. Values are means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Behavioral outcomes of Tcf4+/+ and Tcf4STOP/+ treated with PHP.eB/GFP.
(A–B) Green fluorescence protein (GFP) immunofluorescence staining in sagittal section of P21 wildtype mouse brain that had ICV injection of 1 µl of 8.5 × 1012 vg/ml PHP.eB-hSyn-GFP at P1. Scale bars = 1 mm (A) and 500 µm (B). (C) Treating Tcf4STOP/+ mice with PHP.eB/GFP did not rescue abnormal behavioral phenotypes in open field, object location memory, elevated plus maze, and nest building assay. Values are means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Behavioral outcomes of Tcf4STOP/+::Camk2a-Cre mice.
Total distance traveled for the 30-min testing period in the open field (Tcf4+/+::Camk2a-Cre: n = 13, Tcf4STOP/+::Camk2a-Cre: n = 9), percent time interacting with the novel location object (Tcf4+/+::Camk2a-Cre: n = 8, Tcf4STOP/+::Camk2a-Cre: n = 8), and nest building assay (Tcf4+/+::Camk2a-Cre: n = 8, Tcf4STOP/+::Camk2a-Cre: n = 8). Reinstating Tcf4 expression in Camk2a-expressing neurons rescued memory deficit and abnormal innate behavior in PTHS model mice. Values are means ± SEM. *p < 0.05.
Figure 5.
Figure 5.. Neonatal ICV injection of PHP.eB/Cre partially rescues LFP spectral power in Tcf4STOP/+ mice.
(A) Schematic of local field potential (LFP) recording from the hippocampus of a freely moving mouse. (B) Representative examples of LFP in each experimental group. (C) Power spectrum density of hippocampal LFP analyzed from Tcf4+/+ and Tcf4STOP/+ mice treated with vehicle or PHP.eB/Cre. Inset 1 spans from 15 to 30 Hz. Inset 2 spans from 30 to 50 Hz on x-axis. (D) LFP power analyses of frequency bands ranging from 1 to 50 Hz, (E) delta (1–4 Hz), theta (5–8 Hz), beta (13–30 Hz), and gamma (30–50 Hz) bands (Tcf4+/+ + vehicle: n = 15, Tcf4+/+ + PHP.eB/Cre: n = 14, Tcf4STOP/+ + vehicle: n = 13, and Tcf4STOP/+ + PHP.eB/Cre: n = 14). (F) Top panels: Spectrograms in single LFP sessions of representative experimental groups. Bottom panels: Representative theta power extracted from spectrogram in the top panel. Values are means ± SEM. *p < 0.05, **p < 0.005, ****p < 0.0001.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Tcf4 haploinsufficiency alters LFP spectral power in the theta band.
(A) Example electrode location (red, GFAP; green, NeuN). Scale bar = 100 µm. (B) Representative examples of LFP in Tcf4+/+ and Tcf4STOP/+ mice. (C) Power spectrum density of hippocampal LFP analyzed from Tcf4+/+ and Tcf4STOP/+ mice. Inset 1 spans from 15 to 30 Hz. Inset 2 spans from 30 to 50 Hz on x-axis. (D) LFP power analyses of frequency bands ranging from 1 to 50 Hz, (E) delta (1–4 Hz), theta (5–8 Hz), beta (13–30 Hz), and gamma (30–50 Hz) bands (Tcf4+/+: n = 9 and Tcf4STOP/+: n = 9). (F) Top panels: Spectrograms in a single LFP session of representative experimental groups. Bottom panels: Representative theta power extracted from spectrogram in the top panel. Values are means ± SEM. *p < 0.05.
Figure 6.
Figure 6.. Widespread Cre expression of the forebrain leads to partial upregulation of Tcf4 and partial recovery of selected TCF4-regulated gene expression.
(A) Representative image of in situ hybridization for Cre mRNA in sagittal section of 6-month-old Tcf4STOP/+ mouse that was treated at P1 with PHP.eB/Cre. Scale bar = 1 mm. (B–D) Higher magnification images of boxed regions in panel A. Scale bars = 200 µm. (E) Cre mRNA fluorescence levels of neocortex and CA1 pyramidal cell layer analyzed from individual Tcf4STOP/+ + PHP.eB/Cre mice. The red line and black dotted lines of the violin plot represent median and interquartile ranges of the data, respectively. (F) Relative Tcf4 mRNA expression of the forebrain from vehicle-treated Tcf4+/+ (n = 15), vehicle-treated Tcf4STOP/+ (n = 14), and PHP.eB/Cre-treated Tcf4STOP/+ (n = 15) mice. Tcf4 mRNA expression levels of PHP.eB/Cre-treated Tcf4STOP/+ mice are relatively higher than vehicle-treated Tcf4STOP/+ mice. Relative mRNA expressions of selected TCF4-regulated genes: Grin2a (Tcf4+/+ + Vehicle: 1.0 ± 0.05, n = 15; Tcf4STOP/+ + Vehicle: 0.67 ± 0.02, n = 14; Tcf4STOP/+ + PHP.eB/Cre: 0.85 ± 0.05, n = 14), Mal (Tcf4+/+ + Vehicle: 1.0 ± 0.04, n = 15; Tcf4STOP/+ + Vehicle: 0.70 ± 0.02, n = 14; Tcf4STOP/+ + PHP.eB/Cre: 0.83 ± 0.04, n = 14), Glra3 (Tcf4+/+ + Vehicle: 1.0 ± 0.04, n = 15; Tcf4STOP/+ + Vehicle: 0.80 ± 0.04, n = 14; Tcf4STOP/+ + PHP.eB/Cre: 0.93 ± 0.04, n = 14), and Lpar1 (Tcf4+/+ + Vehicle: 1.0 ± 0.04, n = 15; Tcf4STOP/+ + Vehicle: 0.71 ± 0.03, n = 14; Tcf4STOP/+ + PHP.eB/Cre: 0.84 ± 0.05, n = 14). Values are means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.
Author response image 1.
Author response image 1.. Representative Western blot and quantification for GFP and GAPDH loading control protein from P17 mouse brain lysates.
Quantification represents data from Vehicle- and PHP.eB/Cre-treated Tcf4Stop/+ mice.
See this image and copyright information in PMC

References

    1. Amiel J, Rio M, de Pontual L, Redon R, Malan V, Boddaert N, Plouin P, Carter NP, Lyonnet S, Munnich A, Colleaux L. Mutations in TCF4, encoding a class I basic helix-loop-helix transcription factor, are responsible for Pitt-Hopkins syndrome, a severe epileptic encephalopathy associated with autonomic dysfunction. American Journal of Human Genetics. 2007;80:988–993. doi: 10.1086/515582. - DOI - PMC - PubMed
    1. Bedeschi MF, Marangi G, Calvello MR, Ricciardi S, Leone FPC, Baccarin M, Guerneri S, Orteschi D, Murdolo M, Lattante S, Frangella S, Keena B, Harr MH, Zackai E, Zollino M. Impairment of different protein domains causes variable clinical presentation within Pitt-Hopkins syndrome and suggests intragenic molecular syndromology of TCF4. European Journal of Medical Genetics. 2017;60:565–571. doi: 10.1016/j.ejmg.2017.08.004. - DOI - PubMed
    1. Brennand KJ, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S, Li Y, Mu Y, Chen G, Yu D, McCarthy S, Sebat J, Gage FH. Modelling schizophrenia using human induced pluripotent stem cells. Nature. 2011;473:221–225. doi: 10.1038/nature09915. - DOI - PMC - PubMed
    1. Brzózka MM, Radyushkin K, Wichert SP, Ehrenreich H, Rossner MJ. Cognitive and sensorimotor gating impairments in transgenic mice overexpressing the schizophrenia susceptibility gene Tcf4 in the brain. Biological Psychiatry. 2010;68:33–40. doi: 10.1016/j.biopsych.2010.03.015. - DOI - PubMed
    1. Buzsáki G, Anastassiou CA, Koch C. The origin of extracellular fields and currents--EEG, ECoG, LFP and spikes. Nature Reviews. Neuroscience. 2012;13:407–420. doi: 10.1038/nrn3241. - DOI - PMC - PubMed

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