Pten heterozygosity restores neuronal morphology in fragile X syndrome mice

Abstract

Genetic studies of hippocampal granule neuron development have been used to elucidate cellular functions of Pten and Fmr1. While mutations in each gene cause neurodevelopmental disorders such as autism and fragile X syndrome, how Pten and Fmr1 function alone or together during normal development is not known. Moreover, Pten mRNA is bound by the fragile X mental retardation protein (FMRP) RNA binding protein, but how this physical interaction impinges on phosphatase and tensin homolog protein (PTEN) expression is not known. To understand the interaction of PTEN and FMRP, we investigated the dentate gyrus granule neuron development in Pten and Fmr1 knockout (KO) mice. Interestingly, heterozygosity of Pten restored Fmr1 KO cellular phenotypes, including dendritic arborization, and spine density, while PTEN protein expression was significantly increased in Fmr1 KO animals. However, complete deletion of both Pten and Fmr1 resulted in a dramatic increase in dendritic length, spine density, and spine length. In addition, overexpression of PTEN in Fmr1 KO Pten heterozygous background reduced dendritic length, arborization, spine density, and spine length including pS6 levels. Our findings suggest that PTEN levels are negatively regulated by FMRP, and some Fmr1 KO phenotypes are caused by dysregulation of PTEN protein. These observations provide evidence for the genetic interaction of PTEN and FMRP and a possible mechanistic basis for the pathogenesis of Fmr1-related fragile X neurodevelopmental disorders.

Keywords: Fmr1; Pten; arborization; dentate gyrus; spine density.

Fig. 1.

PTEN protein expression is increased in Fmr1 KO animals. (A) Representative Western blot images of whole hippocampal lysate from Fmr1^KO, Pten^Het, and Pten^Het/Fmr1^KO mice probed for PTEN and FMRP. (B and C) Quantitative graphs initially normalized to actin and then matched relative protein expression in the wild-type mice. **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns for nonsignificance (P > 0.05) calculated using one-way ANOVA with Tukey multiple comparison post hoc test.

Fig. 2.

Genetic deletion of Pten and Fmr1 using retrovirus-mediated Cre expression alters granule neuron morphology. (A) Schematic shows the retroviral coinjection of pRubi and pRubiC-T2A-Cre into hippocampal dentate gyrus on P7, and the representative confocal low magnification (10×) image of dentate gyrus expressing GFP and mCherry in granule cells. (Scale bar, 70 µm.) (B) Schematic shows the experimental timelines: Pups were injected on P7 and all the histological analyses were performed 60 DPI (P67). (C and D) Representative (C) high-resolution confocal images and (D) three-dimensional reconstructed 60 DPI granule neurons from Fmr1^flx/y, Pten^flx/wt, Pten^flx/wt/Fmr1^flx/y, Pten^flx/flx, and Pten^flx/flx/Fmr1^flx/y mice. (Scale bars in C, 50 µm.) Throughout this paper, the wild type/control (Cre⁻) and knockout neurons (Cre⁺) are represented in green (GFP) and red color (mCherry), respectively. (E) Dot plots with each dot representing an individual neuron showing the quantification of the total dendritic length in raw values and (F) normalized values for Fmr1^flx/y, Pten^flx/wt, and Pten^flx/wt/Fmr1^flx/y and (G and H) Pten^flx/flx (Pten KO) and Pten^flx/flx/Fmr1^flx/y (Pten/Fmr1 DKO) animals. (E and F) Pten heterozygous (Pten^flx/wt) and Fmr1 KOs (Fmr1^flx/y) showed a significant increase in the total dendritic length compared to Cre⁻ controls while the Pten^flx/wt/Fmr1^flx/y do not. (G and H) Both Pten KO and Pten/Fmr1 DKO neurons showed increased dendritic length than Cre⁻ controls. Data are represented as the mean ± SEM. **P < 0.01, ****P < 0.0001 and ns for nonsignificance (P > 0.05). Refer to SI Appendix, Table 1 for mean ± SEM, n, and P values.

Fig. 3.

Pten KO and Pten/Fmr1 DKO increased dendritic arborization of granule neuron. Sholl of length analysis of (A) Fmr1^flx/y, (B) Pten^flx/wt, and (C) Pten^flx/wt/Fmr1^flx/y, (D) Pten^flx/flx, and (E) Pten^flx/flx/Fmr1^flx/y KO neurons with their respective Cre⁻ controls. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA with Bonferroni multiple comparison post hoc test.

Fig. 4.

Pten heterozygosity restores the dendritic spine density in Fmr1 KO neurons. (A) Grayscale (Top) and pseudocolored (Bottom; ImageJ fire lookup table) confocal dendritic spine images of all experimental genotypes: Fmr1^flx/y, Pten^flx/wt, Pten^flx/wt/Fmr1^flx/y, Pten^flx/flx, and Pten^flx/flx/Fmr1^flx/y. (B and C) Spine density quantification (B) raw data and (C) normalized values for Fmr1^flx/y, Pten^flx/wt, and Pten^flx/wt/Fmr1^flx/y granule neurons. Pten^flx/wt/Fmr1^flx/y KO neurons showed reduced spine density compared to Fmr1^flx/y KO neurons. (D and E) Quantification of the spine density for Pten ^flx/flx and Pten ^flx/flx/Fmr1^flx/y presented in (D) raw values and (E) normalized data. Compared to Cre⁻ controls, both Pten KO and Pten/Fmr1 DKO neurons showed increased spine density. Data are represented in mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns for nonsignificance (P > 0.05). Refer to SI Appendix, Table 2 for mean spine density ± SEM, P values, number of cells, and animals. (F–I) Quantification of the spine length presented with (F and G) raw dataset and (H and I) normalized values. Dots plots in F and G correspond to Fmr1^flx/y, Pten^flx/wt, and Pten^flx/wt/Fmr1^flx/y and in H and I to Pten^flx/flx and Pten^flx/flx/Fmr1^flx/y animals. (H and I) Pten KO and Pten/Fmr1 DKO cells displayed increased spine length than Cre⁻ neurons. Data are represented in mean ± SEM. ***P < 0.001, ****P < 0.0001, and ns for nonsignificance (P > 0.05). Refer to SI Appendix, Table 2 for mean spine length ± SEM, P values, number of cells, and animals. (Scale bars, 3.72 µm.)

Fig. 5.

PTEN overexpression in Fmr1 KO reduce dendritic arborization and length. (A) The representative confocal image of granule neurons in Pten^flx/wt/Fmr1^flx/y mice expressing pRubiC-T2A-Cre (Cre⁺) and pRubi-GFP-PTEN (Cre⁺ + PTEN) in hippocampal dentate gyrus. (Scale bar, 100 µm.) (B) A 60-DPI three-dimensional reconstruction of Cre⁺ and Cre⁺ + PTEN granule neurons from Pten^flx/wt/Fmr1^flx/y animals. (C) Dot plots showing the quantification of total dendritic length (raw values). Each dot corresponds to an individual neuron. (D and E) Sholl of (D) length and (E) intersection between Cre⁺ and Cre⁺ + PTEN neurons. Data are represented in mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using the t test (two-tailed) and two-way ANOVA with Bonferroni multiple comparison post hoc test.

Fig. 6.

Overexpression of PTEN decreases spine density, spine length, and pS6 levels in Fmr1 KO. (A) Representative confocal image of Cre⁺ (red) and Cre⁺ + PTEN (green) cell body in Pten^flx/wt/Fmr1^flx/y mice with pS6 staining (gray). (Scale bar, 20 µm.) (B) Dot plot showing the quantification of pS6 intensity. (C) Pseudocolored (Left; ImageJ fire lookup table) and grayscale (Right) confocal dendritic spine images of Cre⁺ and Cre⁺ + PTEN in Pten^flx/wt/Fmr1^flx/y. (Scale bars, 3.72 µm.) (D and E) Dot plots showing (D) spine density and (E) spine length quantification. Data are represented in mean ± SEM. *P < 0.05, and ****P < 0.0001 calculated using t test (two-tailed).