BMP Antagonist Gremlin 2 Regulates Hippocampal Neurogenesis and Is Associated with Seizure Susceptibility and Anxiety

Abstract

The Bone Morphogenetic Protein (BMP) signaling pathway is vital in neural progenitor cell proliferation, specification, and differentiation. The BMP signaling antagonist Gremlin 2 (Grem2) is the most potent natural inhibitor of BMP expressed in the adult brain; however its function remains unknown. To address this knowledge gap, we have analyzed mice lacking Grem2 via homologous recombination (Grem2^-/- ). Histological analysis of brain sections revealed significant scattering of CA3 pyramidal cells within the dentate hilus in the hippocampus of Grem2^-/- mice. Furthermore, the number of proliferating neural stem cells and neuroblasts was significantly decreased in the subgranular zone of Grem2^-/- mice compared with that of wild-type (WT) controls. Due to the role of hippocampal neurogenesis in neurological disorders, we tested mice on a battery of neurobehavioral tests. Grem2^-/- mice exhibited increased anxiety on the elevated zero maze in response to acute and chronic stress. Specifically, male Grem2^-/- mice showed increased anxiogenesis following chronic stress, and this was correlated with higher levels of BMP signaling and decreased proliferation in the dentate gyrus. Additionally, when chemically challenged with kainic acid, Grem2^-/- mice displayed a higher susceptibility to and increased severity of seizures compared with WTs. Together, our data indicate that Grem2 regulates BMP signaling and is vital in maintaining homeostasis in adult hippocampal neurogenesis and structure. Furthermore, the lack of Grem2 contributes to the development and progression of neurogenesis-related disorders such as anxiety and epilepsy.

Keywords: BMP signaling; Gremlin 2; anxiety; epilepsy; hippocampal neurogenesis.

Figure 1.

Grem2 regulates BMP signaling in the hippocampus. A, Representative Western blotting images of murine hippocampi protein samples to analyze Smad and Jnk phosphorylation. B, Quantification of blot images shows significantly increase in Smad phosphorylation and modest increase in Jnk phosphorylation in the hippocampi of Grem2^−/− mice. C, PCR analysis of murine hippocampi RNA samples reveals absence of Grem2 RNA and no compensatory changes in the expression of other DAN family BMP antagonists in Grem2^−/− mice. (p)POI, (phosphorylated) protein of interest.

Figure 2.

Grem2^−/− mice have structural deficits in the hilar–CA3 region of the hippocampus. A, B, Representative images of the CA3 (arrowhead) as it enters the DG hilus in WT (A) and Grem2^−/− (B) mice as labeled by NeuN immunofluorescence and examined under 20× magnification. C, Grem2^−/− mice show increased scattering of CA3 pyramidal neurons upon entering the DG hilus (images taken at the bregma −2.055 mm). Extended Data on hippocampal structural deficits are included in Extended Data Figures 2-1–2-3.

Figure 3.

Adult Grem2^−/− mice have proliferative deficits in the SGZ of the DG. A–C, Representative images of murine DG show pHH3⁺, Sox2⁺, Dcx⁺, and TUNEL⁺ immunoreactive cells in WT (A–C) and Grem2^−/− (A’–C’) mice. D, Quantification of Sox2⁺ cells in the DG of WT and Grem2^−/− mice shows a depletion of Sox2⁺ NSCs. E, Quantification of pHH3⁺ cells indicates that Grem2^−/− mice have a decrease in the percentage of Sox2⁺ NSCs that are actively dividing. F, Quantification of Dcx⁺ cells indicates that Grem2^−/− mice have fewer Dcx⁺ immature neuroblasts in the SGZ. G, qPCR analysis of hippocampal RNA confirmed that there are decreased levels of Sox2 and Dcx gene expression in Grem2^−/− mice compared with WT controls (images taken between the bregma −2.35 and −2.48 mm). Extended Data on ventral versus dorsal hippocampal areas are included in Extended Data Figure 3-1.

Figure 4.

Grem2^−/− mice have an anxiety phenotype that becomes more pronounced in stressed conditions. A, B, Mice underwent restraint stress in 50 ml conical tubes that restricted limb movement (A) following an 8 d timeline (B). C, Grem2^−/− mice spent more time on average in the closed arm of the EZM at the baseline, but differences were not significant. With the addition of acute and chronic stress, Grem2^−/− mice spent significantly more time in the closed arms of the EZM. D, There is a significant interaction between sex and genotype in the chronic stress paradigm, with male Grem2^−/− mice showing the greatest anxiety phenotype in response to stress. E–H, Average heat maps of WT and Grem2^−/− mice on the EZM platform at the baseline (E), in the acute stress paradigm (F), males in the chronic stress paradigm (G), and females in the chronic stress paradigm (H; * represents entry point). Extended Data on baseline measurements are included in Extended Data Figure 4-1.

Figure 5.

Grem2^−/− male mice have increased BMP signaling in the DG following chronic stress, which is correlated with decreased neurogenesis. A, qPCR analysis shows sex- and genotype-specific changes in RNA expression of markers of BMP signaling components following stress. Results are presented as logarithmic values of stress/nonstress expression level ratios. B, Representative images of DG of chronically stressed Grem2^−/− and WT mice of both sexes, staining for pSmad1/5/8, Nestin, and Sox2. Extended Data of single channel images of Sox2, Nestin, and pSmad1/5/8 immunofluorescence staining are included in Extended Data Figure 5-1. C, D, Male, but not female, Grem2^−/− mice have significantly higher levels of canonical BMP signaling and fewer Sox2⁺/Nestin⁺ cells following chronic stress compared with WT controls (images taken between the bregma −2.35 and −2.48 mm).

Figure 6.

Grem2^−/− mice suffer from increased susceptibility to and severity of seizures. A, B, Mice were injected with 20 mg/kg of KA with subsequent behavioral observation for 75 min before being killed at 120 min (B). C, Grem2^−/− mice reach generalized tonic seizure 12 min faster on average than WT counterparts. D, Significantly more Grem2^−/− mice reach the highest severity level of seizure (Racine 6) in 75 min. E, Grem2^−/− mice suffered from higher mortality at 120 min postinjection.