A Novel Arhgef6 Mouse Model Shows Focal Volume Loss in the Hippocampus and Deep Cerebellar Nuclei

Background:   Mutations in the ARHGEF6 (Rac/Cdc42 Guanine Nucleotide Exchange Factor (GEF) 6) gene have been previously linked to non-specific X-linked mental retardation (Kutsche et al. 2000).  A null-mutant was previously created and characterized using histology and electrophysiology (Ramakers et al. 2012).  That study found that the gross brain structure appeared normal; however, Golgi staining revealed an increase in both dendritic length and spine density in the hippocampus. Mutations in ARHGEF6 haven also been recently found in autism (Jiang et al. 2013).

Objectives:   To assess the neuroanatomical differences in a novel autism gene candidate, ARHGEF6 with high-resolution structural MRI.

Methods:  

A novel Arhgef6 loss-of-function mouse model was produced from targeted-trap C57BL/6N ES cells (Arhgef6^{tm2e.1(EUCOMM)Wtsi}/Tcp) and maintained on a C57BL/6N background. As a first screen 31 fixed mouse brains were examined.  Sixteen of which were Arhgef6(tm1e.1/y) mice and the other 15 were WT controls (C57BL6/N). The mice were P60 ± 2 days.

MRI Acquisition – A multi-channel 7.0 Tesla MRI scanner was used to acquire anatomical images of the brain. A T2-weighted, 3-D fast spin-echo sequence was used (restricted sampling to a circular region in the two phase encode dimensions). This sequence yielded an image with 40 μm isotropic voxels (3D pixel) in ~14 h.

Data Analysis – To visualize and compare any differences the images from each group are registered together. The goal of the registration is to model how the deformation fields relate to genotype (Lerch et al., 2008). Volume differences are then calculated either in individual voxels or for 159 different segmented regions (Dorr et al. 2008, Ullmann et al. 2013, and Steadman et al. 2014). Multiple comparisons were controlled for using the False Discovery Rate (FDR) (Genovese et al., 2002).

Results:   The total brain volume was unchanged (432 ± 6 mm³ for Arhgef6(-/y) and 432 ± 14 mm³ for WT); however there was a significant difference in the variability in the groups (p=0.02, Levene’s test).  After normalizing for total brain volume, 11 of the 159 regions were found to be significantly different at an FDR of <5%.  Notable decreases were found in the hippocampus (-2.57%, FDR<1%), the inferior and superior colliculi (-3.74 and -3.36, respectively, both FDR <1%), and all three deep cerebellar nuclei, namely the dentate nucleus (-2.65%, FDR=6%), nucleus interpositus (-4.81, FDR <1%), and fastigial nucleus (-4.05, FDR <1%).  These findings can also be seen voxelwise in Figure 1.

Conclusions:   Ramakers et al. reported that Arhgef6 was primarily expressed in the hippocampus, where we also identify significant volume losses. In addition, we also identified focal alterations in the deep cerebellar nuclei, supporting recent evidence implicating cerebellar outputs in autism and mental retardation.