Neurodevelopmental defects in a mouse model of O-GlcNAc transferase intellectual disability

Abstract

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.

Keywords: O-GlcNAcylation; Intellectual disability; Vertebrate development.

Fig. 1.

Genome editing to introduce the OGT^C921Y mutation leads to viable mice. (A) Schematic of the OGT protein with position of the OGT^C921Y variant marked. TPR (blue), TPR-like (TLR; gray) and catalytic (orange) domains are represented. (B) Sequencing of genomic DNA of male OGT^WT and male OGT^C921Y mice confirms the presence of the C921Y point mutation in the transgenic animals. DNA sequence chromatograms for a representative OGT^WT and OGT^C921Y animal are shown alongside the corresponding DNA and amino acid sequences. The G-to-A point mutation for the C921Y variant is marked in red, as are four silent mutations intended to eliminate gRNA recognition sequences. (C) Table showing numbers and percentages of female (OGT^+/+), male (OGT^+/y), female Ogt^C921Y/+ (OGT^C921Y/+) and male Ogt^C921Y/y (OGT^C921Y/y) animals generated from breeding pairs of female Ogt^C921Y/+ and male Ogt^+/y mice. Litter size is shown as mean±s.d.

Fig. 2.

OGT^C921Y mutation causes changes in brain O-GlcNAc homeostasis. (A) Western blot analysis of O-GlcNAc, OGA and OGT levels in adult brain of OGT^WT and OGT^C921Y male mice. Anti-lamin B antibodies raised in rabbit (rLamin B) or mouse (mLamin B) were used as loading controls. Each lane represents independent biological replicates. Molecular masses of markers are shown in kDa. (B) Quantification of total O-GlcNAcylated proteins from the western blot shown in A. (C) Quantification of OGA protein levels from the western blot shown in A. (D) Quantification of OGT protein levels from the western blot shown in A. (E) Quantification of Oga mRNA levels in whole adult brain of OGT^WT and OGT^C921Y male mice by RT-PCR. (F) Quantification of Ogt mRNA levels in whole adult brain of OGT^WT and OGT^C921Y male mice by RT-PCR. Protein and mRNA levels are normalized to the mean of the corresponding OGT^WT replicate set (a.u., arbitrary units). Data in B-F are represented as mean±s.d., n=3 for all genotypes. **P<0.01; ***P<0.001 (two-tailed unpaired t-test used).

Fig. 3.

OGT^C921Y mutation leads to changes in mass and size. (A) Measurement of body weight of 77- to 91-day-old OGT^WT (n=9) and OGT^C921Y (n=7) male mice. (B) Measurement of body length (nose-to-tail length) of 77- to 91-day-old OGT^WT (n=10) and OGT^C921Y (n=10) male mice. (C) Fat mass:body weight ratio (expressed as a percentage) of 6- to 7-month-old OGT^WT (n=5) and OGT^C921Y (n=6) male mice. (D) Lean mass:body weight ratio (expressed as a percentage) of 6- to 7-month-old OGT^WT (n=5) and OGT^C921Y (n=6) male mice. (E) Basal glycemia levels of 6- to 7-month-old OGT^WT (n=5) and OGT^C921Y (n=6) male mice. Data are represented as mean±s.d. *P<0.05; **P<0.01 (two-tailed unpaired t-test used).

Fig. 4.

Microcephaly in OGT-CDG mice. (A) Measurement of skull length of 80- to 91-day-old OGT^WT (n=4) and OGT^C921Y (n=5) male mice. (B) Measurement of skull width of 80- to 91-day-old OGT^WT (n=4) and OGT^C921Y (n=5) male mice. (C) Lateral and superior views of representative microCT three-dimensional reconstructions of the skull of 80- to 91-day-old OGT^WT and OGT^C921Y male animals. Scale bars: 5 mm. (D) Lateral and superior views of a representative microCT three-dimensional reconstruction of a mouse skull presenting 14 landmarks (LM1-LM14) used for Euclidean distance matrix analysis (EDMA). (E) Absolute brain weight of 80- to 91-day-old OGT^WT (n=5) and OGT^C921Y (n=7) male mice, as determined using a precision weighing scale. (F) Brain weight:body weight (BW) ratio (expressed as a percentage) of 80- to 91-day-old OGT^WT (n=5) and OGT^C921Y (n=7) male mice. Data in A,B,E and F are represented as mean±s.d. **P<0.01; ***P<0.001 (two-tailed unpaired t-test used).