MRC DTP: Detecting phenotypes in a mouse model of O-GlcNAc-linked intellectual disability

Protein O-GlcNAcylation is an essential posttranslational modification of Ser/Thr residues on nucleocytoplasmic proteins with N-acetylglucosamine (GlcNAc). It is regulated by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (Fig. 1). OGT catalyses O-GlcNAcylation and possesses a catalytic domain and N-terminal tetratricopeptide repeats (TPRs) that mediate substrate recognition. Ogt is X-linked in vertebrates and is required for mouse embryogenesis. OGT and O-GlcNAc are particularly abundant in neurons and enriched in the postsynaptic density (PSD). O-GlcNAcylation regulates excitatory synapse maturation and is altered in brains of Alzheimer’s patients.

Intellectual Disability (ID) is characterised by substantial limitations in cognitive function and adaptive behaviour, affecting 1-3% of the population. To date, >800 genes (disproportionately X-linked) have been associated with ID, which is often syndromic and associated with developmental delay (DD). Very recently, the van Aalten lab has reported families where missense OGT mutations segregate with ID (Figs. 1). These patients share clinical phenotypes, including DD, behavioural problems and ataxia, suggesting a new syndromic form of ID, OGT-linked Congenital Disorder of Glycosylation (OGT-CDG). Recently we have described the first OGT-CDG patients with mutations in the active site of OGT (N567K23 and N648Y22, Figs. 1, see references).

This project will focus upon the characterisation of two mouse models which carry OGT-CDG mutations in the Ogt gene (OgtN648Y and OgtN567KT) and enable us to test the hypothesis that altered protein O-GlcNAcylation contributes to OGT-CDG phenotypes. These mice have been generated and have been transferred to the SLS animal unit where they are being backcrossed to reach isogenicity.

Building on the expertise in the van Aalten lab, the student will study alterations in O-GlcNAc homeostasis in protein lysates from whole brain tissue/regions (including hippocampus, striatum, cortex, brain stem, cerebellum, and midbrain). Using established O-GlcNAc proteome enrichment strategies, quantitative O-GlcNAc proteomics will be performed on the brain lysates from OGT-CDG mice to quantify changes in the total proteome which may indentify candidate conveyors of the OGT-CDG phenotype for analysis.

Building on the expertise in the McNeilly lab, the student will perform behavioural tests will be performed to assess motor skills (rotarod), learning and memory (novel object recognition) and anxiety (open field and elevated plus-maze) to quantify any cognitive defects in the OGT-CDG mice. Brain morphology, neuronal density and synaptic function will be evaluated in the aforementioned regions by staining for neuronal (NissI, NeuN) and synaptic markers (PSD-95, synaptophysin I, NR2A/NR2B ratio). We will investigate how glial subpopulations contribute to the OGT-CDG phenotypes using specific subtypes markers for astrocytes (GFAP), oligodendrocytes (MOG), and microglia cells (TMEM119). Cellular architecture of individual neurons (neuronal arborisation, spine density and spine morphology) will be studied with Golgi staining. These parameters are commonly disrupted in patients with ID and mouse models recapitulating these diseases. Histology will initially be performed on brains from adult mice (3 months) and repeated at earlier developmental stages.