Work Package 2: Localisation and interactome analysis of dystrophin isoforms in the mouse and human brain

Led by Jennifer Morgan (UCL) 

WP2 aims to determine the localization of dystrophin isoforms in the mouse and adult human brain, transcriptomic changes in selected brain areas that are linked to loss of one or more dystrophin isoforms and identification of candidate dystrophin protein interactors in the mouse brain.  

We found that different dystrophin isoforms were present in different areas of the normal human adult brain, with Dp427c RNA levels being highly expressed in the hippocampus. Dp140, Dp71, and Dp40 showed medium expression levels, with enrichment of Dp71 in the hippocampus and Dp40 in the cerebellum. Dp427m and Dp427p2 were expressed at lower levels in all samples. The corpus callosum, substantia nigra, and nucleus dentate were the areas with the lowest isoform representation. 

In the healthy control mouse brain, Dp427c protein expression was high in the cortex, while Dp427p1 and Dp427p2 were highly expressed in the cerebellum. RNA expression of Dp427c and Dp71 was abundant in the cortex, hippocampus, amygdala and cerebellum. Dp427m and Dp140 were expressed at low levels throughout the brain.  

As expected, there was no Dp427 protein in either Duchenne mouse model (mdx5cv and mdx52), while Dp140 was only deficient in the mdx52 mouse. Dp140 levels were unchanged in the brain of mdx5cv mice.  Dp71 RNA was expressed in the mdx5cv model, while only very low levels were expressed in the mdx52 model.  

We found proteins that interact with Dp427 in different regions of the mouse brain associated with anxiety, and the most significant of these will be validated with immunohistochemistry and Western blot analysis.  We will also analyse interactome data for Dp140 and Dp71.  

We previously performed single cell sequencing on the cortex and hippocampus of 2-week-old healthy control, mdx23 and mdx4cv mice. Approximately 14.5% of cells expressed DMD, regardless of the strain. The percentage of DMD expressing cells was high in the fibrous and protoplasmic astrocytes, germ cells and pericytes. DMD could be related to oligodendrocyte maturation, as the percentage of DMD expressing cells was high in oligodendrocyte precursor cells, but decreased when cells mature to oligodendrocytes.  

Differentially expressed genes identified when comparing wild type with mdx23 mice revealed similar signatures to Parkinson and Huntington disease brains, potentially associated with abnormal hippocampus and cortex morphology.