Eml1 depletion impairs the microtubule cytoskeleton and the binding affinity of its partners in cortical heterotopia

Background:

The layered structure of the cerebral cortex is formed through a complicated sequence of highly controlled stages. During this process, the perturbations of neuronal migration and cell division can result in a rare disorder called cortical heterotopia. Heterotopia patients can have recurrent epileptic seizures, developmental delays, and mild intellectual disabilities. Studying heterotopia has been challenging because human mutations linked to the disease often do not result in heterotopia formation in mouse models. EML1 is a microtubule-binding protein, and it stands out as the first heterotopia-associated gene where mutations lead to heterotopia formation in both humans and mice. In this study, we conducted a comparative proteomic analysis of the Eml1 cKO heterotopic mice cortices and neuronal progenitor primary cells during cerebral cortex development.

Method:

We performed label-free and dimethyl labeling-based quantitative proteomic approaches, and microtubule pelleting assays to understand how Eml1 depletion disrupts protein networks in cortical tissue and neuronal progenitor primary cells during cerebral cortex development.

Results:

Our extensive analysis of Eml1-depleted E15.5 cortices showed dysregulation of multiple synaptic proteins. However, despite achieving high coverage of quantified proteins in our cortex analysis, the proportion of dysregulated proteins was relatively low. EML1 perturbations are closely linked to heterotopia formation with phenotypes in progenitor cells, we hypothesized that deregulations in progenitor cells might be masked in the complexity of cell types at the level of the cortex. To address this, we specifically isolated Pax6+ neuronal progenitor primary cell cultures from control and Eml1 cKO mice at E14.5. This focused approach revealed that Eml1 depletion disrupts various networks of biological processes, including cytoplasmic microtubule organization. Enrichment analysis of downregulated proteins highlighted the significant influence of EML1 on microtubule cytoskeleton organization, microtubules, and structural constituents of the cytoskeleton in neuronal progenitor cells. By using microtubule pelleting assays, we demonstrated that Eml1 depletion dramatically impairs the microtubule cytoskeleton and reduces the binding affinity of its partners.

Conclusion:

Our comprehensive proteomic mapping of cortices and progenitor cells has identified protein signatures specific to Eml1 cKO heterotopic mouse models. These findings offer a valuable resource for investigating the underlying mechanisms involved in heterotopia formation and brain development.