MED27 function is essential for cerebellar development and motor behaviour

Abstract

Figures

I](#awaf237-F1) and [J](#awaf237-F1)), including in the cerebellum. These findings align with the clinical evidence of the loss of GABAergic neurons in several neurodevelopmental disorders including mental retardation,^[6](#awaf237-B6)^ a key feature in MED27 variant patients. To complement our observations of cerebellar abnormalities and GABAergic neuronal loss in *med27* F0 KO zebrafish, we assessed the cerebellar neuronal parvalbumin7 (pvalb7)-positive Purkinje cells which are important inhibitory GABAergic neurons for cerebellar function. We observed a significant reduction in the number o

A](#awaf237-F2) and [B](#awaf237-F2)) and apoptosis ([Fig. 2C](#awaf237-F2) and [D](#awaf237-F2)), suggesting a failure in neurogenesis with defects in progenitor cells differentiating into GABAergic neurons. Interestingly, we also found a significant reduction in the nestin-positive neural stem cells ([Fig. 2E](#awaf237-F2) and [F](#awaf237-F2)), reinforcing that MED27 likely plays a role during early neurogenesis, particularly in regulating the development of early neural progenitors. To confirm this hypothesis, we compared the expression level of specific cerebellar neurogenesis markers^[8]

Full text

In the recent publication in Brain by Maroofian et al.,^1^ the authors provide a comprehensive clinical and radiological description of MED27-related disease. They particularly demonstrated that MED27-related disease manifests brain anomalies along with movement abnormalities. Several reports have also identified MED27 variants in a neurodevelopmental disorder characterized by key features such mental retardation, cerebellar atrophy, spasticity, hypotonia and motor deficits.^2-4^ Wu et al.^3^ showed that homozygous missense variants in the gene MED27 resulted in a significant reduction in MED27 protein expression. However, the pathogenicity of loss-of-function of MED27 and the mechanisms underlying neurodevelopmental defects in MED27-related disease currently remain largely unknown.

We investigated the role of MED27 in neurodevelopment using the zebrafish as a vertebrate model system. The zebrafish genome encodes a single med27 orthologue that is highly conserved with human MED27. We generated a med27 biallelic knockout (F0 KO) zebrafish using the CRISPR-Cas9 system.^5^ We found that med27 F0 KO larvae exhibited a smaller body, eye and head size compared to wild-type control fish (Fig. 1A–D). In light of the neurological phenotypes of patients harbouring MED27 variants, we evaluated various aspects of the morphology and function of the CNS in med27 F0 KO larvae. We investigated motor function in the med27 F0 KO fish using the automated Noldus Ethovision XT behaviour monitoring system. We found that med27 F0 KO exhibited significant motor impairments compared to controls (Fig. 1E and F). Haematoxylin and eosin (H&E) staining of transverse brain sections at 3 days post-fertilization (dpf) revealed structural differences and smaller brain size in med27 F0 KO fish compared to wild-type larvae at 3 dpf and 5 dpf (Fig. 1G and H). Importantly, we observed a marked progressive loss of H&E staining in the cerebellum of med27 F0 KO zebrafish brain compared to controls, suggesting a cellular depletion in the region (Fig. 1G). Taken together, these findings are in line with clinical observations in MED27 variant patients.

Given the brain abnormalities in MED27 patients and med27 F0 KO zebrafish, we next sought to examine the brain neuronal networks. We found a significant reduction in the number of GABAergic neurons in the med27 F0 KO zebrafish brain at 5 dpf (Fig. 1I and J), including in the cerebellum. These findings align with the clinical evidence of the loss of GABAergic neurons in several neurodevelopmental disorders including mental retardation,^6^ a key feature in MED27 variant patients. To complement our observations of cerebellar abnormalities and GABAergic neuronal loss in med27 F0 KO zebrafish, we assessed the cerebellar neuronal parvalbumin7 (pvalb7)-positive Purkinje cells which are important inhibitory GABAergic neurons for cerebellar function. We observed a significant reduction in the number of Purkinje cells within the cerebellum of med27 F0 KO zebrafish brain (Fig. 1K and L). Overall, these data suggest that med27 is involved in cerebellum development, particularly in populating GABAergic neuronal subtypes in vertebrates.

The zebrafish CNS proliferative profile is still very high at 2 dpf and is rapidly downregulated up to 5 dpf.^7^ In 5 dpf med27 F0 KO brains, we strikingly found an increase in proliferating cells (Fig. 2A and B) and apoptosis (Fig. 2C and D), suggesting a failure in neurogenesis with defects in progenitor cells differentiating into GABAergic neurons. Interestingly, we also found a significant reduction in the nestin-positive neural stem cells (Fig. 2E and F), reinforcing that MED27 likely plays a role during early neurogenesis, particularly in regulating the development of early neural progenitors. To confirm this hypothesis, we compared the expression level of specific cerebellar neurogenesis markers^8^ (atoh1a, atoh1b, atoh1c and ptf1a, for Purkinje cell neurogenesis^9,10^). We found that the expression of all these markers is significantly decreased at 2 dpf in med27 F0 KO larvae compared to controls by reverse transcription quantitative PCR (RT-qPCR) (Fig. 2G). These findings suggest that these changes caused by the loss of MED27 arise early on as of 2 dpf with important consequences on cerebellar neurogenesis at later developmental stages. Altogether, our data indicate an important role of MED27 in neurogenesis and cerebellar development.

Supplementary Material

awaf237_Supplementary_Data