Does APC/CCDH1 control the human brain size?: An Editorial Highlight for 'A novel human Cdh1 mutation impairs anaphase-promoting complex/cyclosome (APC/C) activity resulting in microcephaly, psychomotor retardation, and epilepsy' on page 103.

This editorial highlights a study by Rodriguez, Sanchez-Moran et al. (2019) in the current issue of the Journal of Neurochemistry, in which the authors describe a microcephalic boy carrying the novel heterozygous de novo missense mutation c.560A> G; p.Asp187Gly in Cdh1/Fzr1 encoding the APC/C E3-ubiquitin ligase cofactor CDH1. A functional characterization of mutant APC/CCDH1 confirms an aberrant division of neural progenitor cells, a condition known to determine the mouse brain cortex size. These data suggest that APC/CCDH1 may contribute to the regulation of the human brain size.

anaphase‐promoting complex/cyclosome

Cdc20‐homolog‐1/Fizzy‐related protein‐1

6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase‐3

Microcephaly is a brain development disorder resulting in a reduced size of both brain and head (Barkovich et al. 2012). Although the causes of microcephaly are diverse, many of them are genetic, a circumstance that has enabled to understand the physiology of cortical development (Thornton and Woods 2009). Some of these genes are involved in the control of mitotic function suggesting that the disease pathology is associated with aberrant neural progenitor cell proliferation. The anaphase‐promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase essential for the regulation of cell division (Garcia‐Higuera et al. 2008). Depending on the cell cycle stage, APC/C requires binding to either CDC20 or CDH1 (also known as Fzr1) for its full activity. To exit mitosis, APC/C needs to be activated by CDH1, and the APC/CCDH1 complex remains active during the G1 phase of the cell cycle as well as in G0 (postmitotic cells) (Garcia‐Higuera et al. 2008). As postmitotic cells, fully differentiated neurons show a highly active APC/CCDH1 complex, which continuously promotes the degradation of proteins such as 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase‐3 (PFKFB3) (Herrero‐Mendez et al. 2009), ROCK2 (Bobo‐Jimenez et al. 2017), and cyclin B1 (Almeida et al. 2005) to regulate energy metabolism, dendritic integrity, memory, and survival (Bolaños 2016). Interestingly, during mouse brain development, APC/CCDH1 was found to control the division of neural progenitor cells to determine the correct size of the brain cortex (Delgado‐Esteban et al. 2013). Indeed, genetic deletion of Cdh1 specifically during the embryonic stage impairs neurogenesis causing microcephaly (Delgado‐Esteban et al. 2013), thus posing the loss of APC/CCDH1 function as a potential cause of human microcephaly.

Our current knowledge on APC/CCDH1 function in the brain has been built on research performed in cellular and non‐human organismal models. Hence, the actual importance of this ubiquitin ligase in human brain pathophysiology has so far remained uncertain. In the current issue of the Journal of Neurochemistry, Rodríguez et al. (2019) report the first case of a putatively pathogenic, heterozygous missense mutation in Cdh1 causing microcephaly, mental retardation, spasticity, and epilepsy in a 4‐year‐old boy. Assuming a functionally important role in brain development, the authors searched for potentially disease‐causing variants in the Cdh1 gene screening several hundreds of whole exomes, all of which were previously sequenced in individuals with neurodevelopmental disorders of a potentially genetic origin. The heterozygous missense mutation c.560A>G; p.Asp187Gly was verified to have arisen de novo from healthy, non‐consanguineous parents of Spanish descend (Rodriguez et al. 2019). Besides severe antenatal microcephaly, the boy showed psychomotor retardation and refractory epilepsy, well‐known signs of microcephaly. To link the p.Asp187Gly mutation in Cdh1 with the microcephaly phenotype, the authors functionally characterized the mutant protein. The finding that CDH1 protein abundance was substantially lower in the patient’s leucocytes when compared with those isolated from his parents (Rodriguez et al. 2019) strongly suggests an impaired E3 ubiquitin ligase APC/C activity. To confirm this, the authors engineered, by site‐directed mutagenesis, the mutant (c.560A>G) full‐length Cdh1 cDNA. The implementation of mutant Cdh1 in human HEK293T cells confirmed the reduced expression of CDH1 protein (Rodriguez et al. 2019). To ascertain whether APC/C activity was impaired, the authors determined the protein abundances of two well‐known substrates of APC/CCDH1, namely cyclin B1 (Almeida et al. 2005) and PFKFB3 (Herrero‐Mendez et al. 2009). Both protein abundances were found to be significantly increased in cells transfected with mutant Cdh1 when compared with cells expressing wild‐type CDH1 (Rodriguez et al. 2019). Confocal imaging characterization of HEK293T cells expressing either the mutant or the wild‐type CDH1 protein confirmed exclusive nuclear localization of the mutant form (Rodriguez et al. 2019), a condition compatible with low CDH1 protein stability (Nagai et al. 2018). Moreover, analysis of the cell cycle distribution revealed a severe delay in the exit from mitosis in HEK293T cells expressing mutant CDH1 (Rodriguez et al. 2019). Thus, the functional characterization of human cells carrying mutant CDH1 confirms the typical features of APC/CCDH1 loss of function. Finally, the authors expressed either the mutant or the wild‐type CDH1 in mouse neural progenitor cells isolated from a Cdh1‐null genetic background. The analysis of cell cycle revealed an enlargement of the S‐phase in those cells expressing the mutant CDH1 (Rodriguez et al. 2019), that is, the typical feature of neurogenesis impairment causing microcephaly previously reported by the authors via genetic Cdh1 ablation (Delgado‐Esteban et al. 2013). Altogether, these sets of elegant experiments strongly indicate that the novel missense mutation c.560A>G; p.Asp187Gly identified in human Cdh1 results in APC/C loss of activity causing the typical neurogenesis impairment of microcephaly (Fig. 1).

Figure 1

APC/CCDH1(D187G) may cause microcephaly. The novel p.Asp187Gly (D187G) mutation found by Rodriguez, Sanchez‐Moran et al. (2019) in the human Fzr1 gene causes a substantial loss of anaphase‐promoting complex/cyclosome (APC/C) activity. This results in replicative stress of neural progenitor cells (NPCs) leading to impaired neurogenesis (formation of new neurons) during the embryonic stage. Based on the patient’s phenotype, one can hypothesize that this mutation leads to an antenatally reduced size of the brain cortex (microcephaly).

It is intriguing, however, that this heterozygous Cdh1 mutation is sufficient to develop such a severe phenotypic impact (Rodriguez et al. 2019). Primary microcephaly has already been associated with disturbed mitosis, but the underlying mode of inheritance has typically been autosomal recessive (Thornton and Woods, 2009; Faheem et al., 2015). Furthermore, a dominant negative effect is usually not expected in a loss of function pathomechanism. The authors explain that this observation might be ascribed to the low intolerance score of the Cdh1 gene (Firth et al. 2009; Lek et al., 2016) indicating that the loss of function of one mutated allele cannot be compensated by the wild type (Rodriguez et al. 2019). While this may be a suitable explanation, it should be noticed that heterozygous Cdh1‐knockout mice (Cdh1) show no alterations in the cell division of neural progenitors, which are identical to that found in the wild‐type animals (Delgado‐Esteban et al. 2013). These data suggest that the loss of mutant p.Asp187Gly CDH1 protein may not be sufficient to explain the lack of APC/C function. One methodological limitation of this work might be that additional, regulatory variants placed within an intronic region of Cdh1 could not be excluded by whole exome sequencing. Whether there is another functionally relevant splice mutation lingering on the trans allele in this patient remains to be elucidated. Co‐segregation analyses, which are in principle an important step to approach for a variant of unknown significance, were not eligible in this case, as the parents were excluded to be carriers due to the in trio sequencing; thus, it was not necessary to gather further information on siblings.

In conclusion, the study by Rodriguez, Sanchez‐Moran et al. (2019) demonstrates in an excellent way, how a precise interplay of clinical phenotyping, modern sequencing techniques, and functional models can help to identify the molecular genetic cause of even a very rare condition and to understand the underlying molecular mechanisms leading to brain development disorders. Whether the mutant p.Asp187Gly CDH1 protein has any structural feature that impairs its ability to interact with APC/C substrates is another issue remaining to be explored. To elucidate this question and to unambiguously demonstrate the cause–effect relationship between the p.Asp187Gly Cdh1 mutation and microcephaly, it would be needed to characterize a knock‐in mouse genetically engineered to harbor such a mutation in heterozygosity. Addressing this aspect would shed light not only onto the molecular mechanism of CDH1 protein–protein interaction in APC/C function but also onto the regulation of human cortical brain size and, hence, intellectual ability.