Functional characterization of RELN missense mutations involved in recessive and dominant forms of Neuronal Migration Disorders

Background

Cerebral cortex development is a protracted and highly orchestrated process depending, amongst other events, on glutamatergic neuron production from proliferative zones followed by radial migration to reach the appropriate position in the developing cortical plate. In the cortex, neuronal migration happens in an inside-out fashion: later-born neurons bypass the early generated ones and are thus positioned in more superficial regions. Abnormalities in cortical neuronal migration can result in severe cortical malformations affecting cortical lamination and/or folding, and often resulting in neurodevelopmental diseases (Francis and Cappello, 2020).

The glycoprotein Reelin (RELN) has been reported to be involved in several steps of neuronal migration (Jossin, 2020). This extracellular matrix protein is secreted into the extracellular environment where it is cleaved at both its N-terminal and C-terminal domains, and where it homodimerizes (Hattori and Kohno, 2021). In humans, autosomal recessive (homozygous or compound) RELN mutations have been associated with different cortical malformation patterns in eight different families. Additionally, heterozygous mutations in RELN have been identified as risk factors for neuropsychiatric as well as for neurodegenerative disorders, including schizophrenia, Autism Spectrum Disorders (ASD) and Alzheimer’s Disease (Ishii et al, 2016). Although extensive work has been performed to study Reelin’s role in the developing cortex, little is known about how these patient-specific mutations affect Reelin’s secretion, cleavage, and function(s).

In this study, the authors report six patients harboring RELN missense mutations who present a spectrum of cortical malformations associated with abnormal neuronal migration. Then, they perform a set of in vitro and in vivo assays with the goal of assessing the impact of the different mutations on Reelin secretion, processing, and function, as well as to correlate molecular and cellular events with the severity of the pathology observed in the patients.

Main results

First, the authors identified six children presenting compound, maternally-inherited and de novo heterozygous RELN missense mutations (comprising a total of 7 RELN variants). These patients show a variety of cortical malformations including pachygyria (simplified gyrification), polymicrogyria (too many folds that are abnormally small), and periventricular node heterotopia (mispositioned neurons close to the ventricle). Importantly, the authors provide the first evidence of autosomal dominant neuronal migration disorders arising due to de novo heterozygous RELN mutations.

Then, the authors assessed the impact of the different missense mutations on Reelin secretion and processing. To address this question, Riva and colleagues transfected HEK293T cells with plasmids carrying mouse WT-RELN or the different patient mutations. In the first set of experiments, secretion and cleavage were assessed for each mutation individually. Then, a second set of experiments was performed to recapitulate ‘patient conditions’: compound heterozygous mutations were transfected together, and de novo and maternally-inherited heterozygous mutations were co-transfected with WT-RELN. These experiments revealed that most of the mutations (4/7) affect Reelin secretion, resulting in increased intracellular and decreased extracellular levels of Reelin. One mutation resulted in the opposite effect, and thus also showed perturbed Reelin secretion. Additionally, the co-transfection experiments showed that the de novo heterozygous mutations acted as dominant-negative forms, impairing the secretion of the co-transfected WT-RELN.

After determining how RELN mutations affect protein secretion in vitro, the authors assessed the effect of these mutations in Reelin’s activity in vivo. They first chose a previously described assay, in which overexpression of WT-RELN in the mouse developing cortex by in utero electroporation leads to the formation of organized rosette structures in caudal regions of the cortex. These rosettes are composed of cells projecting their processes towards a central RELN-rich cell body-poor inner region. Mutation-containing plasmids were electroporated, and the authors evaluated not only the presence of the rosettes, but also to what degree their organization resembled those observed upon overexpression of WT RELN. Although different effects were observed when electroporating the different mutations, overexpression of 6/7 mutations resulted in either absence of rosette structures, disorganization or mislocalization of the latter, indicating aberrant Reelin function in vivo. Of note, the heterozygous de novo mutations (2/7) acting as dominant-negative forms in vitro and showing the strongest secretion impairment, also showed the strongest phenotype in this assay, completely abolishing the capacity to form rosette-like aggregates.

Finally, the authors assessed neuronal migration upon in utero electroporation of WT and mutant RELN forms in rostral regions of the cortex, where rosettes do not form. As in the previous assay, different mutations impacted neuronal migration in different manners, with 5/7 mutations resulting in delayed migration despite neurons being arrested at different layers of the cortical wall. Interestingly, 2/3 mutations causing polymicrogyria in the patients resulted in neurons trailing in the deep layers of the cortex, hinting at a shared cellular mechanism leading to this pathology.

In conclusion, by combining in vitro and in vivo assays, Riva and colleagues provide an in-depth characterization of the effect of RELN missense patient mutations at the cellular and molecular level. Their results point to Reelin secretion and processing as molecular mechanisms impaired due to patient mutations and elucidate cellular events of cortical development which are likely to contribute to the etiology of the malformations presented by the patients.

Why I choose this paper

Cortical malformations can affect several features of the cortex such as its size (e.g. macrocephaly, microcephaly), folding (e.g. polymicrogyria, pachygyria), or neuronal position (e.g. periventricular heterotopia, subcortical heterotopia). Rather than appearing by themselves, these malformations frequently manifest simultaneously in patients. Additionally, genes responsible for these malformations often show different mutations, affecting different domains of the corresponding protein. Together, these two aspects make it very hard to disentangle which specific protein function(s) may be responsible for a specific pathological trait of the patients’ phenotype, as well as how different mutations affect the protein activity and its correlation with the severity of the disorder. This preprint directly tackles these questions in a direct and systematic fashion, shedding light on RELN’s function and the molecular and cellular mechanisms at play leading to malformations affecting cortical folding.

 

Questions for the authors

Did you consider examining whether homodimerization of RELN is perturbed due to the different patient mutations by using biochemical assays? Following this question, did you test if interaction with any of the known receptors of RELN is perturbed?

In utero electroporation was performed at embryonic day 14.5. Would electroporating at earlier stages, and thus targeting earlier neuronal populations, result in a different phenotype?

Overexpression of WT-RELN leads to the formation of rosettes in caudal levels of the cortex. Is RELN expression pattern different at different rostro-caudal levels of the cortex? What about the expression of its receptors or known interacting partners (e.g. α3β1 integrin)?

Could electroporating all these plasmids have an effect on radial glia directly, given that RELN is expressed underneath a ubiquitous CAG promoter?

References

Francis, F., Cappello, S. (2020). Neuronal migration and disorders – an update. Curr Opin Neurobiol, 66: 57-68.

Hattori, M., Kohno, T. (2021). Regulation of Reelin functions by specific proteolytic processing in the brain. J Biochem, 169(5): 511-516.

Ishii, K., Kubo, K. I., Nakajima, K. (2016). Reelin and neuropsychiatric disorders. Front Cell Neurosci, 10: 229.

Jossin, Y. (2020). Reelin functions, mechanisms of action and signaling pathways during brain development and maturation. Biomolecules, 10(6): 964.

Tags: cerebral cortex, cortical folding, cortical malformations, neuronal migration

Posted on: 27 July 2021

doi: https://doi.org/10.1242/prelights.30161

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