Vincent El Ghouzzi

Genetic primary microcephaly (PM) is a defect in early brain development leading to congenital microcephaly, mostly recessively inherited, and mild-to-moderate intellectual disability. PM has been largely elucidated, thanks to exome and genome sequencing. However, radial microbrain, the most severe form of genetic PM or micrencephaly described in the 1980s, which leads to early lethality or very severe intellectual handicap, remains without a molecular diagnosis. We sought to identify the cause of radial microbrain by analyzing the genotype of children/adults and fetuses with an extremely small brain. We searched for individuals with the smallest head circumference among patients with a confirmed diagnosis of PM included in 2 French and European observational studies coordinated at the Robert Debré Children's Hospital in Paris. Their neurodevelopment and brain imaging were analyzed, as well as next-generation sequencing for a panel of microcephaly genes or exome sequencing. Neuropathologic and immunohistologic analyses of extremely severe microcephalic fetal brains and stage-matched controls were performed. A nonparametric test and Mann-Whitney post-test were used to compare the cortical thickness between groups. We identified 5 individuals (4 female patients, 7 years 10 months–19 years) with a particularly small brain among a series of 50, all suffering from a severe neurodevelopmental disorder with no ability to communicate verbally and, in 3 of them, no ability to walk. Genetic analysis revealed in all individuals the presence of the same homozygous variant c.2953A>G (p.R985G) in the RTTN gene (ROTATIN). The same variant was found in 2 fetuses whose neuropathologic evaluation showed a major reduction in the thickness of the ventricular zone and neuronal heterotopias. The cortical plate was reduced by 70% compared with controls, irrespective of the region considered. Immunostaining with vimentin showed a 50% loss of radial glial columns, characteristic of radial microbrain. Our data show that the homozygous c.2953A>G substitution in RTTN is a recurrent variant responsible for radial microbrain, the most severe form of primary microcephaly. Our combined neurologic, imaging, and histopathologic approaches provide a better understanding of the severity of this condition and its prognosis. ClinicalTrials.gov number: NCT01565005.

GRASP65 is a Golgi-associated peripheral protein encoded by theGORASP1gene and required for Golgi cisternal stacking in vitro. A key role of GRASP65 in the regulation of cell division has also been suggested. However, depletion of GRASP65 in mice has little effect on the Golgi structure and the gene has not been associated with any human phenotype to date. Here, we report the identification of the first human pathogenic variant ofGORASP1(c.1170_1171del; p.Asp390Glufs*18) in a patient combining a neurodevelopmental disorder with neurosensory, neuromuscular, and skeletal abnormalities. Functional analysis revealed that the variant leads to a total absence of GRASP65. The structure of the Golgi apparatus did not show fragmentation, but glycosylation anomalies such as hyposialylation were detected. Mitosis analyses revealed an excess of prometaphases and metaphases with polar chromosomes, suggesting a delay in the cell cycle. These phenotypes were recapitulated in RPE cells in which a similar mutation was introduced by CRISPR/Cas9. These results indicate that loss of GRASP65 in humans causes a novel Golgipathy associated with defects in glycosylation and mitotic progression.

Association genetic studies and genome-scale CRISPR screens have recently identified ARF3 and TMEM251/LYSET/GCAF as Golgi-resident factors essential to brain and skeletal development. Here we discuss how even though the consequences of mutations in these genes affect endosomal and lysosomal compartments, the problem originates in the Golgi complex and may involve either the identity of the carrier vesicles or that of cargo molecules.

The Golgi apparatus (GA) is essential for intracellular sorting, trafficking and the targeting of proteins to specific cellular compartments. Anatomically, the GA spreads all over the cell but is also particularly enriched close to the base of the primary cilium. This peculiar organelle protrudes at the surface of almost all cells and fulfills many cellular functions, in particular during development, when a dysfunction of the primary cilium can lead to disorders called ciliopathies. While ciliopathies caused by loss of ciliated proteins have been extensively documented, several studies suggest that alterations of GA and GA-associated proteins can also affect ciliogenesis. Here, we aim to discuss how the loss-of-function of genes coding these proteins induces ciliary defects and results in ciliopathies.

How the brain develops and achieves its final size is a fascinating issue that questions cortical evolution across species and man’s place in the animal kingdom. Although animal models have so far been highly valuable in understanding the key steps of cortical development, many human specificities call for appropriate models. In particular, microcephaly, a neurodevelopmental disorder that is characterized by a smaller head circumference has been challenging to model in mice, which often do not fully recapitulate the human phenotype. The relatively recent development of brain organoid technology from induced pluripotent stem cells (iPSCs) now makes it possible to model human microcephaly, both due to genetic and environmental origins, and to generate developing cortical tissue from the patients themselves. These 3D tissues rely on iPSCs differentiation into cortical progenitors that self-organize into neuroepithelial rosettes mimicking the earliest stages of human neurogenesis in vitro. Over the last ten years, numerous protocols have been developed to control the identity of the induced brain areas, the reproducibility of the experiments and the longevity of the cultures, allowing analysis of the later stages. In this review, we describe the different approaches that instruct human iPSCs to form cortical organoids, summarize the different microcephalic conditions that have so far been modeled by organoids, and discuss the relevance of this model to decipher the cellular and molecular mechanisms of primary and secondary microcephalies.

Aim To characterize the cortical structure, developmental, and cognitive profiles of patients with WD repeat domain 62 (WDR62)-related primary microcephaly. Method In this observational study, we describe the developmental, neurological, cognitive, and brain imaging characteristics of 17 patients (six males, 11 females; mean age 12y 3mo standard deviation [SD] 5y 8mo, range 5y–24y 6mo) and identify 14 new variants of WDR62. We similarly analyse the phenotypes and genotypes of the 59 previously reported families. Results Brain malformations, including pachygyria, neuronal heterotopia, schizencephaly, and microlissencephaly, were present in 11 out of 15 patients. The mean full-scale IQ of the 11 assessed patients was 51.8 (standard deviation [SD] 12.6, range 40–70). Intellectual disability was severe in four patients, moderate in four, and mild in three. Scores on the Vineland Adaptive Behavior Scales obtained from 10 patients were low for communication and motor skills (mean 38.29, SD 7.74, and 37.71, SD 5.74 respectively). The socialization score was higher (mean 47.14, SD 12.39). We found a significant difference between scores for communication and daily living skills (mean 54.43, SD 11.6; p=0.001, one-way analysis of variance). One patient displayed progressive ataxia. Interpretation WDR62-related cognitive consequences may be less severe than expected because 3 out of 11 of the assessed patients had only mild intellectual disability and relatively preserved abilities of autonomy in daily life. We identified progressive ataxia in the second decade of life in one patient, which should encourage clinicians to follow up patients in the long term.

Endoplasmic reticulum and Golgi stress in microcephaly – Microcephaly is a neurodevelopmental condition characterized by a small brain size associated with intellectual deficiency in most cases and is one of the most frequent clinical sign encountered in neurodevelopmental disorders. It can result from a wide range of environmental insults occurring during pregnancy or postnatally, as well as from various genetic causes and represents a highly heterogeneous condition. However, several lines of evidence highlight a compromised mode of division of the cortical precursor cells during neurogenesis, affecting neural commitment or survival as one of the common mechanisms leading to a limited production of neurons and associated with the most severe forms of congenital microcephaly. In this context, the emergence of the endoplasmic reticulum (ER) and the Golgi apparatus as key guardians of cellular homeostasis, especially through the regulation of proteostasis, has raised the hypothesis that pathological ER and/or Golgi stress could contribute significantly to cortical impairments eliciting microcephaly. In this review, we discuss recent findings implicating ER and Golgi stress responses in early brain development and provide an overview of microcephaly-associated genes involved in these pathways.

Cell division and differentiation are two fundamental physiological processes that need to be tightly balanced to achieve harmonious development of an organ or a tissue without jeopardizing its homeostasis. The role played by the centriolar protein STIL is highly illustrative of this balance at different stages of life as deregulation of the human STIL gene expression has been associated with either insufficient brain development (primary microcephaly) or cancer, two conditions resulting from perturbations in cell cycle and chromosomal segregation. This review describes the recent advances on STIL functions in the control of centriole duplication and mitotic spindle integrity, and discusses how pathological perturbations of its finely tuned expression result in chromosomal instability in both embryonic and postnatal situations, highlighting the concept that common key factors are involved in developmental steps and tissue homeostasis.