Professor Pierre Gressens outlines how the microKin project is providing a larger framework to understand the patho-mechanisms affecting the cortical size
Microcephaly, i.e. the reduction of the head circumference by more than three standard deviations (SD), is a term bringing together several common clinical conditions caused by significant reduction in brain volume. Microcephaly can be syndromic or non-syndromic, genetic or environmental (including CMV, ZIKA infection, alcohol, and irradiation) in origin. Although each specific type of microcephaly is rare, microcephalies affect 2% of the overall population and are often associated with mental retardation. Most likely, microcephalies of environmental and genetic origin share common pathogenetic processes that remain to be identified.1 Among the microcephalies of genetic origin, primary hereditary microcephaly (MCPH) is characterised by a small-sized brain (-3 to -13 SD), not associated with gross anomalies of brain architecture or malformations in other organ systems, suggesting a primary and selective defect in the production of neurons. This defect is prominent in the cortical region of the brain and our studies are focused on this region.
The evolutionary increase in the cortical brain size
The evolutionary increase in the cortical brain size is related to the extension of the subventricular zone. As described in Fig. 1, during early corticogenesis, neuroepithelial cells (NECs) are the first type of progenitors to emerge. This initial pool of progenitors is amplified by symmetric cell divisions. From the onset of neurogenesis, the neural progenitor cells, which include now different sub-types, are distributed into two layers, the ventricular zone (VZ) and the subventricular zone (SVZ). The VZ is formed with radial glial cells (vRGCs), lining the cerebral ventricles and extending long basal processes spanning the whole cortical wall, as shown in Fig.1. vRGCs increasingly undergo asymmetric divisions to self-renew and give rise first to the pioneer neurons of the pre-plate (direct neurogenesis), then to the progenitors forming the SVZ, corresponding either to intermediate progenitor cells (IPC) or basal RGCs (bRGCs). These SVZ progenitors give rise to the cortical neurons (indirect neurogenesis). As shown in Fig. 2, the outcome of vRGC divisions is determined by the orientation of the division axis.2
Although the fundamental aspects of these processes have been conserved during mammalian evolution, adjustments to progenitor cell production have been necessary to allow the remarkable evolutionary expansion of the cerebral cortical surface in large primates including humans. Cortical expansion is in fact associated with the extension of the SVZ to develop an external layer, i.e. the outer SVZ (OSVZ), which comprises IPCs and bRGCs.
bRGCs initially arise from vRGCs by asymmetric division, then divide in the OSVZ, undergoing self-renewing symmetric divisions and asymmetric divisions to generate IPCs and neurons. This massive expansion of OSVZ progenitors is a key anatomical correlate of the progressive increase in cognitive capacities through mammalian evolution. The molecular mechanisms underlying the evolutionary-controlled OSVZ expansion are mostly unknown.3
MCPH: a convenient model to investigate the mechanisms controlling the cortical brain growth
A preliminary functional characterisation of the so far identified 19 microcephaly genes (MCPH1-19) has shown that most MCPH genes encode proteins associated with the centrosome and its environment, or with chromosome condensation, as indicated in Fig. 5. This suggests a primary role of MCPH genes in cell cycle regulation of neural progenitors.4
In MCPH patients, three non-exclusive mechanisms can be hypothesised to explain the dramatic reduction of brain size and the accompanying paucity of neurons:
- A cell cycle impairment, leading to the reduced proliferative capacity of progenitors;
- An imbalance between self-renewing and neurogenic divisions, leading to a smaller pool of progenitors; and
- Excessive death of progenitors and/or early post-mitotic neurons.
Anomalies of the mitotic process can lead to mitotic arrest and subsequent cell death (commonly referred to as mitotic catastrophe). Mouse models of microcephaly in which MCPH genes are mutated have allowed us to unravel some of the underlying molecular defects, such as a lack of co-ordination of centrosome assembly with cell cycle progression or a lack of co-ordination between the microtubule network and mitotic spindle assembly.5
However, despite these significant advances in our understanding of microcephaly, major gaps remain. Based on this limited knowledge, the challenge is to enlarge our knowledge on the pathways by which defects in these genes could lead to aberrant cell divisions and/or the large-scale death of progenitors that attempt to divide under sub-optimal energetic or other conditions, leading to a shortage of adult neurons of the correct type.
The microKin project
The microKin project has been designed to provide a larger framework to understand the patho-mechanisms affecting the cortical size. It benefits from the complementary expertise of the four partners: Pierre Gressens, Inserm France, co-ordinator; Pierre Vanderhaeghen, Université Libre de Bruxelles (ULB), Belgium; Wieland Huttner, Max Planck Institute-Cell Biology and Genetics (MPI-CBG), Germany; Marcos Malumbres, Centro Nacionales de Investigaciones Oncologicas (CNIO), Spain. Hence, this project relies on strategies combining a wide panel of approaches to compare the functions of three MCPH genes – ASPM, CDK5RAP2 and MCPH1 – and to newly address functional interactions between these genes and cell cycle kinases, such as PLK1 and AURKA. These approaches include conditional knockouts or inducible expression of tagged MCPH1, CDK5RAP2, ASPM, Aurora A kinase, PLK1, either in vivo or in neural progenitor cells; pharmacological tools (kinase inhibitors); detailed comparative expression studies in the developing murine and human neocortex; and in vitro models of corticogenesis, based on human iPSCs derived from MCPH patients or mutated using the CRISPR/Cas9 strategy.
These tools are currently used to explore the influence of cell cycle kinase on loading MCPH proteins at the centrosome, the impact of pathways of the energetic metabolism on neural progenitor homeostasis, and to identify further molecular partners of MCPH proteins and cell cycle kinases. Furthermore, we developed strategies to identify genes expressed exclusively in progenitor cells of the OSVZ of humans or both humans and great apes, and thus potentially involved in the amplification of OSVZ progenitors. Interestingly, among the panel of genes showing this restricted pattern, we functionally validated two genes, ARHGAP11b and NOTCH2NL, involving them in the control of the production of OSVZ progenitors.
Of note, these two genes are derived from a duplication of the ancestral ARHGAP11a and NOTCH2 genes. Finally, the consortium has the access and expertise to analyse fetal human brain tissue from control and microcephalic patients, including MCPH patients, as well as human iPSCs (hiPSCs) from MCPH patients, allowing us to specifically address:
- The similarities and differences in behaviour between rodent and human progenitor cells; and
- Cortical morphogenetic defects associated with MPCH, and how these defects could also underpin other types of congenital microcephalies, thus expanding the impact of our findings to the study of other disorders.
In summary, the combined and complementary expertise of the present consortium allows it to address the pathogenesis of MCPH through a very original angle, considering not only the contribution of centrosomal defects to impaired progenitor cell division but also other potential mechanisms, such as mitotic catastrophe, imbalances in energy metabolism, and the control of MCPH protein function by specific kinases that may be of therapeutic relevance to this disease. Together, these studies will provide a larger framework to understand the complex interplay between MCPH genes and other key pathways to maintain the proper progenitor pool homeostasis necessary for the completion of neurogenesis.
This project was selected for funding by the ERA-NET II call in 2015.
1 Woods, C.G. and Basto, R., 2014, DOI: 10.1016/j.cub.2014.09.063 ; Duerinckx, S. and Abramowicz, M., 2018, DOI: 10.1016/j.semcdb.2017.09.015
2 Florio, M. and Huttner, W.B, 2014, DOI: 10.1242/dev.090571
3 Dehay, C. et al. 2015, 10.1016/j.neuron.2014.12.060 ; Sun, T. and Hevner, R.F., 2014, DOI : 10.1038/nrn3707
4 Duerinckx, S. and Abramowicz, M., 2018, DOI: 10.1016/j.semcdb.2017.09.015
5 Woods, C.G. and Basto, R., 2014, DOI: 10.1016/j.cub.2014.09.063 ; Duerinckx, S. and Abramowicz, M., 2018, DOI: 10.1016/j.semcdb.2017.09.015
6 Florio, M. et al., 2015, DOI: 10.1126/science.aaa1975
7 Florio, M. et al., 2018, DOI: 10.7554/eLife.32332 ; Susuki, I.K. et al., 2018, DOI : 10.1016/j.cell.2018.03.067