Mechanisms underpinning human neocortical development and neurodevelopmental disorders: The novel role of Extracellular Matrix.

The human neocortex is the seat of many of the higher cognitive functions that make us human. These include our memory, speech and advanced learning. Our neocortex has greatly expanded during evolution, resulting in an increase in the number of nerve cells (neurons) within it, and an increase in overall size. This expansion was accompanied by folding of the cortical surface, which gives the brain its wrinkled appearance and allowed the expansion of the cortical surface area within the confinement of the developing skull.
Despite their functional importance, we know relatively little about how these folds are formed in development. What is known, is that these cortical folds are very similar between individuals, so much so, that the largest folds are almost identical. This suggests that the formation of the correct number of folds in the correct location is important for their function. In fact, cognitive defects are observed in various neurodevelopmental disorders that result in abnormal cortical folding, i.e. disorders with too much folding, such as polymicrogyria, and not enough folding, such as lissencephaly, suggesting that the regulation of folding during development is crucial.
My work has shown that the extracellular matrix (ECM), the proteins that surround cells in a tissue, has a key role in regulating how cortical folds form. Manipulating the ECM in tissue slices of human fetal neocortex kept in culture induced folding of the cortical plate. This ECM-induced folding was reduced or delayed in neocortex samples with specific neurodevelopmental defects, such as Down syndrome and prenatal methamphetamine exposure. Both of these disorders are known to have developmental delays or defects in the neocortex, which are currently not fully understood. The ECM-induced folding of human neocortex explants can now be used to probe these disorders and others. This is particularly important for the disorders that currently lack suitable animal models. Of these, the most relevant are the disorders that alter cortical folding, as many of the model systems used to study neocortex development naturally lack folding (such as mice).
I predict that there are many more functions of ECM in human neocortex development, folding and related neurodevelopmental disorders. Therefore, the overall aim of my proposal is to investigate how ECM regulates human neocortex development and its related disorders, using the novel human neocortex explant systems I have developed.
The first aim of my proposal is to use my ECM-folding assay to investigate the function of ECM genes that have already been linked to specific neurodevelopmental disorders in patients. Two candidates are the ECM protein perlecan, and the ECM receptor dystrogylcan. Both of these genes are linked to the folding disorder lissencehpaly.
The second aim is to examine the exact composition of the ECM within the developing human neocortex. There is currently very little data on what ECM is expressed in the human neocortex, when it is expressed and where it is located. This is vital information that will help us understand not only the normal function of the ECM in human neocortex development, but also help understand and predict how mutations in ECM genes led to neurodevelopmental defects.
The third aim is to test the function of ECM proteins in human neocortex development, using the human neocortex explant culture system and ECM-folding assay. This will increase our understanding of how ECM regulates the development and folding of the cortex, and its dysregulation.
This fellowship funding would allow me to set up my independent research group, addressing these fundamental questions of how the human neocortex develops and how this development goes awry in developmental disorders. It will uncover the role of the ECM in both of these processes, and increase our knowledge and understanding of the normal neocortex development and folding, and the dysregulation seen in related disorders.

I propose to elucidate the function of the extracellular matrix (ECM) in human neocortex development and neurodevelopmental disorders, using the human fetal neocortex explant systems and ex vivo folding assay I have developed. Aim 1: Determine the function of ECM in human neurodevelopmental disorders, using i) human fetal neocortex tissue with neurodevelopmental disorders, and ii) introducing known ECM perturbations linked to such disorders in healthy human fetal neocortex tissue. Cell behaviour, such as proliferation and migration, and tissue morphology, such as folding, will be analysed using live imaging and immunofluorescence. Atomic force microscopy (AFM) will be used to analyse changes in ECM stiffness alongside cell behaviour. Aim 2: Characterise ECM composition during human neocortex development, using proteomic analysis of the ECM within the different zones of the developing human neocortical wall in combination with AFM to analyse ECM stiffness. This will be performed at several key time points throughout neurogenesis and at the onset of folding (9-22 GW) to assess the dynamics of the ECM network. Aim 3: Elucidate the function of ECM and related signaling on human neocortex development and folding. The ECM candidates identified in Aim 2 will be perturbed/modified in human fetal neocortical explants using ECM-modifying enzymes, signalling pathway inhibitors, electroporation to manipulate gene expression and recombinant ECM proteins. The effect of modifying the ECM will be analysed by AFM, live imaging and immunofluorescence to assess ECM stiffness, cell behaviour, such as proliferation and migration, and tissue morphology, such as folding. Interesting candidates can then be tested in the mouse, to examine the effects of humanizing the ECM during neocortical development. Together, these aims will enable me to elucidate the function of the ECM network in human neocortex development and folding, and how disruption of this leads to neurodevelopmental disorders.

My proposed research will have short and long-term benefits for researchers, both within and outside of my field, clinicians and patients with neurodevelopmental disorders.

Researchers directly working on the project: short term benefits.

The researchers working directly on the aims outlined in my proposal will benefit from the development and advancement of skills in key areas. These include i) scientific development, using cutting-edge technology and tools to study human neocortex development and disorders, such as advanced imaging and atomic force microscopy, and ii) professional development, using courses provided by the university, such as lab management and writing skills. This will enable the enhancement of both the scientific output and career development of these researchers.

Researchers both inside and outside the field: short to mid-term benefits.

Researchers within the wider community will benefit from the findings generated from this research proposal in several ways. First, from the development of tools to study human neocortex development. This includes the refinement of the human fetal neocortex explant and imaging systems developed during my postdoctoral training. In particular, the development of a new explant system using the hydrogels to enable specific regions of the human tissue to be manipulated. Second, from the recourses and findings generated from my proposed research, such as the proteomic analysis of the extracellular matrix in human neocortical development. This information could, for example, be useful for researchers using in vitro systems, such as cerebral organoids or 2D cultures of cortical progenitors. Currently a mix of extracellular matrix is added to these cultures that is not optimized for neocortex development (Matrigel). This could be refined using the proteomic information generated in this proposal, to generate a targeted mix of ECM aimed at potentially increasing the efficiency of neocortical progenitor culture, either for proliferation or differentiation. The techniques and resources generated by this proposed research will be made available to researchers within and outside of the field, directly through collaboration and via publication.

Clinicians and patients: long-term benefits.

There is currently very limited knowledge about the mechanisms underlying many neurodevelopmental disorders. This is particularly so for those disorders that lack suitable model systems, such as the disorders that affect cortical folding. My proposed research aims to address this by increasing our knowledge of how the extracellular matrix drives human neocortical development and, when disrupted, its dysregulation. This is especially relevant for the folding disorder lissencephaly, a reduction in cortical folding, as mutations in extracellular matrix related genes have been found in patients with this disorders. Understanding the mechanisms the drive these disorders will lead to a better understanding of the disorders themselves, allowing clinicians and patients to have more information about the outcomes upon diagnosis, for example on the care that may be required. Most folding disorders are diagnosed late in gestation, due to the relatively late onset of cortical folding (around 20 gestational weeks). Therefore, if key genes can be identified that regulate cortical folding, these can then be used to help classify the type of folding disorder, or in the longer-term could be used to screen for the most severe types of folding disorders.

Finally, understanding the mechanisms that underlie the development of the human neocortex, and the disorders that affect it, could provide vital information to aid the understanding of the mechanisms underlying neurodegenerative disorders. This could be by identifying key pathways to investigate in further studies, or pathways that have the potential to be targeted therapeutically.