Small, but perfectly formed

Primary microcephaly (MCPH) is an autosomal recessive inherited disease characterised by a small, but architecturally normal brain. Although the disease results in mild to moderate, non-progressive mental retardation, development is otherwise generally normal. Dr Jacqueline Bond tells us about her work on MCPH and, in particular, the role of microscopic imaging in helping to understand brain development.

The Leeds Institute of Molecular Medicine (LIMM) is an interdisciplinary research centre dedicated to identifying molecules involved in human diseases and using this knowledge to develop novel therapies and drugs. As part of the medical school based at St James Hospital, researchers at LIMM have access to patients and patient samples enabling first-hand knowledge of disease processes. A strong bench-to-bedside and bedside-to-bench approach aims to translate research into direct benefits for patients.

Our aim, within the Section of Ophthalmology and Neuroscience, is to determine the causes of human inherited neurological disorders, and in doing so, gain insights into the processes of neurogenesis, neuronal homeostasis and function. One of the interesting features of microencephaly (MCPH) is that although the brain is small, it is otherwise normal and there are no other apparent associated developmental abnormalities. The genes involved in MCPH may, therefore, be expected to have a specific role in neurogenesis but not in other developmental processes. A greater understanding of the genes involved in MCPH and the functional aspects of these genes is likely to contribute not only to improved clinical management of patients affected by this disease but also to a greater understanding of brain development in general. 

A number of MCPH genes have been identified, most of which encode centrosomal proteins. Our group, led by Dr Geoffrey Woods, discovered the ASPM (abnormal spindle-like primary microcephaly) gene1 followed by two further genes, CDK5RAP2 and CENPJ2. Mutations in the ASPM gene at the MCPH5 locus on chromosome 1q31.3 are considered the most common cause of MCPH. To date, around 100 mutations have been identified across its 10 kilobase gene. All these mutations cause MCPH but, although we occasionally see families with a more severe phenotype associated with a much smaller brain size and some patients experience epileptic seizures or have reduced height, there are few mutation/phenotype correlations.

It is known that ASPM is expressed in the neuroepithelial layer surrounding the ventricles of the brain and that the ASPM protein localises to the spindle poles during mitosis. ASPM is thought to have a role in controlling the fate of neuroprogenitor cells. A progenitor cell can divide in two ways; symmetrically to make two identical progenitor daughter cells; or asymmetrically when the cleavage furrow in the cell is rotated by 90 degrees, to produce one daughter progenitor cell and one specialised brain cell i.e a neuron. The change in the type of daughter cells is related to the position of cell fate determinants on the cell membrane and the way in which these determinants are allocated to daughter cells during cell division. Normally, ASPM appears to be involved in maintaining symmetrical cell division increasing the number of progenitor cells and providing a high capacity for neuron generation. In MCPH, the maintenance of symmetrical division fails resulting in fewer progenitor cells and a limited capacity to produce neurons. The neurological developmental window is very small, starting at about week five of foetal life and finishing before birth. If insufficient progenitor cells are made during this period, small brain size results. MCPH, therefore, is not linked to any apoptotic or degenerative mechanism but is believed to be a failure in neuron production. 

While it is known that ASPM influences neurogenesis, there are many unanswered questions about exactly how ASPM exerts its effects. For example, how is ASPM turned on and off, and how does over or under expression of ASPM affect brain development? What genes /proteins influence the activity of ASPM? Where does ASPM localise in the cell before mitosis? What influences its migration to the spindle poles?

We believe the structure of ASPM could be important in its function and ongoing work is looking at differences in ASPM structure between interphase and cells undergoing mitosis. We think that ASPM is curled up in interphase and then, as the cell enters mitosis, the protein opens out to become more rod-like. We are also planning to monitor what ASPM does when it gets to the spindle during mitosis.  Does it, for example, form a stable relationship with other proteins or is it a carrier protein that delivers its load and then moves away from the spindle, and, if so, where does it go? We are also trying to identify specific ASPM domains that interact with other cellular components to see how these interactions affect brain development.

At around 410 kilodaltons in size, the ASPM protein is very large making it virtually impossible to transfect intact into cells (generally, immortalised patient cell lines or commercially available human cells lines, such as HeLa cells). However, our approach is to create gene fragment constructs linked to a fluorescent probe for transfection. These are more stable and easier to manipulate. Once inside the cells, protein expression can be identified, and effects on cells can be observed using both fixed and live cell fluorescence microscopy.

Live cell imaging is particularly important in monitoring dynamic events. However, for results to be reliable, it is essential to maintain a constant environment in terms of temperature, humidity, and CO2 especially in extended timelapse studies. A constant environment helps to preserve cell viability, reduces unwanted experimental variables and helps to prevent perturbations, such as focal drift, in the imaging set up.  To help advance its live cell imaging capabilities, LIMM has recently acquired Nikon’s BioStation IM system for timelapse studies. This is an enclosed environmentally controlled microscopy and incubator system equipped with Nikon’s Perfect Focus System (PFS). In BioStation, the incubator is tiny and totally enclosed to maintain a constantly controlled, and optimal environment for cells. Importantly, imaging takes place in situ, eliminating any potential cell stress from environmental changes or physical disturbance, leading to more reliable and consistent results.

BioStation will help us eliminate focal drift, which has caused us problems during previous timelapse studies using other microscope setups. We are planning a series of rapid 4-hour live cell studies using C-terminal constructs to look at protein localisation and migration in the cell. In pilot studies we could easily see the GFP-tagged protein moving to the spindle pole – it was beautiful. We are also planning to use confocal imaging, especially FRET and FRAP, for protein localisation, protein interaction and trafficking studies using our Nikon research level inverted microscope configured for TIRF and four-laser bed confocal imaging. FRAP will be useful for determining what happens to ASPM once it reaches the spindle poles. By inserting a GFP or RFP linked ASPM fragment construct into cells, we can photobleach the spindle pole and then determine, by the reappearance of fluorescence, whether or not further ASPM-protein linked probes migrate into the area, indicating a transitory interaction with the spindle.

While functional gene studies will help to learn more about them process of neurogenesis and MCPH, the problem with MCPH is that it occurs very early in utero and there is very little chance of being able to intervene to influence these events in a developing foetus. There are also no therapies currently available that can prevent or alter the course of the disease. However, the identification of genes involved in MCPH has enabled in utero diagnosis at about 16 weeks gestation and early recognition of the disease may help to maximise developmental potential through supportive care once the infant is born. Knowledge of gene mutations also enables family screening and the identification of MCPH carriers. With genetic counselling this information can help affected families make informed decisions about pregnancies and family planning.

It is clear that there is still a great deal to find out about MCPH. We have affected families that cannot be mapped to know loci, for example, and this indicates there are likely to be further genes for MCPH. We also need to find out more about factors that regulate the transcription and activity of MCPH genes. Besides being of benefit to families affected by MCPH, research contributes to the understanding of brain developmental process in general, which may have an impact on other neurodevelopmental diseases. MCPH has also become a model for the study of human evolutionary brain development and in particular the ratio of brain to body size.  Genes affecting brain size such as ASPM, CDK5RAP2, and CENPJ are thought to have evolved rapidly to result in man’s exceptionally large brain conferring greater adaptability to new situations and hence, great survival success as a species.