How do astrotactin-1 and astrotactin-2 act in the determination of mammalian cell polarity?

The growth, development and repair of the brain requires the migration of neurons so they can shape its sub-structures, make new connections, and repair old ones. Similarly, the growth of other tissues and organs requires the growth of cells in a polarised fashion, to give the distinctive shapes characteristic of body plans and individual organs within them. It has recently been found that a pair of proteins called astrotactins (ASTNs) play critical roles in both these processes.

In the brain, ASTN1 is found on the surface of neurons where it makes connections to the guide tracks ("glial fibres") along which they move. In order for the neurons to move, however, they need to break old connections and then form new ones, and in order to do that ASTN1-mediated contact sites need to be recycled towards the leading edge of the migrating cell. The key molecule enabling that to happen is ASTN2, which is a very similar protein to ASTN1 but which is found in small membrane-defined compartments within the cell rather than sticking out of the cell membrane at its surface, like ASTN1 does.

In other tissues, our collaborators at Johns Hopkins University School of Medicine in the United States recently found that ASTN2 helps control something known as planar cell polarity, which underpins the formation of organs and tissues both in the nervous system and elsewhere in the body.

Clinical and genetic data indicate a key involvement of mutant forms of ASTN1 and ASTN2 in a host of conditions including attention deficit-hyperactivity disorder (ADHD), autism spectrum disorders, schizophrenia and forms of neurodevelopmental delay such as difficulty in learning language. Another recent study showing that different forms of ASTN2 could bring forward by ~5 years the age of onset of Alzheimer's, while effects of ASTNs have also been noted on the immune system and general tissue morphology outside the nervous system.

We are going to use methods which give us a 3D atomic-resolution picture of the structure of ASTN1 and ASTN2 to work out how they carry out their activities controlling normal human development, repair when tissues are damaged, and the suceptibility to conditions like Alzheimer's. Preliminary data from our lab and others suggests that what is critical is a change in the behaviour of ASTN1 and ASTN2 in more acidic conditions - different compartments within the cell have differing acidities and this regulates events within them. We have an excellent starting point because we have crystallised ASTN1 in two different forms, at neutral pH and at acidic pH. We have also made preliminary studies of how they interact but will be able to provide much greater detail from the work we are planning now.

Clearly, we also need to look at living cells and see how ASTN1 and ASTN2 move about within them, affecting each other's activity and location. We will do this as well, which will give a functional context to the work we are doing on the atomic structures of the proteins. The approach we will use is based on fluorescently labelling the proteins and this will enable us both to see the location of the ASTN proteins and to measure the interactions they undergo.

Our work will benefit greatly from ongoing collaboration both with the group at Johns Hopkins and with colleagues local to Oxford. One particularly exciting area to explore further will be the way in which ASTN1 and ASTN2 interact with proteins already known to play leading roles in determining planar call polarity, such as the cell surface receptor proteins Frizzled-3 and Frizzled-6 and Celsr1.

This grant application focuses on the use of structural biology, biophysics and live cell imaging to study the structures of astrotactins (ASTNs) 1 and 2 and the interactions they undergo both with each other and with other proteins within cells and tissues.

Recent data indicate that the role of ASTN1 in controlling cerebellar granule neuron migration depends on ASTN2 which drives its recycling via endocytosis between successive interstitial junctions with glial fibres. In other data, our collaboratoing Project Partners in the Nathans lab (Johns Hopkins) have found a link between ASTN2 and planar cell polarity (PCP) determination. This makes our proposed study extremely timely and its medical importance can be gauged from the involvement of ASTNs - as assessing by largescale clinical and genetic studies - in conditions as diverse as ADHD, schizophrenia and Alzheimer's, beside non-neuronal effects.

Having already crystallised and partially solved the structure of the ASTN2 ectodomain we will complete the X-ray structure determination at neutral and then at acidic pH and then make use of biophysical methods to study its self-interaction. We will also solve the structure of the cytoplasmic ASTN2 N-terminal domain, using crystallography or NMR. The absence of exons 4 and 5 from the NTD has been noted in normal tissue and a PCP variant, respectively. The structure of ASTN1 will then be solved, again using crystallography for the ectodomain and crystallography or NMR for the NTD, also studying its interactions with ASTN2 biophysically. We will use live cell studies with FRET measurement incorporating lifetime imaging to look at the interactions undergone by ASTN1 and ASTN2 in a cellular context, including how this is affected by their cycling through the endosomal system, and also aim to extend our work into investigation of the interaction of ASTNs with proteins which are components of PCP signalling.

It is clear that we are aiming to provide an atomically-detailed description of how ASTN1 and ASTN2 function in what are undoubtedly important physiological processes. Therefore the data we obtain may well have value for commercial private sector scientists seeking to develop novel pharmacological agents against ADHD or Alzheimer's or to promote neural repair. Our data should shape new proposals within R&D efforts and may identify new and more effective strategies for drug design.

Policy makers at a national or international level should also benefit because we will be helping to provide a detailed understanding of why certain genetic variations in ASTN1 and ASTN2 are associated with specific neurodevelopmental and other disorders. This has the potential to shape planning for future funding strategies and the assessment of health provision risk and its management, thus assisting in the effectiveness with which public resources are deployed.

In the third sector, charities promoting research towards an improved understanding of and therapy for diseases such as Alzheimer's and schizophrenia, and for conditions like ADHD and autism, will also benefit because we will be providing tools for understanding in more detail what goes wrong with such pathologies and how they might realistically be ameliorated.

In the wider public, we expect that a detailed description of how basic molecular processes can be understood to underlie high-interest conditions such as the ones relevant to this proposal (Alzheimer's, autism spectrum disorders, schizophrenia etc.) will be of significant benefit in helping an understanding of the roles played by basic scientists and their contribution to the health and wellbeing of the population at large.

This proposal constitutes a ground-breaking area of science in which recent success in the research of the Principal Applicant and named PDRA is teamed with an exciting new discovery in the lab of our Project Partner Jeremy Nathans. We believe it represents a major opportunity for UK MRC-funded science to take the lead in establishing a new level of understanding of what governs neural migration and planar cell polarity and how they interrelate.

The timescale for all the benefits we envisage is potentially relatively short; while pharmacological innovations would necessarily be some way off, the benefit otherwise in shaping thinking about these conditions and how they are caused and might be targeted is, we believe, within a 3-5 year range from the start of the grant.

Finally, as stated under Academic Beneficiaries, there are a number of ways in which scientists will benefit from the research proposed.

- structural and cell biologists working with MACPF/CDC proteins, and those interested in their evolution, will benefit from understanding how the polypeptide fold common to the family is adapted to the control of cell adhesion formation and cell migration.
- neuroscientists interested in the control of neuronal migration will benefit because our work will define the basis on which a novel mechanism determines its polarity.
- developmental biologists working on planar cell polarity (PCP) control will benefit because we will show how the astrotactins play a (previously unimagined) role in PCP.
- medical researchers and geneticists seeking an understanding of the molecular basis of neural repair, neurodevelopmental disorders and the onset of Alzheimer's will benefit because we will provide a molecular understanding of the phenomena observed in humans with variant forms of ASTN1 or ASTN2.
- and academics using live cell imaging techniques and measuring protein-protein interactions within live cells will benefit from the application of FRET-FLIM methods to a complex case involving protein localisation, membrane trafficking and the monitoring of endosome development.

The academic beneficiaries alone are therefore much broader than the expertise of the core team working on the project.