The structural biology of synaptic connectivity: understanding the extracellular organizers of neurotransmission

Neuronal circuits are essentially the biological substrate for all aspects of brain function. And synapses, the connecting points for neurons, hold the key to understanding these circuits. They are continuously remodelled in response to novel experiences, and this is likely the anatomical substrate for learning and formation of long-lasting memories.
Synapses have probably been studied more than any other cellular structure over the past century, with all the tools afforded by neuroscience in its broadest possible sense (from anatomy to genetics, physiology to biochemistry, cell to molecular biology). Structural biology employs a combination of methods, including the use of X-rays and electron beams, to define shapes and mechanisms of action of biological molecules. I aim to apply such techniques to study the special type of protein assemblies that span the so-called "synaptic cleft", the space that separates the outer membranes of two connected neurons. Traditionally, these proteins were studied in isolation, following purification and crystallization. This approach provided a wealth of information regarding the detailed atomic organization of receptors for small molecule neurotransmitters, for example. But if we are to understand how synapses work we must attempt to reach a higher level of complexity, that of multi-molecular assemblies, for the simple reason that in real life proteins never work alone. This is a key goal of my research.
Trans-synaptic protein assemblies are important in (at least) two ways: they provide structural support, physically tying together the pre- and post- synaptic neurons, and they provide avenues for communication between these cells. This latter aspect in particular is poorly characterised, and one of my main hypotheses is that such assemblies are highly dynamic, changing size and possibly shape in response to neuronal activity. From a basic science point of view, understanding such a mechanism should provide a completely novel view into how synaptic signalling actually works.
My work will also have an important impact in medicine, by providing snapshots into the molecular mechanisms that control synaptic stability. In humans, normal healthy aging is marked by variable degrees of neural deterioration and cognitive impairment. These are accompanied by a reduction in synapse numbers in regions of the brain involved in learning, memory and executive functions. Moreover, a malfunction of synaptic signalling and changes in synaptic morphology and number are linked to the majority of psychiatric and neurological disorders, from mental retardation and autism to Alzheimer's disease and addiction. Astonishingly, a recent report from the European Brain Council and the European College of Neuropsychopharmacology states that more than 160 million Europeans (~38% of the population in the 27 EU countries plus Switzerland, Norway and Iceland) suffer from mental disorders. For example, in the UK alone, according to Alzheimer's society, there are currently about 750,000 people suffering form dementia (one in 14 people over 65 years of age, and one in six over 80), costing the society in excess of £17 billion a year. These numbers are likely to go up as life expectancy increases. Surprisingly, recent reports revealed that a number of central nervous system disorders (including certain forms of encephalitis and ataxia) can be treated by immunotherapy. This is because they are triggered by autoantibodies against synaptic proteins. Working together with clinical immunologists and a local company specialized in high-throughput screening, my laboratory will help develop new tools for diagnosis, aiming to identify more conditions that can be tackled in this innovative and relatively straightforward way.

Central nervous system neurons engaged in excitatory synapses are connected by an intricate protein network that spans the synaptic cleft, provides structural support and modulates neurotransmission. The key target of this proposal is to understand the molecular structures, principles of higher order organization and functional mechanisms of two prototypical trans-synaptic assemblies. We will focus on complexes built around type IIa receptor protein tyrosine phosphatases (in particular RPTPsigma) as well as Delta-type glutamate receptors. Our key questions are: What are the rules of assembly in these trans-synaptic complexes? How dynamic are such structures? How do they influence synapse formation, stability and synaptic transmission?
We will combine structural biology techniques (X-ray crystallography, cryo-electron tomography) and quantitative live cell fluorescence microscopy to define the architecture of these complexes in an environment as close as possible to physiological, i.e. a cell membrane context. We will create structure-guided mutant constructs and test them in synapse formation assays and other functional paradigms (electrophysiology in whole cells and brain slices, and mouse models) in collaboration with specialist laboratories.
Furthermore, by exploiting the modular architecture of soluble "synaptic organizer" proteins, we will use structural information to create molecules with novel intermolecular interaction properties through domain recombination. As an alternative, similar tools will be designed by specifically crosslinking nanobodies raised against synaptic receptors. These macromolecular probes may allow the manipulation of the number and properties of excitatory and inhibitory synapses in neuronal circuits and the implementation of novel rules of synaptic communication and homeostatic plasticity, revealing the importance of specific neuronal connections.

Defining the mechanisms of cognitive functions (such as learning, memory, thought, speech or consciousness) in cellular and molecular terms is certainly one of the big challenges in science but also of huge interest to society. We need to satisfy fundamental introspective questions (such as "How does my brain work?") but also hope to become capable of preventing, or delaying, age-related cognitive decline as well as curing neurological and psychiatric disorders.

Although this is essentially a basic science proposal, without doubt the long-term beneficiary of this work is the general public. According to the World Health Organization (http://www.who.int/mental_health/policy/services/integratingmhintoprimarycare/en/index.html), disorders linked to synaptic dysfunction currently affect hundreds of millions of people worldwide. This poses a huge burden to the world's economic output. Without the concerted efforts of academia and industry, this situation is likely to exacerbate considering the predicted increase of life expectancy worldwide. For example, within the UK, research commissioned by the Alzheimer's Society projects that the 2011 number of 750,000 people suffering from dementia may increase to 1 million by 2021 and 1.7 million by 2051.

A detailed understanding of the mechanisms that drive the formation, stability and function of CNS synapses would provide the solid grounding needed to develop such therapies. The crystal structures we will solve have the potential to guide the development of novel drugs, that may trigger fewer side effects compared to those designed using the traditional "silver bullet" approach. While my own laboratory is not focused on drug design, I am actively involved in a collaboration with MRC-Technology and MRC-LMB scientists to exploit our recent crystallographic results on AMPA receptors. Being funded by an MRC grant, there is an added benefit that patents for any therapies designed in this way would be owned by the public, thus ensuring that money spent by the NHS will go directly to a government agency and support further research.

Furthermore, I am particularly excited about the potential impact onto the "synaptic repair" field that may emerge from the use of synthetic synaptic "connectors", molecules that can cross-link pre- and post-synaptic receptors and as a result promote (or indeed reduce, depending on the choice of interactions) the number and/or strength of neuronal synapses.