Regulation of inhibitory synapse function by Neuroligin 2 membrane dynamics, trafficking and phosphorylation

Nerve cells communicate with each other via specialised cell-cell contact sites called synapses. On the presynaptic side the input neuron releases neurotransmitters that activate specialised neurotransmitter receptors located on the receiving neuron at the postsynaptic side. Synapses can have either an excitatory (activating) or an inhibitory (inactivating) role in brain signalling. For the brain to function well, it is important that synapses are formed correctly, i.e. the pre- and postsynaptic side are precisely apposed to each other, and that brain activity is properly regulated. If synapses do not wire correctly during development or go wrong later in life this can lead to devastating neuropsychiatric and neurodegenerative brain disorders like Schizophrenia, autism and Alzheimer's disease, respectively.

The protein family of Neuroligins play a key role in synapse formation and maintenance. They are positioned in the membrane of the postsynaptic side and, by binding the protein Neurexin at the presynaptic side, bridge the synaptic cleft. Neuroligin-2 (NL2) is specifically located at the inhibitory synapse, where it induces clustering of the protein gephyrin to form a scaffold. The main inhibitory neurotransmitter receptor of our central nervous system, the GABA-A receptor, is then stabilised at the synapse by gephyrin and LHFPL4.

Synapses can change their strength in response to changes in neural activity, an important property for key brain functions such as learning and memory, and for maintaining the balance between excitation and inhibition. The strength of the response at a synapse can be regulated by altering the size of the scaffold and the number of attached neurotransmitter receptors. Through its role in stabilising the synapse, NL2 is likely to be a key regulator of the number of GABA-A receptors in the postsynaptic membrane, and thus of inhibitory synapse strength and information processing in the brain. Little is known, however, about the molecular mechanisms that control NL2 number at the synapse, or its dynamic movement into and out of the synapse.

We have initial data suggesting that brain activity causes a change in the amount of NL2 on the cell surface, through its release from the synapse and uptake into the cell (a process called endocytosis). We have also identified a number of novel proteins that interact with NL2 and may be involved with either its stabilisation at the synapse, or regulating whether NL2 taken into the cell is recycled to the synapse or sent for destruction. It is, however, still unknown how brain activity affects these processes, and reciprocally how alterations of NL2 number at the synapse affects its strength. Our research is therefore aimed at answering the following related questions:

- Under what physiologically relevant conditions is the stability of NL2 at the synapse altered?
- How do the various binding partners of NL2 regulate its stability at the synapse, as well as its endocytosis, recycling and degradation?
- How do changes in the amount of NL2 at the synapse affect the strength of the inhibitory synapse?

Ultimately, by gaining molecular insight into how inhibitory synapses are formed and regulated, we will better understand how wiring in the brain is controlled. In addition, since synaptic dysfunction is implicated in many neurodegenerative and neuropsychiatric diseases, our proposed work may also lead to an improved understanding of diseases such as epilepsy, stroke, Alzheimer's disease, Huntington's disease and schizophrenia, and may suggest targets for therapeutic intervention. A detailed understanding of the processes that we study for NL2, will also have implications for other Neuroligins, synaptic membrane proteins, and membrane proteins in non-neuronal cells. Thus, our research may contribute to understanding basic cell biological principles as well as specifically how connections in the brain are formed and maintained.

Controlling the balance between synaptic excitation and inhibition is crucial for the correct operation of the brain. The trans-synaptic adhesion protein Neuroligin-2 (NL2) plays a crucial role in formation and maintenance of the inhibitory synapse by inducing clustering of the scaffold protein gephyrin and binding LHFPL4, which in concert stabilises the inhibitory GABA-A receptor in the postsynaptic domain. Whereas neural activity is known to regulate trafficking of synaptic receptors, little is known about the role of NL2 dynamics in these processes. Our work is aimed at understanding how brain activity affects NL2 stability at the synapse, and uncovering the role of protein modifications and novel protein interaction partners that regulate its membrane dynamics and endocytic sorting. Ultimately, we seek to understand how NL2 contributes to synapse development and maintenance.

We will use a combination of molecular, cell biological, mouse transgenic, imaging (fixed confocal, live and super-resolution) and electrophysiological approaches to determine how NL2 is stabilised at the synapse and how its trafficking (endocytosis, recycling and degradation) is regulated. We will use both dissociated neuronal culture from WT and KO mice, as well as organotypic and acute brain slices that have been genetically manipulated by biolistic (gene gun) transfection or in utero electroporation. Our work will include live cell single molecule tracking of NL2 via Quantum Dots (for which we have developed optimised probes and antibodies) to assess the effect of neural activity and various protein interactions on stabilisation at the synapse and endocytic zones, state-of-the-art super-resolution imaging (SIM, STED, STORM) to determine the positioning of endocytic trafficking compartments and cytoskeletal regulators with respect to the synapse, and patch clamp electrophysiology to study how NL2 stability and trafficking affect inhibitory synapse formation, strength and maintenance.

Understanding brain function on a molecular level is of great benefit to academia and scientific institutes, pharmaceutical industry, the general public and public health.

How will they benefit?

Pharma and Public Health: The annual cost associated to mental and neurological disorders is over £100 billion in the UK, which is 22.6% of all disabilities. The underlying causes for many of these disorders include disrupted membrane protein trafficking and synaptic signalling, the wider topic of our research. The knowledge generated from the proposed work will help to improve our understanding of the mechanisms underlying neuronal development and signalling. Our results will benefit pharmaceutical and biotech sectors working to identify therapeutic targets to combat these disorders. While we do not anticipate that our findings will impact on patient health within the time course of the project, the proposed research will in the long term contribute to UK pharmaceutical industry competitiveness and thus the UK economy by commercial applications, and ultimately improve quality of life and public health.

Academia and science: The multidisciplinary approach of the project ensures that the results will be of interest to a wide scientific audience as well as to specialists in the field. Whereas methods that are applied and developed during the proposed project can be widely applied in other research fields, the knowledge obtained will specifically benefit those with particular interest in protein trafficking, synaptogenesis and neuroplasticity. Collaborators and the wider UK science base will benefit from tools that we develop throughout the project. Dissemination of our data in high-impact journals will contribute to maintaining the high international research profile of the UK, thereby attracting the brightest researchers from all over the world. The researchers on the project will benefit from the high level of scientific and professional training available in the UCL Neuroscience domain. In turn, their training will contribute to the UK academic and scientific community, and to teaching a next generation of scientists. Moreover, they will obtain transferrable skills that can be applied in research and development projects in the pharma and biotech sectors, therefore potentially benefiting the wider UK commercial sector and general public.

What will be done to ensure that they will benefit?

Science and pharma: We aim to publish our work in high-impact international journals to disseminate our findings to academia and the pharmaceutical industry. We will also disseminate our data at earlier stages in the form of poster presentations and invited lectures at universities, pharmaceutical companies and at national and international meetings and symposia. We will also discuss the application of our work with biotech and pharmaceutical industry. Finally, we aim to organise a symposium on membrane protein trafficking in synaptic function to allow for exchange of ideas and data with other experts in the field, as well as write a review on this topic to combine past and current insights in the field.

Public engagement: UCL has an extensive outreach programme with schools, the community and other relevant groups. In order to engage the general public regarding our key findings we will liaise with UCL Media Relations and UCL Public Engagement to discuss means to disseminate our results to a wider public audience. Work from our lab has previously been showcased in the news sections of various research and funding websites, at the Brain Awareness week, and at the houses of parliament as part of the 2011 SET for Britain poster competition. The lab also takes part in the In2ScienceUK program, which offers underprivileged students currently at high school in deprived schools the opportunity to work alongside practising scientists, giving them an insight into scientific research and development.