Biochemical and proteomic approaches to investigating activity-dependent regulation of stargazin in mammalian neurones

Nerve cells (neurones) communicate by releasing chemical messages ?neurotransmitters? that transmit the message to specific message-receptive proteins, ?receptors? in the message-receiving neurone. These events occur in specialized structures, synapses, where the release site and suitable ?receptor? are juxtaposed. It is imperative for compentent neuronal communication that the receptor be trafficked and targeted to the appropriate synapse. Furthermore, the number and stability of the receptors placed in the receptive zone needs to have some degree of ?plasticity? so that it is capable of accommodating different rates of delivery. If messages are transmitted at high frequency ?these might be important messages that need a special response and some mechanisms for recalling the importance of this particular message. In the case of excitatory neurotransmission, glutamate is the neurotransmitter and for moment to moment communication, the ?AMPA receptor? is the responsive element. How do the AMPA receptors get to the appropriate synapse and how are the number and stability of the AMPA receptors altered to interpret and respond appropriately to important messages? These questions form the basis of this project proposal. A family of proteins, the TARPs (Transmembrane AMPAR Regulatory Proteins) have recently been identified and shown to be important components of the protein machines that shuttle AMPARs from their site of assembly to their final destination in the excitatory synapse. But they don?t operate alone, many other proteins known and unknown are also required but what are they? When are they required? And what do they contribute to this whole complex process? This is what we hope to discover here.
To illustrate how important TARPs are consider the stargazer mouse, it fails to make one particular TARP family member,the TARPgamma2 variety. As a consequence the mouse develops absence epilepsy, motor co-ordination, balance and posture difficulties (ataxia) and inner ear problems. Furthermore, the proteins that they help to traffic and target are important in processes such as learning and memory ?dysfunctional TARPs may result in cognitive impairment. Our aim is to identify proteins that assist and regulate TARPs, establish how they know when to perform their tasks and how efficiently and hard they need to work.
This information will be transmitted to the public in oral presentations and posters at Durham?s Brain Awareness Week which I organise, Neuroscience-North East - a conference organized for early-career neuroscientists in which I?m also involved and at the annual Durham Science Fair for local school children.

The excitatory, ionotropic, glutamate-gated AMPA receptors (AMPARs) undergo constitutive rapid cycling at the synapse. Activity-dependent modulation of the rate of delivery and/or removal of AMPARs to/from the synapse underpins synaptic plasticity events such as LTP/LTD and is considered central to cognitive function. Although a raft of AMPAR-interacting proteins has recently been identified, each having a specific, sometimes transient role to play, little is known about the repertoire of proteins involved, nor the molecular mechanisms responsible for the transport to and retention of AMPARs at the synapse. Novel approaches are required if we are to dissect-out specific multi-protein complexes and to identify and assign function to all the individual components. Transmembrane AMPAR Regulatory Proteins (TARPs), e.g. stargazin (TARPgamma2) play a role in AMPAR trafficking and synaptic targeting. TARPs are subject to neuronal activity-dependent post-translational modifications e.g., phosphorylation/glycosylation which appear to be cell compartment specific. How these post-translational modifications of TARPgamma2 arise, how they influence its function, its multi-protein complex formation and AMPAR signaling remain largely unknown. TARPs are expected to be pivotal to AMPAR-mediated brain development, neuronal communication and cognitive functions but might also be implicated in disorders e.g. epilepsy and Alzheimer?s disease and hence may have potential as novel targets for drug design.
Aims and objectives:
(1) We will immunoaffinity purify non-synaptic and synaptic TARP-complexes from mouse cerebellar neurones (TARPgamma2) and glia (TARPgamma4). Applying standard proteomics approaches including 1D-gel and 2D-gel separations followed by MALDI-MS and/or LC-MS-MS analysis we will identify the complement of respective TARP-interacting proteins and begin to construct their proteomic maps.
(2) AMPAR agonists dissociate TARPgamma2-AMPAR interactions. This probably occurs at glutamatergic synapses to either signal transfer of AMPAR from TARPs to the post-synaptic density or initiate AMPAR internalization, recycling or degradation. Procedures will be applied as in (1) to evaluate whether the repertoire of TARP-associated proteins is affected when TARPs are AMPAR-associated (+CNQX) versus AMPAR-dissociated (+glutamate).
(3) Using TARPgamma2-expressing cultured mouse cerebellar granule cells (mCGCs) pharmacologically manipulated to regulate neuronal activity and signalling we will investigate by immunoblotting, cell surface labeling, protein turnover assays etc., how the post-translational status, function and, by fluorescence confocal microscopy, cellular location of TARPgamma2 and associated-AMPARs is affected by neuronal activity.
(4) We will virally infect TARPgamma2 -/- mCGCs with modified GFP-TARPgamma2 expression constructs and by a proteomics approach as in (1) and (2) determine how post-translational modifications and TARPgamma2 domains influence the assembly of TARPgamma2-multiprotein complexes and their function.