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The role of ubiquitination in stability and plasticity of the GABAergic synapse

Periodic Reporting for period 1 - UbiGABA (The role of ubiquitination in stability and plasticity of the GABAergic synapse)

Reporting period: 2015-05-01 to 2017-04-30

Neurons are polarised cells that communicate via specialised cell-cell contact sites called synapses. The formation and stabilisation of these synapses is essential for processes such as learning and memory. Signal transmission at these sites is triggered through presynaptically released neurotransmitters that act on postsynaptic receptors. Depending on the type of synapse, these signals are either excitatory or inhibitory. The main inhibitory synaptic receptor in the brain is the GABAA Receptor (GABAAR), which is stabilised in the synapse by the intracellular protein gephyrin and the transmembrane adhesion protein neuroligin-2 (NL2). Synapses constantly change in response to activity in the brain and to balance excitatory and inhibitory activity. Their signalling strength can be regulated by adapting the number of postsynaptic receptors. This involves diffusion of GABAARs into and out of synapses at the membrane, and shuttling between cell surface and intracellular compartments. In addition, changes in the amount of gephyrin and NL2 at the inhibitory synapse lead to (de)stabilisation of GABAARs. Internalised receptors are either re-inserted (recycled) into the membrane or targeted for lysosomal degradation. In many neurological and psychiatric diseases, including epilepsy, Autism Spectrum Disorders and schizophrenia, regulation of inhibitory signalling is compromised, but the underlying mechanisms are still poorly understood. Thus, understanding how GABAAR trafficking and synaptic stabilisation is regulated is critical for tackling these disorders. The main objective was to increase our knowledge of the mechanisms that regulate trafficking of GABAARs, gephyrin and NL2, and thereby inhibitory signalling. Initially we focussed on a protein modification called ubiquitination, a well-known mechanism that regulates protein trafficking and turnover. In this process, ubiquitin ligases attach the small protein ubiquitin to a target protein. For membrane proteins, this drives endocytosis followed by recycling or degradation. On intracellular substrates, polyubiquitin chains target the protein for degradation. Ubiquitin can also be removed by de-ubiquitinating enzymes. We aimed to characterise the protein interactions important for regulating ubiquitination and de-ubiquitnation of GABAARs, gephyrin and NL2 and hence the functioning of the inhibitory synapse.
We found that NL2 can be endocytosed and mono-ubiquitinated, while an NL2 mutant that cannot be ubiquitinated is internalised slower in cell lines. In neurons, the NL2 ubiquitin mutant displayed a slightly higher mobility on the neuronal membrane, indicating reduced stability, but we found that this was not due to altered interaction with gephyrin. We could not find a correlation between NL2 ubiquitination and lysosomal degradation, and inhibiting the lysosome did not result in increased accumulation of NL2, suggesting that degradation is not the main outcome of NL2 internalisation. Instead, we found that internalised NL2 is localised to endosomal compartments, where it interacts with proteins that may regulate its recycling. We also investigated the consequences of interrupting NL2 recycling for the stability of the inhibitory synapse and found that altered trafficking of NL2 affects the stability and function of the inhibitory synapse. We focussed on studying the behaviour of GABAARs at the synapse in response to the above described pathways of NL2 stabilisation and trafficking, and assessed how alterations in NL2 at the synapse affect synaptic clustering of GABAARs as well as inhibitory signalling. Our studies indicate that altered trafficking of NL2 affects stability of GABAARs. We also found that gephyrin can be poly-ubiquitinated, which targets it for degradation. We also assessed the effects of the de-ubiquitinase OTUD4 on gephyrin ubiquitination but progress was severely hindered by the propensity of this protein to aggregate and induce cell death upon overexpression. Due to these technical challenges, we focussed instead on studying the behaviour of gephyrin at the synapse in response to the above described pathways of NL2 trafficking, and assessed how alterations in NL2 at the synapse affects synaptic clustering of gephyrin.
We are currently preparing a manuscript describing the role of protein complexes important for regulating NL2 trafficking and phospho-regulation. This research has contributed better understanding how the brain forms and maintains connections, and regulates their strength and activity. Neurological disorders that involve an altered balance of excitatory and inhibitory synaptic activity and defects in protein trafficking include epilepsy, stroke, Huntington’s disease, Parkinson’s disease, schizophrenia and autism. Research on these diseases may thus directly benefit from the outcome of our research.Whereas the proposed action mainly comprises fundamental research, our results will add to the knowledge base that may in the longer term form the basis for the identification by the pharmaceutical and biotech sector of drugable targets in neuropsychiatric and neurological disorders. Part of my data has already been disseminated via a poster presentation at scientific meetings. During the MSCA I have also actively collaborated with other research groups, leading to 1 accepted publication (H. Augustin et al, Development, 2017), and 1 to follow. The project also gave me the opportunity to extend my scientific network, particularly with the European Neuroscience community, by attending international meetings and courses, including the 5th European Synapse meeting (Bristol, UK, 2015), the 10th FENS Forum of Neuroscience (Copenhagen, Denmark, 2016), and a course in ‘Principles and applications of Fluorescence Microscopy’ (Institut Pasteur, Paris, France, 2016). Over the time course of the project I was also involved in training a next generation of scientists, including 3 MSc students and 2 BSc students. The outcome of their projects included insights into the interaction of NL2 with inhibitory scaffold proteins and the role of phosphorylation therein. Each of these students are now pursuing their own career in the life sciences, we 2 of them currently enrolled in a PhD program. Therefore this MSCA has contributed to their development and future role in research as well as the transfer of skills within the European life science community. As a public engagement activity, I have mentored a student in the In2Science program, which offers underprivileged students in their final years at high school in deprived areas the opportunity to work alongside practising scientists for a 2-week period, giving them an insight into scientific research and development. The aim of the schedule is to raise the participation of bright underprivileged young people into Science degrees at top Universities. Finally, the project has enabled me increase my transferable research skills, including but not limited to: expanding my knowledge of major questions in the field of molecular neuroscience, further experience in communicating the outcomes of my research, building collaborations, managing a multi-factorial project, and student supervision. I also obtained experience in state-of-the-art techniques and skills including Single Particle Tracking with Quantum Dots, and super resolution imaging, which, together with the network that I have built will contribute to my further career.