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Chemical biology approach to study the role of PDZ domain-mediated interactions in synaptic plasticity

Final Report Summary - NEUROCHEMBIOTOOLS (Chemical biology approach to study the role of PDZ domain-mediated interactions in synaptic plasticity)

Complex and dynamic protein-protein interactions are the base of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a prototypical example of protein-based network whose nanoscale structure and composition determines cellular function. The dynamic regulation of PSD composition and receptor movement into or out of the PSD is the base of current molecular theories of learning and memory. Over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions in the synaptic targeting and anchoring of glutamate receptors (AMPARs, NMDARs). In particular, synaptic scaffolding proteins from the PSD-95 family, which possess multiples PDZ domain copies, play a key role in stabilizing glutamate receptors at the PSD. However, the mechanisms that dynamically govern their respective synaptic retention remain poorly understood partly due to the lack of tools that would allow disruption and control of endogenous specific interactions.

The aim of this project is to design and exploit original tools to better understand the role of PDZ domain-mediated interactions in the context of synaptic plasticity by employing a chemical biology approach associating synthesis, biophysical measurements and live cell studies. In particular, two different aspects have been addressed: 1) development of efficient and specific competing biomimetic ligands that integrate the multivalent nature of these interactions and 2) spatio-temporal control of the disruption of these PDZ domain-mediated interactions. The specific targets comprise on the one hand the main synaptic PDZ domain-containing scaffolding proteins, and on the other AMPA and NMDA receptor complexes. Finally, these tools will complement common biological approaches by allowing us to study localized and dynamic macromolecular events between endogenous proteins.

During the project we have produced a collection of biomimetic peptide-based tools derived from PDZ domain-binding motifs of proteins found in the post synapse of excitatory neurons. We have also isolated and determined the endogenous binding targets of the biomimetic ligands. Then, we have determined by fluorescence-based methods the binding affinity of the designed molecules for different PDZ domains of the isolated endogenous proteins. Finally, we have studied by advanced imaging techniques and electrophysiology the biomimetic ligands and their effects in complex cellular environments. Initially, we have worked in heterologous cells and later in neuronal primary cell cultures and in brain slices.

The results can be classified into two categories:
- Development and characterization of original biomimetic tools.
- Exploitation of original biomimetic tools in complex cellular environments

1.1 Development and characterization of original biomimetic tools:
The main results under this category include:
- Establishment and improvement of synthetic approaches and methods to produce a broad range of biomimetic peptide-based tools.
- Generation of a collection of biomimetic ligands for biophysical and cellular characterization of PDZ domain mediated interactions.
- Production of a collection of caged biomimetic ligands for cellular characterization of PDZ domain mediated interactions with high spatial and temporary resolution.
- Development of a fluorescence polarization assay to characterize high affinity peptide-based ligands.
- Biophysical characterization of the biomimetic tools.

1.2 Exploitation of original biomimetic tools in complex cellular environments:
- Identification of the endogenous targets of the biomimetic ligands.
- Development and optimization of a cell-based assay to study protein-protein interactions by using biomimetic ligands.
- Identification of a phosphorylation state of a unique Tyr at position782 in Nlg1 that regulates Gephyrin binding in vitro

On the one hand, our synthetic strategy takes advantage of well-known reactions to obtain a repertoire of complex biomimetic molecules. Thus, we anticipate that the combination of our synthetic methods, ligation techniques and caging approach will be of high interest to the broad chemical biology researcher’s community.
On the other hand, we expect that the new developed tools will provide us with precious information for the elucidation of the molecular mechanisms underlying synaptic events such as synaptic formation, stabilization and plasticity. The results obtained for biomimetic ligands (WT and mutants) of Gephyrin binding motif of Neuroligin-1 (Nlg1) allowed us to demonstrate that the phosphorylation state of a unique Tyr at position782 in Nlg1 regulates Gephyrin binding in vitro. This result has a relevant biological implication as Nlgs can form both excitatory and inhibitory synapses. The balance between these two kinds of synapses is regulated by the expression levels of the excitatory and inhibitory scaffolding proteins PSD-95 and gephyrin, respectively. This mechanism may play an important role in controlling the excitation/inhibition balance, a crucial parameter in pathologies such as autism and mental retardation (Giannone at al., Cell Reports 2013).
In general, the disruption of the transient macromolecular complexes constituted by scaffold proteins and their various binding partners by biomimetic ligands is contributing to the better understanding of synapse maturation and receptor cycling, elements that are critical for the synaptic stabilization or release of receptors as well as the implications of receptors mobility in synaptic functions. These findings could also result in socio-economic long term benefits as diseases involving cognitive impairment exhibit a rising prevalence and cost for society. By attempting to better identify the specificity determinants with our biomimetic approach, we also expect to define new starting points to help create therapeutic agents. This is a realistic expectative as peptides targeting PDZ domains have already shown their neuroprotective ability in ischemia models (Cook, Nature, 2012).