Spatial and temporal control of synapse formation through the neurexin/neuroligin adhesion complex – Synapse-2Dt

To temporally control the formation of synapses, we labeled neurons with neurexin aggregates, and observed the accumulation of post-synaptic markers, including neuroligin, the scaffolding protein PSD-95 and AMPA-type glutamate receptors. Using fluorescent nanoparticles, we characterized a diffusion/trapping mechanism controlling the synaptic delivery of neuroligin-1 and AMPA receptors. We also showed that the recruitment of PSD-95 requires tyrosine phosphorylation of neuroligin-1. Finally, we developed a comprehensive biophysical model of AMPA receptor trafficking to synapses. This first part of the work has enabled a better understanding of the biological function of neurexin and neuroligin adhesion molecules in synaptic assembly.

• AMPA receptors are rapidly recruited at neurexin/neuroligin adhesions, suggesting a role of these receptors in the initiation of synapses.
• We built a unified model of AMPA receptor trafficking to synapses, allowing a better understanding of synaptic plasticity processes.
• Neurexin binding induces tyrosine phosphorylation of neuroligin-1, a mechanism which plays a role in the assembly of excitatory versus inhibitory synapses. This mechanism opens avenues for identifying a tyrosine kinase important in controlling the excitation / inhibition balance, a major parameter in neurological pathologies.
• We developed micro-patterned substrates coated with neurexin or SynCAM to spatially control the formation of pre-or post-synapses, a system which provide opportunities for high-throughput screening of compounds involved in synaptic differentiation.
• We have developed an automated acquisition platform to achieve sptPALM, combining super resolution microscopy with photo-activation and high-throughput monitoring of individual proteins.
• We have developed several software solutions for the quantification of the organization and molecular dynamics, using microscopy data gathered from the super-resolution localization of individual molecules.

To spatially control the formation of synapses, we developed a biomimetic system of synaptogenesis by culturing neurons on micro-patterned substrates formed of islets coated with neurexin or SynCAM. This system allows us to induce respectively pre-or post-synapses at multiple perfectly identified locations, offering screening perspectives.
These new systems will provide the basis for high content screening of pharmacological compounds, peptides, or siRNAs that could affect the initial stages of synapse formation. Knowing that pathological mutations of neurexins and neuroligins have been implicated in autism, mental retardation, and schizophrenia, this work could eventually lead to the identification of new therapeutic targets for these neurodevelopmental disorders.

• Recruitment of AMPA receptors at neurexin/neuroligin adhesions via membrane diffusion and trapping through PSD-95 (Mondin et al. Journal of Neuroscience, 2011).
• Unified model of AMPA receptor trafficking at synapses, including both vesicular recycling and diffusion/trapping processes (Czöndör et al. Proc. Natl. Acad. Sci. USA 2012).
• Tyrosine phosphorylation of neuroligin preferentially controls the assembly of excitatory versus inhibitory synapses (Giannone et al. Cell Reports 2013).
• Novel micropatterned substrates to control synaptic differentiation (Czondör et al. Nat. Comm. 2013 and Patent Thoumine/Czöndör/Garcia PCT/FR2012/051688, 2012).
• Wavelet-segmentation based image analysis methods to localize indvidual molecules Patent Sibarita JB. (CNRS / Univ. Bordeaux Segalen), N°12166450.2, Europe/US, 2012. Articles : Izeddin et al, Optics Express 2012. Kechkar et al., Plos One 2013)

Scientific context:
The making of connections between neurons (synapses) is fundamental to brain development and function. Although some progress has already been achieved in understanding the roles of adhesion proteins in synapse development, major issues are still unresolved, in particular regarding: 1) the kinetics and molecular mechanisms leading from initial cell-cell contact to a mature functional synapse; 2) the intracellular signalling pathways associated with axon/dendrite adhesion; and 3) the specificity and function of the various synaptic components recruited, both at the pre- and the post-synapse.

In particular, the actual assays to probe synapse development are based on ‘manual’ and completely random observations of synapse formation, and thus unable to provide enough statistics on these processes. Furthermore, the coexistence of many adhesion systems in parallel with varying developmental expression profiles and thus complex stoechiometries, make it very difficult to dissect the function of each adhesion protein. Thus, cellular chip systems to induce synapses “on demand” at controlled time and locations, using bio-mimetic systems with specific adhesion proteins, would be highly advantageous to address these fundamental issues of neurobiology.

In parallel, recent studies, including our own work (Heine et al., PNAS 2008; Saint-Michel et al., Biophys J 2009), have shown that the neurexin/neuroligin complex played critical roles in synapse initiation, maturation, and function. For example, knockout studies revealed an essential function of neurexins in coupling calcium channels to the pre-synaptic machinery and in maintaining normal post-synaptic transmission. Conversely, neuroligin-knockout mice die shortly after birth as a result of reduced network activity in brainstem centers that control respiration, and show selectively altered synaptic responses. Furthermore, pathological mutations in neuroligin genes, leading to impaired addressing of neuroligins at the synapse, are related to autism and X-linked mental retardation in humans. Thus, understanding the molecular mechanisms of neurexin/neuroligin function at the synapse, and developing screening tests of neuroligin-dependent post-synaptic differentiation, may lead in the future to the identification of new therapeutic targets for such brain disorders. Finally, co-culture and microsphere assays using primary neurons show that neuroligin and neurexin molecules are extremely potent in initiating bona fide pre- and post-synapses, respectively.

Objectives
Our objective is to exploit the synaptogenic properties of the neurexin/neuroligin adhesion complex to control in time and space the formation of synapses, and thus achieve high resolution probing of synapse development and function. To this aim, we will work in an interdisciplinary fashion on the development of new biological, physico-chemical and quantitative imaging tools to study synaptogenesis.

Partners and tasks:

The project involves three partners:
1. O. Thoumine, a specialist of neuronal biology and cell adhesion
2. CYTOO SA, a start-up company specialized in the development and production of micro-patterned substrates
3. JB. Sibarita, a specialist of developments in microscopy and quantitative imaging

We will establish three main tasks:
1. To develop new micro-patterned substrates for synaptogenesis (CYTOO/Thoumine).
2. To develop new quantitative imaging methods for high throughput screening of synaptogenesis (Sibarita/Thoumine)
3. To investigate the molecular mechanisms underlying synapse development mediated by the neurexin/neuroligin complex (Thoumine)