Proteolytic processing of the synaptic cell adhesion molecule neurexin-3beta and the neurobiological functions of the resulting cleavage products

Neurexins are key synaptic cell adhesion molecules involved in synapse specification and maturation. They couple synaptic activity and cell adhesion to regulate activity-dependent synapse development. Recently, neurexin processing by alpha- and gamma- secretases has been reported. In the present study, we characterized the sites at which Nrxn3beta is processed by alpha- and gamma- secretases, identified the enzymes responsible for the shedding, characterised the fragments resulting from the cleavage and investigated the potential functions of generated fragments. We show that Nrxn3beta is shed at two sites- one processed by ADAM17, the other by ADAM10. The shedding generates two soluble ectodomain (sNrxn3beta) fragments and two C-terminal fragments (CTFs). Each CTF is further processed by gamma-secretase to generate one intracellular domain (ICD) and multiple P3-like or Abeta-like fragments. We show that the production of the main sNrxn3beta generated by ADAM10 affects axonal bouton development and spinogenesis during adult newborn neuron development in vivo. Moreover, we demonstrate that sNrxn3beta affects the expression of genes associated with regulation of synaptic vesicle exocytosis (Rab3a), NMDA receptor subunit composition (Grin2c) and metalloprotease inhibition (TIMP-1). Furthermore, we report first evidence that the ICD generation might influence the levels of a protein responsible for the NMDA delivery to dendritic sites (KIF17). The investigation of properties of neurexin P3-like and Abeta-like fragments revealed a tendency of Abeta-like fragments to form oligomers and exert a toxic effect in primary cortical neurons. Our study shed light on the molecular pathways regulated by neurexin cleavage fragments and their possible implications in different pathologies.

Structural basis and biophysical properties of synaptic plasticity studied by atomic force microscopy

Alexandre Yersin

In this thesis, we studied two systems important for synaptic plasticity, one presynaptic and another postsynaptic. The protein complex composed of VAMP 2, SNAP-25 and syntaxin 1 (SNARE complex) is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. In order to better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope (AFM) and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces, dissociation kinetics of the SNAREs and led us to propose a sequence of formation for the complex. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein nSec1 injected into the AFM chamber prevented the SNARE complex formation. Finally we measured the effect of tetanus toxin protease (TeTx) on the SNARE complex and its activity by on-line registration during TeTx injection. These experiments reveal that AFM is a valuable tool to investigate complicate interactions among a protein complex containing up to four components. Trafficking of glutamate receptors AMPA is closely related to postsynaptic induction of long-term potentiation. In a second part of this thesis, we used AFM to explore the distribution of AMPA receptors on-line at the surface of living cells in correlation with cellular viscoelastic properties. First, we showed that AFM permits to detect specifically AMPA receptors at the surface of living neurons and we proved that the specificity of detection is stable during time. Moreover, we were able to visualize variations in AMPA receptor density on surface in response to different stimulations. Finally, AFM also allowed us to study local viscoelastic properties of the cell and their modifications occurring during AMPA receptor trafficking. Our measurements reveal that AMPA receptors are situated at the cell surface on local stiffness maxima. We have noticed that an excitatory stimulation removes these stiffness maxima before producing internalization of receptors. Our results suggest also that AMPA receptors may be constituted of two subpopulations depending on the cell stiffness. A "hard" subpopulation may be preferentially internalized after an excitatory stimulation whereas a "soft" one seems to remain at the cell surface.

EPFL2004

Structural basis and biophysical properties of synaptic plasticity studied by atomic force microscopy

Alexandre Yersin

EPFL2004