Periodic Reporting for period 3 - SynLink (Molecular Structure and Engineering of Synaptic Organizer Proteins in Health and Disease)
Periodo di rendicontazione: 2023-03-01 al 2024-08-31
Synapses are the specialized cellular junctions that form the basic units of communication between neuronal cells. Given the variety of network-dependent functions that synapses need to support, a fundamental question is how their properties are specified at the molecular level.
Membrane-anchored and soluble “synaptic organizer proteins” form adhesive interactions that mediate synapse formation and differentiation. However, a structural and mechanistic understanding of how they recruit and organize the molecular machinery for neurotransmission is largely lacking. Simultaneously, dysfunction of synapses and loss of neurons are hallmarks of neurodegenerative disease that underlie a persistent deterioration of cognitive functions. The properties of synaptic organizer proteins to form and functionalize synapses could be exploited as a mechanism for synaptic repair to reverse neuronal degeneration.
The overall objectives of this proposal are (i) to reveal the structural basis for trans-synaptic molecular nanocolumn formation by determining the structures of complexes of synaptic organizer proteins and neurotransmitter receptors, and (ii) to leverage insights into the structure and function of soluble synaptic organizers for generating engineered variants that can remodel synapses with the potential for restoring neuronal circuitry and cognition in animal models of Alzheimer’s disease (AD), the most common form of dementia associated with early defects in synaptic function.
To achieve these aims, our team combines techniques of structural biology (X-ray crystallography, cryo-electron microscopy and biophysical interaction analysis), protein engineering (combinatorial screening using yeast surface display), and cellular neuroscience (neuronal culture, electrophysiology, advanced imaging and mouse models). Our results will elucidate fundamental principles of synaptic signalling and pave the way for disease-modifying therapies that focus on recovery of synaptic connectivity and function.
Membrane-anchored and soluble “synaptic organizer proteins” form adhesive interactions that mediate synapse formation and differentiation. However, a structural and mechanistic understanding of how they recruit and organize the molecular machinery for neurotransmission is largely lacking. Simultaneously, dysfunction of synapses and loss of neurons are hallmarks of neurodegenerative disease that underlie a persistent deterioration of cognitive functions. The properties of synaptic organizer proteins to form and functionalize synapses could be exploited as a mechanism for synaptic repair to reverse neuronal degeneration.
The overall objectives of this proposal are (i) to reveal the structural basis for trans-synaptic molecular nanocolumn formation by determining the structures of complexes of synaptic organizer proteins and neurotransmitter receptors, and (ii) to leverage insights into the structure and function of soluble synaptic organizers for generating engineered variants that can remodel synapses with the potential for restoring neuronal circuitry and cognition in animal models of Alzheimer’s disease (AD), the most common form of dementia associated with early defects in synaptic function.
To achieve these aims, our team combines techniques of structural biology (X-ray crystallography, cryo-electron microscopy and biophysical interaction analysis), protein engineering (combinatorial screening using yeast surface display), and cellular neuroscience (neuronal culture, electrophysiology, advanced imaging and mouse models). Our results will elucidate fundamental principles of synaptic signalling and pave the way for disease-modifying therapies that focus on recovery of synaptic connectivity and function.
During this first half of the project, our team has focused on the necessary methodological developments.
We have perfected our expression platform for large-scale mammalian protein production, based on our previously published lentiviral plasmid suite, by introducing numerous improvements to enable production of heteromeric proteins and protein complexes. We are now using this technology for the production of all our soluble and membrane target proteins. A second development is the implementation of a unique workflow for synthetic Nb discovery starting from highly diverse (~1012 unique sequences) in vitro RNA libraries, based on ribosome display, yeast display, and magnetic and fluorescence-enabled cell sorting.
These methods are enabling engineering of transient, low-affinity neuronal ligand-receptor interactions. The ability to increase the affinity between two interaction surfaces is key to future structural studies of transient protein-protein interactions, which are abundant in the synaptic cleft. The strategy is based on introducing sequence variability in interaction interfaces using genetic techniques, followed by screening using our yeast display platform.
We are using the synthetic Nb procedure to identify binders against a variety of postsynaptic and presynaptic signaling proteins, which will enable the rapid testing of new synthetic synaptic organiser protein designs.
Finally, we are working on screening proteins putatively interacting with ionotropic glutamate receptors (iGluRs). We have confirmed a number of candidate molecules, and are in the process of quantifying the interaction strength of these complexes, before turning to their structure determination.
We have perfected our expression platform for large-scale mammalian protein production, based on our previously published lentiviral plasmid suite, by introducing numerous improvements to enable production of heteromeric proteins and protein complexes. We are now using this technology for the production of all our soluble and membrane target proteins. A second development is the implementation of a unique workflow for synthetic Nb discovery starting from highly diverse (~1012 unique sequences) in vitro RNA libraries, based on ribosome display, yeast display, and magnetic and fluorescence-enabled cell sorting.
These methods are enabling engineering of transient, low-affinity neuronal ligand-receptor interactions. The ability to increase the affinity between two interaction surfaces is key to future structural studies of transient protein-protein interactions, which are abundant in the synaptic cleft. The strategy is based on introducing sequence variability in interaction interfaces using genetic techniques, followed by screening using our yeast display platform.
We are using the synthetic Nb procedure to identify binders against a variety of postsynaptic and presynaptic signaling proteins, which will enable the rapid testing of new synthetic synaptic organiser protein designs.
Finally, we are working on screening proteins putatively interacting with ionotropic glutamate receptors (iGluRs). We have confirmed a number of candidate molecules, and are in the process of quantifying the interaction strength of these complexes, before turning to their structure determination.
The anticipated scientific insights will reveal how glutamatergic neurotransmitter receptors are recruited to the synapse by their interaction with other synaptic proteins, in an activity-dependent fashion. So far, there are no structural insights into this phenomenon, while it is crucial in the light of the fundamental process of neuronal signal transmission. Secondly, these insights will be translated into the construction of synthetic synaptic organiser molecules, which are conceptually novel reagents for restoring synapse function in disease. In the context of the relative failure of immunotherapies in the treatment of dementias such as Alzheimer’s Disease (AD), our strategy could present a new therapeutic avenue.