How does the autism-related protein Shank contribute to the regulation of neuronal spine plasticity?
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
Neuronal spines are stable structures that can also rapidly rearrange in response to synaptic activation. The fine balance between plasticity and stability supports learning and memory, while even a small upset of the balance leads to neurological diseases such as autism spectrum disorder (ASD). Spine plasticity is controlled by a complex network of molecular interactions in the postsynaptic density (PSD) and its proximity that are poorly understood.
Shank3 forms a structural part of the PSD core supporting synaptic receptors. Its deletion and mutations are associated with different forms of ASD, and Shank3 effect on spine and PSD structure and dynamics was demonstrated in cultured neurons. We have discovered a new SPN domain in Shank3 and solved the domain structure and demonstrated its interaction with Ras-family GTPases. This unexpected direct connection of Shank3 to Ras and Rap signalling pathways lead to an exciting hypothesis of Shank3 involvement in signalling processes that we followed up in non-neuronal cells to demonstrate a novel mechanism of integrin adhesion receptor regulation. This PhD proposal focuses on Shank3 signalling in neurons.
We hypothesise that Shank3 connection to Ras-family GTPases serves as a novel molecular mechanism of spine regulation. We will use a combination of structural analysis and microscopy experiments in neurons to test this hypothesis.
1. How does Shank3 interact with Ras GTPases?
We will use NMR and X-ray crystallography to solve the structures of Shank3 SPN complexes with GTPases. We will use isothermal calorimetry (ITC) to define whether other Shank3 domains affect the interactions. We will design mutants that modulate Shank3 interactions in cells, and probes to monitor the interactions in cells by fluorescence.
2. How does Shank3 interaction with Ras GTPases affect spine plasticity?
We will transfect Shank3 mutants and fluorescent probes into cortical neuronal cultures at different stages of development and monitor the protein localisation and interactions, and the structural effects on spines by fluorescent microscopy as we have done previously. We will then use transmission electron microscopy and SBFSEM to further quantify the dependence of spine, as well as PSD, morphology and number on Shank3 interactions with GTPases.
Strategic Research Priorities
The project fits into BBSRC research priority 2 - bioscience for health, investigating fundamental neuronal mechanisms that control development of central nervous system early in life and maintenance of the healthy state throughout the lifespan. The project focuses on the synaptic plasticity and homoeostasis that support learning and memory, often deteriorating with age. The project, by furthering our understanding of the molecular basis of neuronal spine remodelling, will provide insight to the development of therapeutic strategies and agents to correct age- or disease-related neurodegeneration. The project fully meets the requirements or the priority 4 - exploring new ways of working by delivering training in the wide range research methods spanning from molecules to neuronal cells and giving student practical skills in operating latest instruments of the research facilities of Liverpool and Newcastle. The student will work alongside teams of structural biologists in the NMR Centre (Liverpool) and neurobiologists in IoN (Newcastle), as well as interacting with the international collaborative research teams and visiting their laboratories in Germany and France. The training in the project will address the current shortage of multidisciplinary experts that have both molecular and cellular knowledge required to modern biology research and translation of fundamental knowledge into new therapies.
Shank3 forms a structural part of the PSD core supporting synaptic receptors. Its deletion and mutations are associated with different forms of ASD, and Shank3 effect on spine and PSD structure and dynamics was demonstrated in cultured neurons. We have discovered a new SPN domain in Shank3 and solved the domain structure and demonstrated its interaction with Ras-family GTPases. This unexpected direct connection of Shank3 to Ras and Rap signalling pathways lead to an exciting hypothesis of Shank3 involvement in signalling processes that we followed up in non-neuronal cells to demonstrate a novel mechanism of integrin adhesion receptor regulation. This PhD proposal focuses on Shank3 signalling in neurons.
We hypothesise that Shank3 connection to Ras-family GTPases serves as a novel molecular mechanism of spine regulation. We will use a combination of structural analysis and microscopy experiments in neurons to test this hypothesis.
1. How does Shank3 interact with Ras GTPases?
We will use NMR and X-ray crystallography to solve the structures of Shank3 SPN complexes with GTPases. We will use isothermal calorimetry (ITC) to define whether other Shank3 domains affect the interactions. We will design mutants that modulate Shank3 interactions in cells, and probes to monitor the interactions in cells by fluorescence.
2. How does Shank3 interaction with Ras GTPases affect spine plasticity?
We will transfect Shank3 mutants and fluorescent probes into cortical neuronal cultures at different stages of development and monitor the protein localisation and interactions, and the structural effects on spines by fluorescent microscopy as we have done previously. We will then use transmission electron microscopy and SBFSEM to further quantify the dependence of spine, as well as PSD, morphology and number on Shank3 interactions with GTPases.
Strategic Research Priorities
The project fits into BBSRC research priority 2 - bioscience for health, investigating fundamental neuronal mechanisms that control development of central nervous system early in life and maintenance of the healthy state throughout the lifespan. The project focuses on the synaptic plasticity and homoeostasis that support learning and memory, often deteriorating with age. The project, by furthering our understanding of the molecular basis of neuronal spine remodelling, will provide insight to the development of therapeutic strategies and agents to correct age- or disease-related neurodegeneration. The project fully meets the requirements or the priority 4 - exploring new ways of working by delivering training in the wide range research methods spanning from molecules to neuronal cells and giving student practical skills in operating latest instruments of the research facilities of Liverpool and Newcastle. The student will work alongside teams of structural biologists in the NMR Centre (Liverpool) and neurobiologists in IoN (Newcastle), as well as interacting with the international collaborative research teams and visiting their laboratories in Germany and France. The training in the project will address the current shortage of multidisciplinary experts that have both molecular and cellular knowledge required to modern biology research and translation of fundamental knowledge into new therapies.
Organisations
| Description | The work has so far focused on further understanding the structure of the N-terminal region of the Shank3 protein to help further understanding of how this protein has a regulatory function in dendritic spines. The N-terminal region of Shank3 is composed of two domains, the Shank/Prosap N-terminal domain (SPN) and Ankyrin Repeat (ANK) domains that are associated through a polar interface. We have found that some significant autism-associated mutations that are found in this N-terminal region cause significant de-stabalisation of the N-terminal domains. Therefore we have hypothesised that the stability of the N-terminal region is critical for Shank3 neuronal function. NMR analysis of the very N-terminal domain, the SPN domain, has shown that it is intrinsically unstable when it is not in contact with the ANK domain, particularly in salt concentrations below 0.5M. NMR analysis, using mutations designed to disrupt the SPN-ANK interface , has shown that the separation of the SPN domain leads to its destabilisation. Moreover, single point mutations have dramatic effects on the N-terminal region, such as the autism-associated P141A, which causes destabilisation of the interface and reduces overall stability of the N-terminal region. These findings were confirmed through melting temperature data measured by CD and nanoDSF. From these findings, we propose an 'open-closed' model of the N-terminal region, where the SPN domain dynamically dissociates from the ANK domain, simultaneously leading to self-association and activation of the Shank3 binding sites. These two effects need to be taken into consideration when analysing autism-related mutations of Shank3. In addition, the previously solved interaction between the Shank3 N-terminal region and the Ras-family GTPases has been explored further and in association with our collaborators we have shown another interaction between the Shank3 N-terminal and the active K-Ras GTPase. |
| Exploitation Route | These findings contribute to the overall understanding of the Shank3 protein and this knowledge can be used to further determine regulatory mechanisms disrupted in the dendritic spines of neurons, that could contribute to disorders such as autism spectrum disorders. The structural information learnt can be aligned with observations seen in cell culture with mutations of Shank3 to fully understand how these mutations may be working in cells. In addition, this detailed structural knowledge of the domains of Shank3 could help later on with the development of potential drugs to help with neuronal disorders. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |