Physiological consequences of Protocadherin-10 sumoylation on neuronal function.

Project Information

SUMO-PCDH10

Grant agreement ID: 845466

DOI

10.3030/845466

Project closed

EC signature date 28 March 2019

Start date 1 June 2019

End date 31 August 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 171 473,28

EU contribution

€ 171 473,28

Coordinated by

HUMANITAS MIRASOLE SPA
Italy

Periodic Reporting for period 1 - SUMO-PCDH10 (Physiological consequences of Protocadherin-10 sumoylation on neuronal function.)

Reporting period: 2019-06-01 to 2021-05-31

Summary of the context and overall objectives of the project

Synapses are the basic functional units of the central nervous system (CNS) where the neuronal transmission takes place. Dendritic spines, representing the postsynaptic compartment of excitatory synapses, are small membrane protrusions enriched in actin. Synapse physiology and maturation is regulated by the orchestrated activity of plenty of proteins, including enzymes, receptors, scaffolding and cell adhesion molecules (CAM). Protocadherin-10 (Pcdh10) is a synaptic CAM highly expressed in the CNS, both in the pre- and post-synaptic compartments. At the molecular level, Pcdh10 targets the postsynaptic scaffolding protein PSD-95, in its ubiquitinated form, to the proteasome and promotes synaptic elimination. Interestingly, an altered copy number of the Pcdh10 gene associates with the development of Autism Spectrum Disorder (ASD) in humans. Accordingly, mice lacking one copy of Pcdh10 (Pcdh10+/-) display a severe impairment in social behaviors. However, the molecular mechanisms by which Pcdh10 function impacts synapse assembly and stability are still unclear. Our preliminary data identified Pcdh10 as a potential target of sumoylation. Sumoylation is an essential post-translational modification critical to several cellular signaling pathways. It consists in the covalent but reversible enzymatic conjugation of the Small Ubiquitin-like MOdifier (SUMO) protein to specific lysine residues of substrate proteins. In neurons, sumoylation plays a key role in controlling several neuronal functions including presynaptic release and dendritic spine maturation. Here, we hypothesize that sumoylation of Pcdh10 is crucial for synapse maturation.
Thus, the overall goal of my research project is to unveil the physiopathological consequences of Pcdh10 sumoylation in neurons.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

First of all, we validated Pcdh10 as novel SUMO substrate in mammalian brain. Next, we aimed to understand the molecular mechanisms controlling Pcdh10 function in neurons. We demonstrated that Pcdh10 is a SUMO target in vitro and in vivo and that its sumoylation is activity-dependent and developmentally regulated. Using site-directed mutagenesis, we point-mutated the predicted K831 SUMO site into an arginine (K831R), generating a non-sumoylatable form of Pcdh10. Interestingly, preventing Pcdh10 sumoylation at the K831 residue does not perturb the stability of Pcdh10 and its subcellular localization. Prior to unraveling the functional consequences of sumoylation in regulating Pcdh10 function and dysfunction, we first investigated the physiological role of Pcdh10 in the assembly and function of cortical synapses. To this end, we silenced Pcdh10 expression in cortical neurons at 7 days-in-vitro (DIV7) using a specific shRNA (shRNA-Pcdh10) and evaluated the effects on synapse development and physiology at DIV15. Combining advanced microscopy and electrophysiology approaches we demonstrated that the loss of Pcdh10 expression leads to an increase in both dendritic spines and excitatory synapses density, which correlates with higher frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs). Taking advantage of super-resolution Stimulated emission depletion (STED) microscopy, we performed detailed measurements of dendritic spine architecture, including spine head and neck. Neurons lacking Pcdh10 displayed shorter spines characterized by a larger head, indicating that Pcdh10 is essential in maintaining the proper dendritic spine morphology. We confirmed these results in vivo using In Utero Electroporation (IUE), further supporting the role of Pcdh10 in controlling excitatory synapse formation and function.
Conversely, neither the number and architecture of inhibitory synapses nor the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) were impaired in Pcdh10 silenced neurons, indicating that Pcdh10 is essential in the physiology of excitatory but not inhibitory synapses.
Interestingly, rescue experiments demonstrated that reintroducing the non sumoylatable form of Pcdh10 failed in rescuing the dendritic spine density in Pcdh10-silenced neurons. Conversely, the WT and K831R forms of Pcdh10 properly restored the mEPSCs frequency defects induced by Pcdh10 knockdown.
Altogether, our results demonstrate that sumoylation is an essential mechanism controlling the Pcdh10 physiological role in neurons and that perturbing such process compromises excitatory synapse formation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Autism affects brain functions and leads to behavioral, social and cognitive impairments, representing a major health and economic burden worldwide. To date, numerous genes have been associated with the development of autism and significant research efforts have been devoted to decipher their role in the pathogenesis of this disorder. However, the function of several autism-related genes remains unknown, thus preventing the development of effective therapies. Here, we uncover the role of the autism-related protein Pcdh10 in regulating excitatory synapse formation and function. Furthermore, we demonstrate that sumoylation of Pcdh10 is essential in controlling the density of dendritic spines suggesting a possible involvement of Pcdh10 sumoylation in modulating the synaptic strength.
The regulation of synaptic strength, spine architecture and density are key processes for the control of neuronal functions and are altered in several forms of autism. Thus, my results, by providing key information on the physiological role of Pcdh10 sumoylation, highlight sumoylation as a novel regulatory mechanism in excitatory synapse maturation and function. This pioneers a novel and stimulating topic in the neuroscience field aiming at uncovering the role of sumoylation on the etiology of mental disorders.

Image promoting the SUMO-PCDH10 project