Super-resolution of PSD95 remodelling during synaptic plasticity
by Valérie Clavet-Fournier
Date of Examination:2021-01-14
Date of issue:2021-07-01
Advisor:Dr. Katrin Willig
Referee:Prof. Dr. Siegrid Löwel
Referee:Dr. Jeong Seop Rhee
Referee:Prof. Dr. Fred Wolf
Referee:Prof. Dr. Ralf Heinrich
Referee:Dr. Oliver Schlüter
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EnglishStrengthening and weakening of synapses are a fundamental information processing unit within the neuronal circuit of the brain. It has been shown that the most abundant scaffold protein of the postsynaptic density (PSD), PSD95, undergoes structural remodeling during long-term potentiation (LTP). With superresolution microscopy techniques, it was recently shown that the nanoorganization of PSD95 often appears in clusters or perforations, which has been revealed in living mice using in vivo STED microscopy. Moreover, recent evidence suggests that also other synaptic proteins show a clustered nanoorganization and that pre- and postsynaptic proteins and glutamate AMPA receptors align in so-called nanocolumns trans-synaptically. However, the impact of these nanoorganization on synaptic strength as well as their changes after activity remains ellusive. Therefore, my project aims to investigate the remodeling of pre- and postsynaptic structures after chemical LTP induction using STED nanoscopy. I performed live-cell STED imaging of endogenous PSD95 assemblies for up to 2h after chemical LTP induction of CA1 hippocampal neurons to reveal the morphological changes of PSD95 organization at nanoscale resolution. Moreover, I explored the assembly of the active zone protein Bassoon and AMPA receptor nanodomains in correlation with structural changes of PSD95 assemblies after LTP in hippocampal culture neurons using immunohistochemistry and two-color STED microscopy. For different time-points, before and after LTP, I analyzed the area of PSD95, AMPA receptor and Bassoon assemblies. The shape of their nanoorganization was analyzed qualitatively by classification of different shapes of protein assemblies and quantitatively by calculating a filling factor of these proteins per synapse. My data shows that the PSD95 area undergoes an increase with a delay of 1h following LTP induction and that this change is accompanied by the formation of segmented 2 and later perforated PSD95. Furthermore, the area of PSD95 assembly increases simultaneously with AMPA receptor nanodomain and Bassoon assembly. Bassoon assemblies show a similar shape as PSD95 organizations, and their remodeling is correlated after LTP induction. Finally, the increase of the number of AMPA receptor nanodomain is correlated with the formation of perforated and segmented 2 PSD95 2h following LTP stimulation and as well with the activation of silent synapses. Therefore, remodeling of PSD95 is accompanied by an increase of the number and the area of AMPA receptor cluster, as well as an increase of the area of Bassoon assembly. Thus, those results suggest that the structural plasticity of PSD95 is an important feature to improve the synaptic strength of the synapse.
Keywords: PSD95; STED; Long-term potentiation; AMPA; Bassoon