Periodic Reporting for period 1 - SYNarch (Understanding the nanoscale synaptome architecture of the brain)
Reporting period: 2022-07-01 to 2024-06-30
In the brain, neurons are connected by billions of synapses. The postsynaptic density (PSD) is a densely packed structure located beneath the postsynaptic membrane of excitatory synapses. PSD proteins play essential roles in synaptic structure and function, and their dysfunction is linked to more than 100 brain disorders, including schizophrenia, depression, autism, and intellectual disabilities. Key to PSD protein function is their assembly into complexes and supercomplexes, which are known to vary in composition and spatial distribution within synapses, contributing to synapse diversity.
The Grant lab developed a pipeline for mapping synaptic complexes using high-speed confocal microscopy and advanced image analysis, revealing brain-wide synapse molecular diversity that changes throughout the lifespan in mice. At higher resolution, super-resolution microscopy showed that PSD95 proteins form nanoscale structures within synapses, adding nanoarchitecture as a further, as yet little understood, level to synapse diversity. These nanoscale arrangements are crucial for synaptic transmission and plasticity. Disease-associated mutations disrupt PSD supercomplex structures and synaptome architecture, particularly in models of autism and schizophrenia, but their impact on nanoarchitecture is unknown. The SYNarch project aimed to employ key PSD proteins to map diversity in synapse nanoarchitecture across regions of the mouse brain, providing a baseline for investigations into how mutations affect this fundamental level of synaptic protein organization, with implications for major brain disorders.
During SYNarch, a method was developed to evaluate synaptic nanoarchitecture using Förster resonance energy transfer (FRET) between PSD95 molecules, which was combined with the synaptome mapping pipeline. This new combination of resolution and scale uncovered the diversity of synapse nanoarchitecture across brain regions and lifespan in mice, and probed the impact of a schizophrenia mutation.
The Grant lab developed a pipeline for mapping synaptic complexes using high-speed confocal microscopy and advanced image analysis, revealing brain-wide synapse molecular diversity that changes throughout the lifespan in mice. At higher resolution, super-resolution microscopy showed that PSD95 proteins form nanoscale structures within synapses, adding nanoarchitecture as a further, as yet little understood, level to synapse diversity. These nanoscale arrangements are crucial for synaptic transmission and plasticity. Disease-associated mutations disrupt PSD supercomplex structures and synaptome architecture, particularly in models of autism and schizophrenia, but their impact on nanoarchitecture is unknown. The SYNarch project aimed to employ key PSD proteins to map diversity in synapse nanoarchitecture across regions of the mouse brain, providing a baseline for investigations into how mutations affect this fundamental level of synaptic protein organization, with implications for major brain disorders.
During SYNarch, a method was developed to evaluate synaptic nanoarchitecture using Förster resonance energy transfer (FRET) between PSD95 molecules, which was combined with the synaptome mapping pipeline. This new combination of resolution and scale uncovered the diversity of synapse nanoarchitecture across brain regions and lifespan in mice, and probed the impact of a schizophrenia mutation.
I first developed and evaluated the FRET method for mapping synapse nanoarchitecture. Histological sections of brain were prepared from genetically modified mice that express PSD95-Halo, in which the key PSD protein PSD95 is linked to the self-labeling Halo tag protein, which forms a covalent bond with its ligands. Labelling PSD95-Halo using a mixture of Halo tag ligands conjugated with either of two different fluorophores, I succeeded in detecting FRET between the fluorophores. This indicates detection of the association between PSD95 molecules on synapses at the nanoscale; similarly, interaction between PSD95 and a further key synaptic protein, SAP102, was detected. Then, I optimized the experimental settings, including the combination and concentration of fluorophores for the labeling and optical settings of spinning disk confocal microscopy to maximize the dynamic range of FRET detection. Collaborating with Grant lab computational scientists, I next developed a computational pipeline to detect fluorescence and calculate the FRET efficiency of individual synapses labeled with Halo tag ligands. Using this pipeline, I conducted a comprehensive analysis of synapses in whole mouse brain at 2 weeks, 4, 12 and 18 months of age, uncovering differences in synaptic nanoarchitecture, as reported by FRET efficiency, across brain regions and lifespan. That the adult (4 months of age) mouse brain shows such regional diversity in FRET efficiency is illustrated in the figure. An analysis of the impact of the Psd93 mutation, a schizophrenia model, on this architecture is underway.
The SYNarch project has advanced current understanding of synapses in terms of the nature and diversity of interactions that occur between PSD95 proteins at the nanoscale. The project has built a baseline that can now be extended, using the high-resolution, wide-scale methods developed herein, beyond PSD95 and SAP102 to encompass further synaptic proteins and combinations thereof. It is anticipated that this will help uncover the relationship between synapse nanoarchitecture and brain function at a very fundamental level, including synaptic transmission efficiency. Furthermore, application of SYNarch methods in mouse models (analysis in the Psd93 schizophrenia model is underway) will add a new layer to our current understanding of the synapse pathology underlying neuropsychiatric disorders, which may have significant social and economic impact.