Periodic Reporting for period 4 - FunctionalProteomics (Proteomic fingerprinting of functionally characterized single synapses)
Reporting period: 2023-04-01 to 2024-09-30
Chemical synapses in the central nervous system are extremely diverse: their size, their molecular composition and their functional properties vary orders of magnitudes. The structural and molecular mechanisms underlying the functional heterogeneity of synapses have been in the forefront of cellular neuroscience for many decades, resulting in a considerable progress in our understanding. However, the major shortcoming is the lack of high-resolution methods that allow the molecular and structural analysis of functionally characterized synapses. One of the major aims of the current proposal was to develop a high-resolution multiplexed molecular method that allows the quantitative analysis of dozens of molecules in functionally characterized single hippocampal synapses at resolutions ~40 nm.
Understanding the molecular mechanisms of synaptic functional diversity is essential for understanding the dynamic properties of neuronal networks during behavior. In addition, changes in synaptic function have been shown to play major roles in many psychiatric and neurodegenerative disorders, but the exact molecular alterations are currently unknown. Revealing the molecular underpinnings of diverse function in healthy neuronal networks will provide the foundation of understanding changes in different diseases states.
The overall objective of the application is to reveal causal links between the quantity of different proteins and defined functional properties of synapses and to investigate the functional properties and the proteomics of synapses made by functionally characterized nerve cells in behaving animals.
Understanding the molecular mechanisms of synaptic functional diversity is essential for understanding the dynamic properties of neuronal networks during behavior. In addition, changes in synaptic function have been shown to play major roles in many psychiatric and neurodegenerative disorders, but the exact molecular alterations are currently unknown. Revealing the molecular underpinnings of diverse function in healthy neuronal networks will provide the foundation of understanding changes in different diseases states.
The overall objective of the application is to reveal causal links between the quantity of different proteins and defined functional properties of synapses and to investigate the functional properties and the proteomics of synapses made by functionally characterized nerve cells in behaving animals.
Using electron microscopy replica immunolabelling, we have provided evidence for distinct nanoscale arrangements of presynaptic release sites and voltage-gated Ca2+ channels in two distinct cerebellar synapses. We have also shown that the distinct nanoscale distributions of Ca2+ channels around docked vesicles are responsible for distinct release probabilities (Rebola et al., 2019, Neuron).
We have demonstrated large functional diversity of synapses made by hippocampal CA1 pyramidal cells and fast-spiking GABAergic interneurons. The large variability in EPSC amplitude is the consequence of variable numbers of functional release sites between the connected nerve cells. Molecular analysis of the functionally characterized synapses has revealed that synapses with the same number of release sites contain variable amounts of Munc13-1 molecules; a key vesicle docking/priming factor. These Munc13-1 molecules are arranged in nanoclusters within the active zones, the number of which equals that of functional release sites. These nanoclusters have variable sizes and contain different numbers of Munc13-1 molecules (Karlocai et al., 2021, eLife).
We have developed a quantitative, high-resolution, multiplexed immunolocalization method that allows the proteomic analysis of functionally characterized single synapses with a resolution of about 40 nm (Holderith et al., 2020, Cell Rep).
Using pharmacological, physiological and computer modelling approaches, we have demonstrated that the unreliability of a given hippocampal synapse is the consequence of synaptic vesicles that are not capable of fusing with the plasma membrane (fusion incompetent) rather than the low fusion probability of fusion-competent vesicles (Aldahabi et al., 2024, PNAS).
We have demonstrated large functional diversity of synapses made by hippocampal CA1 pyramidal cells and fast-spiking GABAergic interneurons. The large variability in EPSC amplitude is the consequence of variable numbers of functional release sites between the connected nerve cells. Molecular analysis of the functionally characterized synapses has revealed that synapses with the same number of release sites contain variable amounts of Munc13-1 molecules; a key vesicle docking/priming factor. These Munc13-1 molecules are arranged in nanoclusters within the active zones, the number of which equals that of functional release sites. These nanoclusters have variable sizes and contain different numbers of Munc13-1 molecules (Karlocai et al., 2021, eLife).
We have developed a quantitative, high-resolution, multiplexed immunolocalization method that allows the proteomic analysis of functionally characterized single synapses with a resolution of about 40 nm (Holderith et al., 2020, Cell Rep).
Using pharmacological, physiological and computer modelling approaches, we have demonstrated that the unreliability of a given hippocampal synapse is the consequence of synaptic vesicles that are not capable of fusing with the plasma membrane (fusion incompetent) rather than the low fusion probability of fusion-competent vesicles (Aldahabi et al., 2024, PNAS).
Our development of a new high resolution localization method will allow us to address several key unanswered questions in synaptic neuroscience including the molecular nanoscale arrangements of pre- and postsynaptic molecules and their alterations in distinct disease conditions. Specifically, building on our results demonstrating quantitative Munc13-1 heterogeneity of functional release sites, we will address the question of how other presynaptic docking and priming molecules are localized with respect to the different Munc13-1 nanoclusters. We will also investigate the nanoscale arrangements of presynaptic molecules in the postmortem brain of schizophrenia subject. In addition, we will investigate potential changes in the molecular composition of functional release sites in distinct animal models of neurodevelopmental disorders, allowing us to perform in vitro electrophysiological and two-photon imaging experiments to investigate the functional consequences of potential alterations. Finally, we will perform molecular and functional analysis of identified synapses that are made by pre- and postsynaptic nerve cells the function of which had been monitored during behavior.