Periodic Reporting for period 1 - LIPSYNING (Eat me microglia: lipid scrambling as a signal for synaptic pruning)
Reporting period: 2016-09-01 to 2018-08-31
The development of the nervous system is associated with the generation of excess neuronal synapses that is followed by their removal, a process known as synaptic pruning. In the primate cortex, for example, 70% of connections are lost within the first six months of life. Why are so many synapses lost, what determines which synapses are eliminated, what are the molecular mechanisms involved, and what are the consequences of not getting it right? Over the recent years, several studies have presented microglial phagocytosis as a path for synapse elimination. Neural activity plays a role in synaptic pruning, but the neuronal “eat-me” signals that mediate phagocytic recognition and engulfment of synapses remain to be identified. We hypothesized that cell surface exposure of the lipid phosphatidylserine (PtdSer) was a key “eat-me” signal for synaptic pruning during development. Therefore, we aimed to define the role of PtdSer in synapse-microglia interaction and to assess the morphological, circuit maturation and behavioral effects of impaired PtdSer exposure in phospholipid scramblase-deficient brains. We proposed to use novel custom-made tools to observe PtdSer exposure without interfering with PtdSer-dependent cellular interactions and mouse models with disrupted PtdSer exposure to study how PtdSer contributes to circuit refinement. The identification of an “eat-me” signal sheds the light on what distinguishes synapses destined to be eliminated from those that survive and is a step towards the understanding why the majority of synapses are turned over during brain development before the final connectome emerges. As aberrant brain wiring during development is known to be defective in a wide range of neurodevelopmental disorders, understanding how circuits are formed and refined during developmental period is critical to understand their aetiology and initiate the development of the therapy based on molecular mechanisms of disease. Our findings of this project identified the molecular pathway that is essential for axonal pruning in developing brain, indicating for the first time caspase-3 – Xkr-8 – PtdSer axis in the elimination of superfluous projections to obtain structurally and functionally mature brain.
In this project we investigated the role of phosphatidylserine (PtdSer) scrambling in synaptic pruning. We used immunohistochemical detection of phosphatidylserine-specific opsonin MFG-E8 to identify the extent and the localization of PtdSer in developing brain. We further used conditional pyramidal neuron knock-out of Xkr8 phospholipid scramblase to define the role of PtdSer scrambling for synapse elimination. We used region-specific IHC labelling for cortical and thalamic projections to assess pruning specificity in Xkr8 cKO brains. Morphological findings were confirmed by electrophysiology of acute hippocampal slices and fMRI analysis of the whole brain connectome. We found that PtdSer was preferentially exposed on synaptic structures and promoted microglia-synapse interaction. PtdSer exposure was developmentally upregulated and required the activity of Xkr8 phospholipid scramblase. Conditional Xkr8 knock-out in excitatory neurons led to diminished axonal bouton trogocytosis and insufficient elimination of excitatory synapses. These morphological aberrations were followed by abnormal electrophysiological profiles of Xkr8-deficient neurons that exhibited increased spontaneous activity and the failure of functional synaptic maturation. Finally, Xkr8 deficiency led to increased global connectivity that was maintained into adulthood. Altogether we demonstrated that mammalian synaptic pruning requires developmental PtdSer exposure via Xkr8 scramblase activity, identifying the crucial cascade to expose an „eat-me“ signal on unnecessary synapses for their developmental removal.
This interdisciplinary project employed a wide range of approaches spanning from molecular analysis to the measurements of neuronal activity and global brain function. The application of ex vivo and in vivo systems ensured comprehensive inquiry into molecular pathways guiding brain development, which is essential for contemporary neuroscientific studies. These methods were successfully integrated into the toolkit of the Department of Neurobiology and Biophysics of Life Sciences Center of Vilnius University and strengthened molecular neuroscience research there.
The findings of this project established a base for future investigations of neurodevelopmental refinement of brain circuitry. The unique tools developed in this project will be used to dissect further mechanisms of synaptic maturation in developing brain. Aberrant brain wiring during development is known to be defective in a wide range of neurodevelopmental disorders and understanding how circuits are formed and refined during this period will be critical for explaining their aetiology, in order to alleviate societal challenges associated with neurodevelopmental and neuropsychiatric disorders, such as autism or schizophrenia.
Scientific outcomes of this project were presented at multiple scientific conferences, including local (Conferences of Lithuanian Neuroscience Society, BaltLASA Conference of Baltic Laboratory Animal Science Association, Conference of Lithuanian Stem Cell Association), European (EMBO Microglia, FENS Forum 2020, Neuronus IBRO Neuroscience Forum) and international events (IBRO World Congress, Society for Neuroscience Global Connectome). Importantly, in addition to the dissemination of the results within the scientific community, this project included wide public engagement through different media, such as TV programs, radio interviews, the articles in the newspapers and journals, and school visits to meet both pupils and their teachers. It is estimated that the audience of the public outreach activities of this project exceeded 150 000.
The findings of this project established a base for future investigations of neurodevelopmental refinement of brain circuitry. The unique tools developed in this project will be used to dissect further mechanisms of synaptic maturation in developing brain. Aberrant brain wiring during development is known to be defective in a wide range of neurodevelopmental disorders and understanding how circuits are formed and refined during this period will be critical for explaining their aetiology, in order to alleviate societal challenges associated with neurodevelopmental and neuropsychiatric disorders, such as autism or schizophrenia.
Scientific outcomes of this project were presented at multiple scientific conferences, including local (Conferences of Lithuanian Neuroscience Society, BaltLASA Conference of Baltic Laboratory Animal Science Association, Conference of Lithuanian Stem Cell Association), European (EMBO Microglia, FENS Forum 2020, Neuronus IBRO Neuroscience Forum) and international events (IBRO World Congress, Society for Neuroscience Global Connectome). Importantly, in addition to the dissemination of the results within the scientific community, this project included wide public engagement through different media, such as TV programs, radio interviews, the articles in the newspapers and journals, and school visits to meet both pupils and their teachers. It is estimated that the audience of the public outreach activities of this project exceeded 150 000.