Periodic Reporting for period 1 - DiverseSynapse (Revealing the Landscape of Synaptic Diversity by Cell type- and Synapse-specific Proteomics and Transcriptomics)
Período documentado: 2022-08-01 hasta 2025-01-31
Neurons are highly polarized and use compartmentalized information processing at synapses for communication with other neurons. Synapses differ in shape, size, and function and exhibit ongoing plasticity during development and modification of neural circuits. This plasticity is essential for correct wiring of the nervous system and its ability to encode, maintain and remember facts, procedures and experiences. While the structural diversity of synapses is largely known, their potential molecular diversity is much less well understood. Molecules residing at synapses have been identified by diverse approaches but the complement of proteins at individual synapse types and their stoichiometric relationships with other molecules is unknown. Current classification mostly relies on neurotransmitter/receptor phenotypes, leading to broad descriptors like “excitatory” or “inhibitory” synapses. This relatively coarse understanding of molecular diversity of synapses is mostly due to technical limitations associated with the small size of synapses and difficulties in purifying them, particularly those associated with different cell types. As the function of the synapse and its ability to change is largely determined by the molecules (mRNAs, proteins, lipids) inhabiting it, it is essential to understand the molecular diversity of synapses. Indeed, the quality, quantity and interactions of proteins at synapses largely determine the physiological properties of synaptic transmission and on a larger scale, the organization and function of neuronal circuits. The aims of this project are to determine the transcriptomes and proteomes of genetically-identifiable synaptic populations in different brain areas, to determine the molecular diversity in these same synapse populations using transcriptomic analysis of individual synapses, and to assess how these synaptic proteomes, transcriptomes and the transcriptomic diversity respond to plasticity. We use fluorescence-activated synaptosome sorting (FASS) to purify different synaptic populations from different brain areas, and next generation RNA sequencing (RNA-NGS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), optimized for quantitative transcriptomic and proteomic profiling of very small amounts of sorted synapses. We further develop a method, SynDrops, for the transcriptomic analysis of individual synapses. These studies will reveal the diversity within synapse types and across brain areas and states, and allow the field to probe “diseased” synapses in the future.
To investigate the proteomic landscape of synaptic diversity across brain regions and cell types we prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with FASS, we isolated and analyzed the proteomes of 18 different synapse types by LC-MS/MS. We discovered 1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different synapse types. With these data we created an interactive web tool (https://syndive.org/(se abrirá en una nueva ventana)) allowing to query the abundance and localization of individual proteins in individual synapse types. We identified shared synaptic protein modules and proteomic hotspots for synapse specialization, found unique and common features of the striatal dopaminergic proteome and discovered the proteome signatures that relate to the functional properties of different interneuron classes. This work provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit level experiments (Van Oostrum et al., 2023).
We next compared transcriptomes and proteomes of cell type-specific synaptosomes from different hippocampal subregions and subfield CA1 strata of a transgenic line. We purified synaptosomes from those microdissected regions using FASS, providing sufficient material for downstream analysis of both the proteome (LC-MS/MS) and the transcriptome (RNA-NGS) from the same source. We identified over 15,000 mRNAs and 10,000 proteins, revealing thousands with local enrichment (e.g. classes of glutamate receptors, voltage-gated potassium channels, myelin-associated molecules, adhesion molecules). Synaptosome analysis further identified specific enrichment of molecules from collagen, ribosome, solute carrier, and receptor families at different synapses formed along CA1 neurons. By integrating mRNA and protein data, we defined clusters of co-regulated molecules such as adhesion and neurofilament proteins and transporter mRNAs and found subsets of mRNA-protein pairs with strong correlations and anti-correlations in their abundance variation. Our findings comprise a rich resource on the molecular landscape of the hippocampus and its synapses (accessible at syndive.org) and highlight the coordinated organization of transcripts and proteins between regions, neuronal compartments and synapses (Kaulich et al., 2024).
To characterize the transcriptomic composition of individual synapses, we developed SynDrops, a droplet microfluidic approach which involves sorting and purifying fluorescently labelled synaptic particles in a cell-type-specific manner. Individual synaptic particles are encapsulated into picoliter droplets containing the necessary components to convert synaptic RNAs to cDNA, and adding a unique molecular barcode to the RNAs within each droplet. After sequencing the mixed contents of these droplets, we use bioinformatics to reconstruct the RNA content of individual synapses based on their molecular barcodes. The method is currently in the final state of refinement. In species mixing experiments, we have already validated the single synapse resolution of the method. Using the method, we are generating unbiased transcriptomic profiles of hundreds of thousands of individual synapses. This will allow us to identify RNAs frequently found in synapses and RNAs frequently found together.
We also examine how specific synaptic proteomes and transcriptomes respond to altered synaptic activity and plasticity. We established a complementary primary neuronal cell culture system, allowing us to profile transcriptome remodeling in the synapse after pharmacological manipulations. Preliminary data reveal that the synaptic transcriptome also reflects different activity states with dozens of genes upregulated during homeostatic downscaling. We are pursuing additional sorting strategies where the activity level of the synapse can also be monitored. Our approach reveals new insights into the transcriptomic diversity of synapse types and states and can be flexibly adapted to profile synapse remodeling during any form of plasticity or after any behavioral manipulation.
We next compared transcriptomes and proteomes of cell type-specific synaptosomes from different hippocampal subregions and subfield CA1 strata of a transgenic line. We purified synaptosomes from those microdissected regions using FASS, providing sufficient material for downstream analysis of both the proteome (LC-MS/MS) and the transcriptome (RNA-NGS) from the same source. We identified over 15,000 mRNAs and 10,000 proteins, revealing thousands with local enrichment (e.g. classes of glutamate receptors, voltage-gated potassium channels, myelin-associated molecules, adhesion molecules). Synaptosome analysis further identified specific enrichment of molecules from collagen, ribosome, solute carrier, and receptor families at different synapses formed along CA1 neurons. By integrating mRNA and protein data, we defined clusters of co-regulated molecules such as adhesion and neurofilament proteins and transporter mRNAs and found subsets of mRNA-protein pairs with strong correlations and anti-correlations in their abundance variation. Our findings comprise a rich resource on the molecular landscape of the hippocampus and its synapses (accessible at syndive.org) and highlight the coordinated organization of transcripts and proteins between regions, neuronal compartments and synapses (Kaulich et al., 2024).
To characterize the transcriptomic composition of individual synapses, we developed SynDrops, a droplet microfluidic approach which involves sorting and purifying fluorescently labelled synaptic particles in a cell-type-specific manner. Individual synaptic particles are encapsulated into picoliter droplets containing the necessary components to convert synaptic RNAs to cDNA, and adding a unique molecular barcode to the RNAs within each droplet. After sequencing the mixed contents of these droplets, we use bioinformatics to reconstruct the RNA content of individual synapses based on their molecular barcodes. The method is currently in the final state of refinement. In species mixing experiments, we have already validated the single synapse resolution of the method. Using the method, we are generating unbiased transcriptomic profiles of hundreds of thousands of individual synapses. This will allow us to identify RNAs frequently found in synapses and RNAs frequently found together.
We also examine how specific synaptic proteomes and transcriptomes respond to altered synaptic activity and plasticity. We established a complementary primary neuronal cell culture system, allowing us to profile transcriptome remodeling in the synapse after pharmacological manipulations. Preliminary data reveal that the synaptic transcriptome also reflects different activity states with dozens of genes upregulated during homeostatic downscaling. We are pursuing additional sorting strategies where the activity level of the synapse can also be monitored. Our approach reveals new insights into the transcriptomic diversity of synapse types and states and can be flexibly adapted to profile synapse remodeling during any form of plasticity or after any behavioral manipulation.
In our project, we developed robust methodological pipelines for proteomic and transcriptomic analysis of synaptosome populations from different cell-types and brain regions (Van Oostrum et al., 2023, Kaulich et al., 2024). Our yet to be finalized SynDrops protocol will allow for transcriptomic profiling of individual synapses. We see a great application potential of these protocols in proteome and transcriptome studies in neuroscience and beyond, also addressing questions related to synaptic malfunctioning in diseases. Further on, our Syndive platform is a valuable tool for researchers interested in synaptic protein abundance and localization.