Proteomics-based synapse characterization: From proteins to circuits.

The highly heterogeneous nature of neuronal cell types and their connections presents a major challenge to the characterization of neural circuits at the protein level. New approaches now enable an increasingly sophisticated dissection of cell type- and cellular compartment-specific proteomes, as well as the profiling of the protein composition of specific synaptic connections. Here, we provide an overview of these approaches and discuss how they hold considerable promise toward unravelling the molecular mechanisms of neural circuit formation and function. Finally, we provide an outlook of technological developments that may bring the characterization of synaptic proteomes at the single-synapse level within reach.

Hydrop enables droplet-based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads.

Single-cell RNA-seq and single-cell assay for transposase-accessible chromatin (ATAC-seq) technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost and pave the way for more specialized multiome assays, custom droplet microfluidics may provide solutions complementary to commercial setups. We developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost noncommercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 7996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA's high capture rate to analyze a small population of fluorescence-activated cell sorted neurons from the Drosophila brain, confirming the protocol's applicability to low input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi) omics protocols.

Scientists are now able to determine the order of chemical blocks, or nucleic acids, that make up the genetic code. These sequencing tools can be used to identify which genes are active within a biological sample. They do this by extracting and analysing open chromatin (regions of DNA that are accessible to the cell's machinery), or sequences of RNA (the molecular templates cells use to translate genes into working proteins). Initially, most sequencing tools could only provide an 'averaged-out' profile of the genes activated in bulk pieces of tissue which contain multiple types of cell. However, advances in technology have led to new methods that can extract and analyse open chromatin or RNA from individual cells. First, the cells are separated, via a technique called microfluidics, into tiny droplets of water along with a single bead that carries a unique barcode. The cell is then broken apart inside the droplet and the barcode within the bead gets released and attaches itself to the genetic material extracted from the cell. All the genetic material inside the droplets is then pooled together and sequenced. Researchers then use the barcode tags to identify which bits of RNA or DNA belong to each cell. Single-cell sequencing has many advantages, including being able to pinpoint precise genetic differences between healthy and abnormal cells, and to create cell atlases of whole organisms, tissues and microbial communities. But existing methods for extracting chromatin are very expensive, and there were no openly available tools for processing thousands of cells at speed. Furthermore, while several single-cell RNA sequencing tools are already freely available, they are not very sensitive or practical to use. Here, De Rop et al. have developed a new open-source platform called HyDrop that overcomes these barriers. The method entails a new type of barcoded bead and optimised elements of existing microfluidics protocols using open-source reagents. These changes created a more user-friendly workflow and increased sensitivity of sequencing at no additional cost. De Rop et al. used their new platform to screen the RNA and open chromatin of thousands of individuals cells from the brains of mice and flies. HyDrop outperformed other open-source methods when working in RNA-sequencing mode. It also provides the first open-source tool for sequencing open chromatin in single cells. Further improvements are expected as researchers tweak the platform, which for now provides an affordable alternative to existing methods.

Contribution of GABAergic interneurons to amyloid-ß plaque pathology in an APP knock-in mouse model.

The amyloid-ß (Aß) peptide, the primary constituent of amyloid plaques found in Alzheimer's disease (AD) brains, is derived from sequential proteolytic processing of the Amyloid Precursor Protein (APP). However, the contribution of different cell types to Aß deposition has not yet been examined in an in vivo, non-overexpression system. Here, we show that endogenous APP is highly expressed in a heterogeneous subset of GABAergic interneurons throughout various laminae of the hippocampus, suggesting that these cells may have a profound contribution to AD plaque pathology. We then characterized the laminar distribution of amyloid burden in the hippocampus of an APP knock-in mouse model of AD. To examine the contribution of GABAergic interneurons to plaque pathology, we blocked Aß production specifically in these cells using a cell type-specific knock-out of BACE1. We found that during early stages of plaque deposition, interneurons contribute to approximately 30% of the total plaque load in the hippocampus. The greatest contribution to plaque load (75%) occurs in the stratum pyramidale of CA1, where plaques in human AD cases are most prevalent and where pyramidal cell bodies and synaptic boutons from perisomatic-targeting interneurons are located. These findings reveal a crucial role of GABAergic interneurons in the pathology of AD. Our study also highlights the necessity of using APP knock-in models to correctly evaluate the cellular contribution to amyloid burden since APP overexpressing transgenic models drive expression in cell types according to the promoter and integration site and not according to physiologically relevant expression mechanisms.