Biology subjects

Vasek, M. J.

Publications and source records attributed to Vasek, M. J..

5 recordsLinked to original sources

bioRxiv2023

Evaluation of gliovascular functions of Aqp4 readthrough isoforms

Aquaporin-4 (AQP4) is a water channel protein that links astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, that preferentially localizes around the BBB through interaction with the scaffolding protein -syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel mouse AQP4 line (AllX) to increase relative levels of the readthrough variant above the [~]15% of AQP4 in the brain of wildtype (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and -syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to wildtype, and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. Increased dose of AQP4x enhanced perivascular localization of - syntrophin and AQP4, while total protein expression of the two were unchanged. However, at 100% readthrough, AQP4x localization and formation of higher-order complexes was disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for increased endothelial cell vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/549379v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18f8f32org.highwire.dtl.DTLVardef@25afeorg.highwire.dtl.DTLVardef@a3e0e1org.highwire.dtl.DTLVardef@100f2e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Microglia perform local protein synthesis at perisynaptic and phagocytic structures

Recent studies have illuminated the importance of several key signaling pathways in regulating the dynamic surveillance and phagocytic activity of microglia. Yet little is known about how these signals result in the assembly of phagolysosomal machinery near targets of phagocytosis, especially in processes distal from the microglial soma. Neurons, astrocytes, and oligodendrocytes locally regulate protein translation within distal processes. Therefore, we tested whether there is regulated local translation within peripheral microglia processes (PeMPs). We show that PeMPs contain ribosomes which engage in de novo protein synthesis, and these associate with a subpool of transcripts involved in pathogen defense, motility, and phagocytosis. Using a live slice preparation, we further show that acute translation blockade impairs the formation of PeMP phagocytic cups, the localization of lysosomal proteins within them, and phagocytosis. Collectively, these data argue for a regulated local translation in PeMPs and indicate a need for new translation to support dynamic microglial function.

neuroscience↗

bioRxiv2020

CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses.

Quaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3 UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocytes territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

neuroscience↗

bioRxiv2019

Transposon-mediated, cell type-specific transcription factor recording in the mouse brain

Transcription factors (TFs) play a central role in the regulation of gene expression, controlling everything from cell fate decisions to activity dependent gene expression. However, widely-used methods for TF profiling in vivo (e.g. ChIP-seq) yield only an aggregated picture of TF binding across all cell types present within the harvested tissue; thus, it is challenging or impossible to determine how the same TF might bind different portions of the genome in different cell types, or even to identify its binding events at all in rare cell types in a complex tissue such as the brain. Here we present a versatile methodology, FLEX Calling Cards, for the mapping of TF occupancy in specific cell types from heterogenous tissues. In this method, the TF of interest is fused to a hyperactive piggyBac transposase (hypPB), and this bipartite gene is delivered, along with donor transposons, to mouse tissue via a Cre-dependent adeno-associated virus (AAV). The fusion protein is expressed in Cre-expressing cells where it inserts transposon "Calling Cards" near to TF binding sites. These transposons permanently mark TF binding events and can be mapped using high-throughput sequencing. Alternatively, unfused hypPB interacts with and records the binding of the super enhancer (SE)-associated bromodomain protein, Brd4. To demonstrate the FLEX Calling Card method, we first show that donor transposon and transposase constructs can be efficiently delivered to the postnatal day 1 (P1) mouse brain with AAV and that insertion profiles report TF occupancy. Then, using a Cre-dependent hypPB virus, we show utility of this tool in defining cell type-specific TF profiles in multiple cell types of the brain. This approach will enable important cell type-specific studies of TF-mediated gene regulation in the brain and will provide valuable insights into brain development, homeostasis, and disease.

neuroscience↗

bioRxiv2019

Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

In situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

genomics↗