Viral vectors for neuronal cell type-specific visualization and manipulations

Abstract Alzheimer’s disease (AD) is marked by neurofibrillary tangles and senile plaques comprising amyloid β (Aβ) peptides. However, specific contributions of different cell types to Aβ deposition remain unknown. Non-coding microRNA (miRNA) play important roles in AD by regulating major proteins involved, like Aβ precursor protein (APP) and β-site APP-cleaving enzyme (BACE1), two key proteins associated with Aβ biogenesis. MiRNAs typically silence protein expression via binding specific sites in 3’- untranslated region (3’UTR) mRNA. MiRNA regulates protein levels in a cell-type specific manner; however, mechanism of miRNA’s variable activities remains unknown. We developed “miRNA-associated native protein expression” (miRnape) assays to determine a natural "UTR limit" for a miRNA’s function in a particular cell type. We report that miR-298 treatment reduced native APP protein levels in an astrocytic but not in a neuronal cell line. From miR-298’s effects on APP-3’UTR activity and native protein levels, we infer that APP 3’-UTR length could explain the differential miR-298’s activity. Such truncated, but natural, 3’-UTR found in a specific cell type provides an opportunity to regulate native protein levels by particular miRNA. Thus, miRNA’s effect tailoring to a specific cell type bypassing another undesired cell type with a truncated 3’-UTR would potentially advance translational research.

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Complexity and graded regulation of neuronal cell-type–specific alternative splicing revealed by single-cell RNA sequencing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013056118 ◽

2021 ◽

Vol 118 (10) ◽

pp. e2013056118

Author(s):

Huijuan Feng ◽

Daniel F. Moakley ◽

Shuonan Chen ◽

Melissa G. McKenzie ◽

Vilas Menon ◽

...

Keyword(s):

Alternative Splicing ◽

Single Cell ◽

Neuronal Cell ◽

Cell Types ◽

Gabaergic Neurons ◽

Cell Type ◽

Differential Splicing ◽

Single Cell Rna Sequencing ◽

Hierarchical Levels ◽

Cell Type Specific

The enormous cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell-type–specific gene-regulatory programs at the molecular level. Although prevalent in the brain, the contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here, we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single-cell RNA-sequencing data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class. These programs consist of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2, and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs contribute to splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which may provide a molecular mechanism to specify neuronal identity and function that are orthogonal to established classifications based on transcriptional regulation.

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Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

10.1101/2021.08.03.454921 ◽

2021 ◽

Author(s):

Sruti Rayaprolu ◽

Sara Bitarafan ◽

Ranjita Betarbet ◽

Sydney N Sunna ◽

Lihong Cheng ◽

...

Keyword(s):

Mouse Brain ◽

Neurological Diseases ◽

Neuronal Cell ◽

Cell Types ◽

Proteomic Profiling ◽

Cell Type ◽

Specific Expression ◽

Cell Type Specific ◽

The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

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Single nucleus analysis of the chromatin landscape in mouse forebrain development

10.1101/159137 ◽

2017 ◽

Cited By ~ 2

Author(s):

Sebastian Preissl ◽

Rongxin Fang ◽

Yuan Zhao ◽

Ramya Raviram ◽

Yanxiao Zhang ◽

...

Keyword(s):

Neuronal Cell ◽

Cell Types ◽

Cellular Heterogeneity ◽

Regulatory Sequences ◽

Specific Cell ◽

Cell Type ◽

Tissue Samples ◽

Forebrain Development ◽

Cell Type Specific ◽

Accessible Chromatin

ABSTRACTGenome-wide analysis of chromatin accessibility in primary tissues has uncovered millions of candidate regulatory sequences in the human and mouse genomes1–4. However, the heterogeneity of biological samples used in previous studies has prevented a precise understanding of the dynamic chromatin landscape in specific cell types. Here, we show that analysis of the transposase-accessible-chromatin in single nuclei isolated from frozen tissue samples can resolve cellular heterogeneity and delineate transcriptional regulatory sequences in the constituent cell types. Our strategy is based on a combinatorial barcoding assisted single cell assay for transposase-accessible chromatin5 and is optimized for nuclei from flash-frozen primary tissue samples (snATAC-seq). We used this method to examine the mouse forebrain at seven development stages and in adults. From snATAC-seq profiles of more than 15,000 high quality nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell-types in foetal and adult forebrains. We further define cell-type specific cis regulatory sequences and infer potential master transcriptional regulators of each cell population. Our results demonstrate the feasibility of a general approach for identifying cell-type-specific cis regulatory sequences in heterogeneous tissue samples, and provide a rich resource for understanding forebrain development in mammals.

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An extended toolkit for production and use of RVdG-CVS-N2c rabies viral vectors uncovers hidden hippocampal connections

10.1101/2021.12.23.474014 ◽

2021 ◽

Author(s):

Anton Sumser ◽

Maximilian Joesch ◽

Peter Jonas ◽

Yoav Ben-Simon

Keyword(s):

Viral Vectors ◽

Molecular Tools ◽

Cell Type ◽

Large Collection ◽

Neuronal Connectivity ◽

Retrograde Labeling ◽

Neuronal Populations ◽

Wide Range ◽

Cell Type Specific ◽

Cortical Microcircuit

From the large collection of molecular tools used to investigate neuronal connectivity, envA-pseudotyped rabies viral vectors (RVdGenvA) uniquely enable cell-type specific, trans-synaptic retrograde labeling. However, widespread use of the powerful and flexible method is to date hindered by low-yield and cumbersome production pipelines. Here, we report the development of new cell lines, which significantly reduce production time while increasing viral titer and eliminating background contamination from native-coat particles. We further show that RVdGenvA-CVS-N2c vectors produced using this system retain their enhanced retrograde-trafficking when compared with SAD-B19 vectors, allowing us to uncover undescribed cortico-hippocampal connections and to monitor activity in a cortical microcircuit of behaving animals. Along with new suites of AAV and RVdG-CVS-N2c vectors, developed to enable retrograde labeling from a wide range of neuronal populations and tailored for diverse experimental requirements, we present here an optimal system for mapping, manipulating and imaging of neuronal circuits.

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