Identification of a circadian output circuit for rest:activity rhythms in Drosophila.

Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain and report here that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay.

Extracellular vesicles (EVs) have attracted enormous attention for their diagnostic and therapeutic potential. However, it has proven challenging to achieve the sensitivity to detect individual nanoscale EVs, the specificity to distinguish EV subpopulations, and a sufficient throughput to study EVs among an enormous background. To address this fundamental challenge, we developed a droplet-based optofluidic platform to quantify specific individual EV subpopulations at high throughput. The key innovation of our platform is parallelization of droplet generation, processing, and analysis to achieve a throughput (∼20 million droplets/min) more than 100× greater than typical microfluidics. We demonstrate that the improvement in throughput enables EV quantification at a limit of detection = 9EVs/µL, a >100× improvement over gold standard methods. Additionally, we demonstrate the clinical potential of this system by detecting human EVs in complex media. Building on this work, we expect this technology will allow accurate quantification of rare EV subpopulations for broad biomedical applications.

Caspase-Activated Oligonucleotide Probe.

Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resolution. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biological applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.

Efficient Synthesis of Light-Triggered Circular Antisense Oligonucleotides Targeting Cellular Protein Expression.

Light-activated ("caged") antisense oligonucleotides are powerful molecules for regulating gene expression at submicron spatial resolution through the focal modulation of endogenous cellular processes. Cyclized caged oligos are particularly promising structures because of their inherent stability and similarity to naturally occurring circular DNA and RNA molecules. Here, we introduce an efficient route for cyclizing an antisense oligodeoxynucleotide incorporating a photocleavable linker. Oligo cyclization was achieved for several sequences in nearly quantitative yields through intramolecular copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Caging stability and light activation were characterized by FRET efficiency, denaturing gel assay, and melting temperature measurements. Finally, a cyclized caged oligo was designed to target gfap, and it gave a tenfold reduction in glial fibrillary acidic protein upon photoactivation in astrocytes.

Serotonergic neuron regulation informed by in vivo single-cell transcriptomics.

Despite the recognized importance of the dorsal raphe (DR) serotonergic (5-HT) nuclei in the pathophysiology of depression and anxiety, the molecular components/putative drug targets expressed by these neurons are poorly characterized. Utilizing the promoter of an ETS domain transcription factor that is a stable marker of 5-HT neurons (Pet-1) to drive 5-HT neuronal expression of YFP, we identified 5-HT neurons in live acute slices. We isolated RNA from single 5-HT neurons in the ventromedial and lateral wings of the DR and performed single-cell RNA-Seq analysis identifying >500 G-protein coupled receptors (GPCRs) including receptors for classical transmitters, lipid signals, and peptides as well as dozens of orphan-GPCRs. Using these data to inform our selection of receptors to assess, we found that oxytocin and lysophosphatidic acid 1 receptors are translated and active in costimulating, with the α1-adrenergic receptor, the firing of DR 5-HT neurons, while the effects of histamine are inhibitory and exerted at H3 histamine receptors. The inhibitory histamine response provides evidence for tonic in vivo histamine inhibition of 5-HT neurons. This study illustrates that unbiased single-cell transcriptomics coupled with functional analyses provides novel insights into how neurons and neuronal systems are regulated.

Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes.

We show that the transfer of the adult ventricular myocyte (AVM) transcriptome into either a fibroblast or an astrocyte converts the host cell into a cardiomyocyte. Transcriptome-effected cardiomyocytes (tCardiomyocytes) display morphologies, immunocytochemical properties, and expression profiles of postnatal cardiomyocytes. Cell morphology analysis shows that tCardiomyoctes are elongated and have a similar length-to-width ratio as AVMs. These global phenotypic changes occur in a time-dependent manner and confer electroexcitability to the tCardiomyocytes. tCardiomyocyte generation does not require continuous overexpression of specific transcription factors; for example, the expression level of transcription factor Mef2c is higher in tCardiomyocytes than in fibroblasts, but similar in tCardiomyocytes and AVMs. These data highlight the dominant role of the gene expression profile in developing and maintaining cellular phenotype. The transcriptome-induced phenotype remodeling-generated tCardiomyocyte has significant implications for understanding and modulating cardiac disease development.

High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95% mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Intron retention facilitates splice variant diversity in calcium-activated big potassium channel populations.

We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX. i17a siRNA treatment followed by STREX protein immunocytochemistry demonstrated both reduced levels and altered subcellular distribution of STREX-containing BKCa channel protein. Selective reduction of i17a-BKCa or STREX-BKCa mRNAs induced similar changes in the burst firing properties of hippocampal neurons. Collectively, these data show that STREX splice variant regulation via cytoplasmic splicing and intron retention helps generate STREX-dependent BKCa current diversity in hippocampal neurons.

Withdrawn as duplicate: Commentary: "Zooming in" on Glioblastoma: Understanding Tumor Heterogeneity and its Clinical Implications in the Era of Single-Cell Ribonucleic Acid Sequencing.

This article has been withdrawn due to an error that caused the article to be duplicated. The definitive version of this article is published under DOI 10.1093/neuros/nyab288.

Photoactivatable Circular Caged Oligonucleotides for Transcriptome In Vivo Analysis (TIVA).

Light activation is an effective way to impart spatiotemporal control over oligonucleotide probes that are widely applied for gene expression regulation and target function investigation. Among the major oligonucleotide caging strategies, cyclization with a photocleavable linker is an elegant design, which affords both atom efficiency and stability in many biological environments. Here, we introduce an improved protocol for circular oligonucleotide synthesis requiring only one round of HPLC purification. With a series of poly-U oligonucleotide strands of different sizes and backbone modifications, the pre-photolysis caging stability and post-photolysis target binding affinity were studied through a denaturing gel assay and melting temperature measurements. A 14U 2'-OMe RNA probe was selected, with strong potential application in transcriptome in vivo analysis (TIVA) for mRNA isolation.

Mammalian cell transfection: the present and the future.

Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microscope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective.

Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background.

Messenger RNA (mRNA) isolated from single cells can generate powerful biological insights, including the discovery of new cell types with unique functions as well as markers potentially predicting a cell's response to various therapeutic agents. We previously introduced an oligonucleotide-based technique for site-selective, photoinduced biotinylation and capture of mRNA within a living cell called transcriptome in vivo analysis (TIVA). Successful application of the TIVA technique hinges upon its oligonucleotide probe remaining completely inert (or "caged") to mRNA unless photoactivated. To improve the reliability of TIVA probe caging in diverse and challenging biological conditions, we applied a rational design process involving iterative modifications to the oligonucleotide construct. In this work, we discuss these design motivations and present an optimized probe with minimal background binding to mRNA prior to photolysis. We assess its caging performance through multiple in vitro assays including FRET analysis, native gel comigration, and pull down with model mRNA transcripts. Finally, we demonstrate that this improved probe can also isolate mRNA from single living neurons in brain tissue slices with excellent caging control.

Lamin B2 Levels Regulate Polyploidization of Cardiomyocyte Nuclei and Myocardial Regeneration.

Heart regeneration requires cardiomyocyte proliferation. It is thought that formation of polyploid nuclei establishes a barrier for cardiomyocyte proliferation, but the mechanisms are largely unknown. Here, we show that the nuclear lamina filament Lamin B2 (Lmnb2), whose expression decreases in mice after birth, is essential for nuclear envelope breakdown prior to progression to metaphase and subsequent division. Inactivating Lmnb2 decreased metaphase progression, which led to formation of polyploid cardiomyocyte nuclei in neonatal mice, which, in turn, decreased myocardial regeneration. Increasing Lmnb2 expression promoted cardiomyocyte M-phase progression and cytokinesis and improved indicators of myocardial regeneration in neonatal mice. Inactivating LMNB2 in human iPS cell-derived cardiomyocytes reduced karyokinesis and increased formation of polyploid nuclei. In primary cardiomyocytes from human infants with heart disease, modifying LMNB2 expression correspondingly altered metaphase progression and ploidy of daughter nuclei. In conclusion, Lmnb2 expression is essential for karyokinesis in mammalian cardiomyocytes and heart regeneration.

Control of cytokinesis by ß-adrenergic receptors indicates an approach for regulating cardiomyocyte endowment.

One million patients with congenital heart disease (CHD) live in the United States. They have a lifelong risk of developing heart failure. Current concepts do not sufficiently address mechanisms of heart failure development specifically for these patients. Here, analysis of heart tissue from an infant with tetralogy of Fallot with pulmonary stenosis (ToF/PS) labeled with isotope-tagged thymidine demonstrated that cardiomyocyte cytokinesis failure is increased in this common form of CHD. We used single-cell transcriptional profiling to discover that the underlying mechanism of cytokinesis failure is repression of the cytokinesis gene ECT2, downstream of ß-adrenergic receptors (ß-ARs). Inactivation of the ß-AR genes and administration of the ß-blocker propranolol increased cardiomyocyte division in neonatal mice, which increased the number of cardiomyocytes (endowment) and conferred benefit after myocardial infarction in adults. Propranolol enabled the division of ToF/PS cardiomyocytes in vitro. These results suggest that ß-blockers could be evaluated for increasing cardiomyocyte division in patients with ToF/PS and other types of CHD.

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.

Neuron-specific mRNA complexity responses during hippocampal apoptosis after traumatic brain injury.

In an effort to understand the complexity of genomic responses within selectively vulnerable regions after experimental brain injury, we examined whether single apoptotic neurons from both the CA3 and dentate differed from those in an uninjured brain. The mRNA from individual active caspase 3(+)/terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling [TUNEL(-)] and active caspase 3(+)/TUNEL(+) pyramidal and granule neurons in brain-injured mice were amplified and compared with those from nonlabeled neurons in uninjured brains. Gene analysis revealed that overall expression of mRNAs increased with activation of caspase 3 and decreased to below uninjured levels with TUNEL reactivity. Cell type specificity of the apoptotic response was observed with both regionally distinct expression of mRNAs and differences in those mRNAs that were maximally regulated. Immunohistochemical analysis for two of the most highly differentially expressed genes (prion and Sos2) demonstrated a correlation between the observed differential gene expression after traumatic brain injury and corresponding protein translation.

Gene expression profile for schizophrenia: discrete neuron transcription patterns in the entorhinal cortex.

BACKGROUND: Several lines of evidence indicate the altered function of the temporal lobe, including the hippocampus and entorhinal cortex (EC), is associated with schizophrenia. We used single-cell gene expression technologies to assess coordinate changes in the expression of multiple genes, including neuronal signaling and synaptic-related markers in EC layer II stellate neurons. METHODS: We used a single-neuron microdissection technique coupled with linear antisense RNA amplification and high density/candidate gene arrays to assess coordinate changes in gene expression. The expression and relative abundance of more than 18,000 messenger RNAs were assessed from EC layer II stellate neurons from postmortem samples of schizophrenic and age-matched control brains. Results of this initial screen were used to perform a more specific secondary messenger RNA screen for each subject. RESULTS: Data disclosed marked differences in expression of various G-protein-coupled receptor-signaling transcripts, glutamate receptor subunits, synaptic proteins, and other transcripts. Results of secondary screening showed significant decreases in levels of G-protein subunit i(alpha)1, glutamate receptor 3, N-methyl-D-aspartate receptor 1, synaptophysin, and sensory nerve action potentials 23 and 25 in the stellate neurons of schizophrenic patients. We observed down-regulation of phospholemman (a phosphoprotein associated with anion channel formation) messenger RNA and protein levels in layer II/III stellate neurons in the population with schizophrenia. CONCLUSIONS: These results provide a preliminary expression profile of schizophrenia in defined neuronal populations. Understanding the coordinated involvement of multiple genes in human disease provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention.

Single-cell molecular biology: implications for diagnosis and treatment of neurologic disease.

The continued discovery of basic pathologic mechanisms underlying neuropsychiatric illnesses will be critical to the development of improved diagnostic tests and more targeted therapeutic strategies. Molecular biological methods capable of evaluating gene expression at the single-cell level have great potential for advancing our knowledge of these processes. This review describes two techniques that are providing new insights into the intracellular regulation of ribonucleic acid trafficking and processing. These technologies promise to accelerate our understanding of both normal and abnormal molecular processes within neurons, and they have the potential for direct application to the study of human neurologic disease.