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We argue that the study of single-cell subcellular organelle omics is needed to understand and regulate cell function. This requires and is being enabled by new technology development.

As the Brain Research through Advancing Neurotechnologyies, better known as the BRAIN Initiative moves forward at a rapid pace increasing attention has focused on neuroethics. The National Institut...

Analysis of single-cell transcriptomes shows the single-cell heterogeneity between cells within a population which is vital to our understanding of normal function and disease development. To obtain single-cell transcriptome profiling, however, the poly-A RNA must be accurately isolated from the target cell. We developed a single-cell analysis procedure called transcriptome in vivo analysis (TIVA), which will allow accurate characterization of targeted cell-specific transcriptomes from live tissue. This is accomplished using a RNA capture molecule called TIVA tag that captures the transcriptome of selected cells in their natural microenvironment. An important aspect of the TIVA approach is that the tag is delivered into the cytoplasm of live cells using cell-penetrating peptides (CPPs). Once the TIVA tag is in the cellular cytoplasm, it binds to mRNA after photoactivation of the compound. Using CPPs in combination with photoactivation is the first noninvasive access method for accurately isolating single-cell mRNA from live single cells in tissues in their natural microenvironment.

Analysis of single-cell transcriptomes shows the single-cell heterogeneity between cells within a population which is vital to our understanding of normal function and disease development. To obtain single-cell transcriptome profiling, however, the poly-A RNA must be accurately isolated from the target cell. We developed a single-cell analysis procedure called transcriptome in vivo analysis (TIVA), which will allow accurate characterization of targeted cell-specific transcriptomes from live tissue. This is accomplished using a RNA capture molecule called TIVA tag that captures the transcriptome of selected cells in their natural microenvironment. An important aspect of the TIVA approach is that the tag is delivered into the cytoplasm of live cells using cell-penetrating peptides (CPPs). Once the TIVA tag is in the cellular cytoplasm, it binds to mRNA after photoactivation of the compound. Using CPPs in combination with photoactivation is the first noninvasive access method for accurately isolating single-cell mRNA from live single cells in tissues in their natural microenvironment.

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.

The prefrontal cortex (PFC) plays a key role in higher order cognitive functions and psychiatric disorders such as autism, schizophrenia, and depression. In the PFC, the two major classes of neurons are the glutamatergic pyramidal (Pyr) cells and the GABAergic interneurons such as fast-spiking (FS) cells. Despite extensive electrophysiological, morphological, and pharmacological studies of the PFC, the therapeutically utilized drug targets are restricted to dopaminergic, glutamatergic, and GABAergic receptors. To expand the pharmacological possibilities as well as to better understand the cellular and network effects of clinically used drugs, it is important to identify cell-type-selective, druggable cell surface proteins and to link developed drug candidates to Pyr or FS cell targets. To identify the mRNAs of such cell-specific/enriched proteins, we performed ultra-deep single-cell mRNA sequencing (19 685 transcripts in total) on electrophysiologically characterized intact PFC neurons harvested from acute brain slices of mice. Several selectively expressed transcripts were identified with some of the genes that have already been associated with cellular mechanisms of psychiatric diseases, which we can now assign to Pyr (e.g., Kcnn2, Gria3) or FS (e.g., Kcnk2, Kcnmb1) cells. The earlier classification of PFC neurons was also confirmed at mRNA level, and additional markers have been provided.

Single-cell analysis has contributed greatly to gaining a better understanding of human brain function and has implications for neurodegenerative and neuropsychiatric disorders. Long noncoding RNAs (lncRNAs) acting, in part, as epigenetic regulators exist in brain cells in high abundance exhibiting a large diversity that play important roles in neural development, function, and neurodegenerative disease. Due to lncRNA tissue-type and cell-type specific expression characteristics, it is important to analyze lncRNA at single-cell resolution. In this chapter, we highlight a method named scTISA (single-cell transcription in situ with antisense RNA amplification), which is applicable to fixed single cells and can yield polyA+ lncRNAs and mRNAs data at the same time.

Ethanol (EtOH) abuse induces significant mortality and morbidity worldwide because of detrimental effects on brain function. Defining the contribution of astrocytes to this malfunction is imperative to understanding the overall EtOH effects due to their role in homeostasis and EtOH-seeking behaviors. Using a highly controllable

Single-cell analysis has contributed greatly to gaining a better understanding of human brain function and has implications for neurodegenerative and neuropsychiatric disorders. Long noncoding RNAs (lncRNAs) acting, in part, as epigenetic regulators exist in brain cells in high abundance exhibiting a large diversity that play important roles in neural development, function, and neurodegenerative disease. Due to lncRNA tissue-type and cell-type specific expression characteristics, it is important to analyze lncRNA at single-cell resolution. In this chapter, we highlight a method named scTISA (single-cell transcription in situ with antisense RNA amplification), which is applicable to fixed single cells and can yield polyA

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.

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.

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.

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.

This Perspective discusses the development of the linear amplified RNA amplification technique over the last 25 years, and future applications of this important and versatile methodology., It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Summary: A number of mitochondrial diseases arise from single-nucleotide variant (SNV) accumulation in multiple mitochondria. Here, we present a method for identification of variants present at the single-mitochondrion level in individual mouse and human neuronal cells, allowing for extremely high-resolution study of mitochondrial mutation dynamics. We identified extensive heteroplasmy between individual mitochondrion, along with three high-confidence variants in mouse and one in human that were present in multiple mitochondria across cells. The pattern of variation revealed by single-mitochondrion data shows surprisingly pervasive levels of heteroplasmy in inbred mice. Distribution of SNV loci suggests inheritance of variants across generations, resulting in Poisson jackpot lines with large SNV load. Comparison of human and mouse variants suggests that the two species might employ distinct modes of somatic segregation. Single-mitochondrion resolution revealed mitochondria mutational dynamics that we hypothesize to affect risk probabilities for mutations reaching disease thresholds. : Morris et al. use independent sequencing of multiple individual mitochondria from mouse and human brain cells to show high pervasiveness of mutations. The mutations are heteroplasmic within single mitochondria and within and between cells. These findings suggest mechanisms by which mutations accumulate over time, resulting in mitochondrial dysfunction and disease. Keywords: single mitochondrion, single cell, human neuron, mouse neuron, single-nucleotide variation

Summary Ethanol (EtOH) abuse induces significant mortality and morbidity worldwide because of detrimental effects on brain function. Defining the contribution of astrocytes to this malfunction is imperative to understanding the overall EtOH effects due to their role in homeostasis and EtOH-seeking behaviors. Using a highly controllable in vitro system, we identify chemical signaling mechanisms through which acute EtOH exposure induces a modulatory feedback loop between neurons and astrocytes. Neuronally-derived purinergic signaling primed a subpopulation of astrocytes to respond to subsequent acute EtOH exposures (SEastrocytes: signal enhanced astrocytes) with greater calcium signal strength. Generation of SEastrocytes arose from astrocytic hemichannel-derived ATP and accumulation of its metabolite adenosine within the astrocyte microenvironment to modulate adenylyl cyclase and phospholipase C activity. These results highlight an important role of astrocytes in shaping the overall physiological responsiveness to EtOH and emphasize the unique plasticity of astrocytes to adapt to single and multiple exposures of EtOH.

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.

Assays examining the open-chromatin landscape in single cells require isolation of the nucleus, resulting in the loss of spatial/microenvironment information. Here we describe CHEX-seq (CHromatin EXposed) for identifying single-stranded open-chromatin DNA regions in paraformaldehyde-fixed single cells. CHEX-seq uses light-activated DNA probes that binds to single-stranded DNA in open chromatin. In situ laser activation of the annealed probes’ 3’-Lightning Terminator™ in selected cells permits the probe to act as a primer for in situ enzymatic copying of single-stranded DNA that is then sequenced. CHEX-seq is benchmarked with human K562 cells and its utility is demonstrated in dispersed primary mouse and human brain cells, and immunostained cells in mouse brain sections. Further, CHEX-seq queries the openness of mitochondrial DNA in single cells. Evaluation of an individual cell’s chromatin landscape in its tissue context enables “spatial chromatin analysis”.One Sentence SummaryA new method, CHEX-seq (CHromatin eXposed), identifies the open-chromatin landscape in single fixed cells thereby allowing spatial chromatin analysis of selected cells in complex cellular environments.

The NIH Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative is focused on developing new tools and neurotechnologies to transform our understanding of the brain, and neuroethics is an essential component of this research effort. Coordination with other brain projects around the world will help maximize success.

Cells are complex machines whose behaviors arise from their internal collection of dynamically interacting organelles, supramolecular complexes, and cytoplasmic chemicals. The current understanding of the nature by which subcellular biology produces cell-level behaviors is limited by the technological hurdle of measuring the large number (>103 ) of small-sized (

There is a growing appreciation of the extent of transcriptome variation across individual cells of the same cell type. While expression variation may be a byproduct of, for example, dynamic or homeostatic processes, here we consider whether single-cell molecular variation per se might be crucial for population-level function. Under this hypothesis, molecular variation indicates a diversity of hidden functional capacities within an ensemble of identical cells, and this functional diversity facilitates collective behavior that would be inaccessible to a homogenous population. In reviewing this topic, we explore possible functions that might be carried by a heterogeneous ensemble of cells; however, this question has proven difficult to test, both because methods to manipulate molecular variation are limited and because it is complicated to define, and measure, population-level function. We consider several possible methods to further pursue the hypothesis that variation is function through the use of comparative analysis and novel experimental techniques.

Summary 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.

Neuroscience presents important neuroethical considerations. Human neuroscience demands focused application of the core research ethics guidelines set out in documents such as the Belmont Report. Various mechanisms, including institutional review boards (IRBs), privacy rules, and the Food and Drug

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

The advent of single cell transcriptome analysis has permitted the discovery of cell-to-cell variation in transcriptome expression of even presumptively identical cells. We hypothesize that this variability reflects a many-to-one relation between transcriptome states and the phenotype of a cell. In this relation, the molecular ratios of the subsets of RNA are determined by the stoichiometric constraints of the cell systems, which underdetermine the transcriptome state. Furthermore, the variability is, in part, induced by the tissue context and is important for system-level function. This theory is analogous to theories of literary deconstruction, where multiple 'signifiers' work in opposition to one another to create meaning. By analogy, transcriptome phenotypes should be defined as subsets of RNAs comprising selected RNA systems where the system-associated RNAs are balanced with each other to produce the associated cellular function. This idea provides a framework for understanding cellular heterogeneity in phenotypic responses to variant conditions, such as disease challenge.

Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3′ and 5′ terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (Φ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

Chen et al. (2017) demonstrate whole-genome amplification of single-cell genomic DNA using linear nucleic acid amplification. This provides reliable single-nucleotide variation (SNV) detection across the single-cell genome, facilitating an understanding of cell-to-cell similarities and distinctions.

SummaryThough 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.

Background Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. Results Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5–10 molecules. Conclusions Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3300-3) contains supplementary material, which is available to authorized users.

Single cell sequencing is currently in its relative infancy although an unprecedented amount of information is already being generated. These techniques are providing new insight into intercellular variability as well as identification of previously unrecognized drug targets. As more groups are gaining an interest in this fruitful technique, new sample preparation techniques, sequencing platforms, and bioinformatics tools are being developed which only improve the quantity and quality of data generated in these studies. Great advancements in harvest (in vivo pipette), sample preparation, and sequencing (Illumina HiSeq 2500/MiSeq, Ion Torrent PGM, Pacific Biosciences RS) are allowing for previously untestable questions to be answered and for expanded accessibility of these technologies.

Summary Elk-1 is a member of the Ets family of transcription factors, which are identified by a conserved Ets DNA-binding domain that mediates transcriptional regulation at Ets sequence--containing promoters. The activation domain of Elk-1 is important for executing its physiologic functions and contains many phosphorylation sites targeted by various MAP kinases following exposure to cell stressors or mitogenic stimuli. The different combinations of phosphorylated sites allow specificity of cellular responses mediated through redundant signaling pathways activated by distinct stimuli. Through phosphorylation of S383, mitogen-activated protein kinase (MAPK)--activating stimuli have been shown to regulate various processes important in carcinogenesis through transcriptional regulation in various cell lines, including proliferation. Phosphorylation at the T417 site (pT417), but not the S383 site, is involved in neuronal apoptosis induced through dendritic signaling mechanisms and associates with neuronal lesions in many Lewy body diseases. This points to distinct roles for these different phosphorylation sites in pathophysiologic pathways. However, the S383 site remains the best characterized in the context of normal function and carcinogenesis in cell lines, and less is known about the biochemistry of other phosphorylation sites, particularly in more biochemically relevant models. Here, we show that Elk-1 pT417 is present in epithelial cell nuclei of various normal and cancer tissues and that the number of pT417-positive cells correlates with differentiation grade of colonic adenocarcinomas. This nuclear localization and correlation with tumor differentiation in adenocarcinoma suggests a potentially important transcriptional and biochemical role of this phosphorylation site in carcinogenesis of this tumor type.

Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate the measurement transfer functions to be linear above ~5-10 molecules. Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.