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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.

Background RNA localization involves cis-motifs that are recognized by RNA-binding proteins (RBP), which then mediate localization to specific sub-cellular compartments. RNA localization is critical for many different cell functions, e.g., in neuronal dendrites, localization is a critical step for long-lasting synaptic potentiation. However, there is little consensus regarding which RNAs are localized and the role of alternative isoforms in localization. A comprehensive catalog of localized RNA can help dissect RBP/RNA interactions and localization motifs. Here, we utilize a single cell sub-cellular RNA sequencing approach to profile differentially localized RNAs from individual cells across multiple single cells to help identify a consistent set of localized RNA in mouse neurons. Results Using independent RNA sequencing from soma and dendrites of the same neuron, we deeply profiled the sub-cellular transcriptomes to assess the extent and variability of dendritic RNA localization in individual hippocampal neurons, including an assessment of differential localization of alternative 3′UTR isoforms. We identified 2225 dendritic RNAs, including 298 cases of 3′UTR isoform-specific localization. We extensively analyzed the localized RNAs for potential localization motifs, finding that B1 and B2 SINE elements are up to 5.7 times more abundant in localized RNA 3′UTRs than non-localized, and also functionally characterized the localized RNAs using protein structure analysis. Conclusion We integrate our list of localized RNAs with the literature to provide a comprehensive list of known dendritically localized RNAs as a resource. This catalog of transcripts, including differentially localized isoforms and computationally hypothesized localization motifs, will help investigators further dissect the genome-scale mechanism of RNA localization. Electronic supplementary material The online version of this article (10.1186/s12915-019-0630-z) contains supplementary material, which is available to authorized users.

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.

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.

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

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.

During early avian development, primordial germ cells (PGC) are highly migratory, moving from the central area pellucida of the blastoderm to the anterior extra-embryonic germinal crescent. The PGCs soon move into the forming blood vessels by intravasation and travel in the circulatory system to the genital ridges where they participate in the organogenesis of the gonads. This complex cellular migration takes place in close association with a nascent extracellular matrix that matures in a precise spatio-temporal pattern. We first compiled a list of quail matrisome genes by bioinformatic screening of human matrisome orthologs. Next, we used single cell RNA-seq analysis (scRNAseq) to determine that PGCs express numerous ECM and ECM-associated genes in early embryos. The expression of select ECM transcripts and proteins in PGCs were verified by fluorescent in situ hybridization (FISH) and immunofluorescence (IF). Live imaging of transgenic quail embryos injected with fluorescent antibodies against fibronectin and laminin, showed that germinal crescent PGCs display rapid shape changes and morphological properties such as blebbing and filopodia while surrounded by, or in close contact with, an ECM fibril meshwork that is itself in constant motion. Injection of anti-β1 integrin CSAT antibodies resulted in a reduction of mature fibronectin and laminin fibril meshwork in the germinal crescent at HH4-5 but did not alter the active motility of the PGCs or their ability to populate the germinal crescent. These results suggest that integrin β1 receptors are important, but not required, for PGCs to successfully migrate during embryonic development, but instead play a vital role in ECM fibrillogenesis and assembly.

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.

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.

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.

The building blocks of complex biological systems are single cells. Fundamental insights gained from single-cell analysis promise to provide the framework for understanding normal biological systems development as well as the limits on systems/cellular ability to respond to disease. The interplay of cells to create functional systems is not well understood. Until recently, the study of single cells has concentrated primarily on morphological and physiological characterization. With the application of new highly sensitive molecular and genomic technologies, the quantitative biochemistry of single cells is now accessible.

RNA precursors give rise to mRNA after splicing of intronic sequences traditionally thought to occur in the nucleus. Here, we show that intron sequences are retained in a number of dendritically-targeted mRNAs, using microarray and Illumina sequencing of isolated dendritic mRNA as well as in situ hybridization. Many of the retained introns contain ID elements, a class of SINE retrotransposon. A portion of these SINEs confers dendritic targeting to exogenous and endogenous transcripts showing the necessity of ID-mediated mechanisms for the targeting of different transcripts to dendrites. ID elements are capable of selectively altering the distribution of endogenous proteins, providing a link between intronic SINEs and protein function. As such, the ID element represents the first common dendritic targeting element to be found across multiple RNAs. Retention of intronic sequence is a more general phenomenon then previously thought and plays a functional role in the biology of the neuron, partly mediated by co-opted repetitive sequences.

OBJECTIVE Temperature and nutrient homeostasis are two interdependent components of energy balance regulated by distinct sets of hypothalamic neurons. The objective is to examine the role of the metabolic signal insulin in the control of core body temperature (CBT). RESEARCH DESIGN AND METHODS The effect of preoptic area administration of insulin on CBT in mice was measured by radiotelemetry and respiratory exchange ratio. In vivo 2-[18F]fluoro-2-deoxyglucose uptake into brown adipose tissue (BAT) was measured in rats after insulin treatment by positron emission tomography combined with X-ray computed tomography imaging. Insulin receptor–positive neurons were identified by retrograde tracing from the raphe pallidus. Insulin was locally applied on hypothalamic slices to determine the direct effects of insulin on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates. RESULTS Injection of insulin into the preoptic area of the hypothalamus induced a specific and dose-dependent elevation of CBT mediated by stimulation of BAT thermogenesis as shown by imaging and respiratory ratio measurements. Retrograde tracing indicates that insulin receptor–expressing warm-sensitive neurons activate BAT through projection via the raphe pallidus. Insulin applied on hypothalamic slices acted directly on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates. The hyperthermic effects of insulin were blocked by pretreatment with antibodies to insulin or with a phosphatidylinositol 3–kinase inhibitor. CONCLUSIONS Our findings demonstrate that insulin can directly modulate hypothalamic neurons that regulate thermogenesis and CBT and indicate that insulin plays an important role in coupling metabolism and thermoregulation at the level of anterior hypothalamus.

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.

Brown adipocytes (BAs) are specialized for adaptive thermogenesis and, upon sympathetic stimulation, activate mitochondrial uncoupling protein (UCP)-1 and oxidize fatty acids to generate heat. The capacity for brown adipose tissue (BAT) to protect against obesity and metabolic disease is recognized, yet information about which signals activate BA, besides β3-adrenergic receptor stimulation, is limited. Using single-cell transcriptomics, we confirmed the presence of mRNAs encoding traditional BAT markers (i.e., UCP1, expressed in 100% of BAs Adrb3, expressed in50% of BAs) in mouse and have shown single-cell variability (1000-fold) in their expression at both the mRNA and protein levels. We further identified mRNAs encoding novel markers, orphan GPCRs, and many receptors that bind the classic neurotransmitters, neuropeptides, chemokines, cytokines, and hormones. The transcriptome variability between BAs suggests a much larger range of responsiveness of BAT than previously recognized and that not all BAs function identically. We examined the in vivo functional expression of 12 selected receptors by microinjecting agonists into live mouse BAT and analyzing the metabolic response. In this manner, we expanded the number of known receptors on BAs at least 25-fold, while showing that the expression of classic BA markers is more complex and variable than previously thought.

The interactions between various RNA-binding proteins (RBPs) and the RNA transcripts they bind strongly influence posttranscriptional control of gene expression in vertebrates. The hundreds of vertebrate RBPs that have been identified within the genome, often with multiple RNA recognition motifs, are capable of recognizing specific target RNA sequences mediating the maturation, movement, and translational state of their RNA cargoes. To identify the cargoes associated with a specific RBP, we have developed a technique called antibody-positioned RNA amplification (APRA), which positions an oligonucleotide with a degenerate priming sequence in proximity to the RNAs sequestered by a specific RBP. The conjugation of the priming oligonucleotide to the antibody by itself does not interfere with the antibody’s intrinsic affinity for the target RBP epitope, thus enabling RNA targets to be reverse-transcribed and amplified via a T7 bacteriophage RNA polymerase promoter sequence located upstream of the degenerate priming sequence in the oligonucleotide. By identifying the mRNA transcripts associated with the RBP in situ, we may be able to ascertain the significance of their temporal expression and physiological activities within the vast transcriptional networks regulating functional responses to stimuli.

The sensitivity of new transcriptomic techniques is rapidly improving to the point that single-cell molecular analysis is now becoming commonplace. However to obtain accurate transcriptome data, the initial experimental steps must strive to maintain the natural environment of cell and always get set in motion under in vivo conditions. Achieving these critical experimental parameters is technically challenging for investigators and currently the most frequently used molecular techniques experimentally commence with tissues or cells in artificial environments or under in vitro conditions. Here we review an innovative experimental approach that is called transcriptome in vivo analysis (TIVA) that was designed to overcome theses well-known limitations. The TIVA methods permit cell-specific transcriptome capture from viable intact heterogeneous tissues. Cell-penetrating peptides (CPPs) are used to deliver multifunctional transcriptome-capture tags (TIVA tags) to the cytoplasm of the cell under in vivo conditions. The TIVA capture tag enables investigators to target and isolate cell-specific transcriptomes in their natural microenvironments. The combination of maintaining in vivo conditions and selective cell-specific transcriptome capture provides investigators with the opportunity to yield the most biologically accurate and informative transcriptome data hitherto.

Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

Multiple nuclear transcription factors including E-26-like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.

The nuclear transcription factor E-26-like protein 1 (Elk-1) is thought to impact neuronal differentiation [Sharrocks, A. D. (2001) Nat. Rev. Mol. Cell Biol. 2, 827–837], cell proliferation [Sharrocks, A. D. (2002) Biochem. Soc. Trans. 30, 1–9], tumorigenesis [Chai, Y. L., Chipitsyna, G., Cui, J., Liao, B., Liu, S., Aysola, K., Yezdani, M., Reddy, E. S. P. & Rao, V. N. (2001) Oncogene 20, 1357–1367], and apoptosis [Shao, N., Chai, Y., Cui, J., Wang, N., Aysola, K., Reddy, E. S. P. & Rao, V. N. (1998) Oncogene 17, 527–532]. In addition to its nuclear localization, Elk-1 is found throughout the cytoplasm, including localization in neuronal dendrites [Sgambato, V., Vanhoutte, P., Pages, C., Rogard, M., Hipskind, R., Besson, M. J. & Caboche, J. (1998) J. Neurosci. 18, 214–226], raising the possibility that Elk-1 may have alternative extranuclear functions in neurons. Using coimmunoprecipitation and reciprocal coimmunoprecipitation from adult rat brain, we found an association between Elk-1 protein and the mitochondrial permeability transition pore complex (PTP), a structure involved in both apoptotic and necrotic cell death. Electron microscopy in adult rat brain sections confirmed this association with mitochondria. Elk-1 was also identified from purified mitochondrial fractions by using Western blotting, and Elk-1 increased its association with mitochondria following proapoptotic stimuli. Consistent with a role for Elk-1 in neuron viability, overexpression of Elk-1 in primary neurons decreased cell viability, whereas Elk-1 siRNA-mediated knockdown increased cell viability. This decrease in viability induced by Elk-1 overexpression was blocked with application of a PTP inhibitor. These results show an association of the nuclear transcription factor Elk-1 with the mitochondrial PTP and suggest an additional extranuclear function for Elk-1 in neurons.

To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo . Specific regions of the 3′ and 5′ UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.

Dendrites are specialized extensions of the neuronal soma that contain components of the cellular machinery involved in RNA and protein metabolism. Several dendritically localized proteins are associated with the precursor-mRNA (pre-mRNA) splicing complex, or spliceosome. Although some spliceosome-related, RNA-binding proteins are known to subserve separate cytoplasmic functions when moving between the nucleus and cytoplasm, little is known about the pre-mRNA splicing capacity of intact dendrites. Here, we demonstrate the presence and functionality of pre-mRNA-splicing components in dendrites. When isolated dendrites are transfected with a chicken δ-crystallin pre-mRNA or luciferase reporter pre-mRNA, splicing junctions clustered at or near expected splice sites are observed. Additionally, in vitro synaptoneurosome experiments show that this subcellular fraction contains a similar complement of splicing factors that is capable of splicing chicken δ-crystallin pre-mRNA. These observations suggest that pre-mRNA-splicing factors found in the dendroplasm retain the potential to promote pre-mRNA splicing.

Genomic microarrays are rapidly becoming ubiquitous throughout a wide variety of biological disciplines. As their use has grown during the past few years, many important discoveries have been made in the fields of central nervous system (CNS) injury and disease using this emerging technology. In addition, single-cell mRNA amplification techniques are now being used along with microarrays to overcome many of the difficulties associated with the cellular heterogeneity of the brain. This development has extended the utility of gene expression profiling and has provided researchers with exciting new insights into the neuropathology of CNS injury and disease at a molecular and cellular level. New methodological, standardization, and statistical techniques are currently being developed to improve the reproducibility of microarrays and facilitate the analysis of large amounts of data. In this review, we will discuss the application of these techniques to experimental, clinically relevant models of traumatic brain injury.

Microarray technology has become a common tool for developing expression profiles. Initially used in the analysis of cells lines and homogeneous tissues, this platform has been applied to more diverse tissues, such as the brain. Several neural disorders have already been profiled by microarrays using relatively large amounts of tissue. This data has unveiled many genes with differential expression between normal and diseased tissue that could potentially be used as gene markers for these afflictions. Because of the heterogeneity of the CNS, it is likely that small differences between gene expression in these studies would be enhanced by the sampling of a subset of cells based on these newly characterized gene markers. Subtraction of normal, unaffected cells from the sample may also result in a more accurate profile of a diseased cell. Expression profile studies from several neuropathological states are presented, with emphasis placed on those studies using small samples of cellular material and those using specialized methods of cell isolation and RNA amplification.

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 (prionandSos2) demonstrated a correlation between the observed differential gene expression after traumatic brain injury and corresponding protein translation.

The cellular and the inter-connective complexity of the central nervous system (CNS) necessitate's analysis of functioning at both the system and single cell levels. Systems neuroscience has developed procedures that facilitate the analysis of multicellular systems including multielectrode arrays, dye tracings and lesioning assays, and at the single cell level there have been significant strides in assessing the physiology and morphology of individual cells. Until recently little progress had been made in understanding the molecular biology of single neuronal cells. This review will highlight the development of PCR and aRNA procedures for analysis of mRNA abundances in single cells. Also, other procedures for the analysis of protein abundances as well as the association of RNA with proteins will also be summarized. These procedures promise to provide experimental insights that will help unravel the functional mechanisms regulating the cellular components of the CNS.

Synaptogenesis and synaptic plasticity demonstrate the ability of neurons to mature and respond to various stimuli. Both of these events require protein synthesis, in particular, localized translation within dendrites. Dendrites localize specific mRNAs in proximity to dendritic spines and have the capacity to actively translate these mRNAs within various 'hotspots' in the dendritic space. Rates of dendritic translation are stimulated and inhibited by various agents, suggesting that several signaling pathways that modulate localized protein synthesis may exist within the dendrite. This review will cover several suggested pathways for regulation of dendritic translation and propose a correlation between deregulated dendritic translation and 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.

The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.

Traumatic brain injury (TBI) elicits a complex sequence of putative autodestructive and neuroprotective cellular cascades. It is hypothesized that the genomic responses of cells in the injured brain serve as the basis for these cascades. Traditional methods for analyzing differential gene expression following brain trauma demonstrate that immediate early genes, cytokines, transcription factors, and neurotrophic factors can all participate in the brain's active and directed response to injury, and may do so concurrently. It is this complexity and multiplicity of interrelated molecular mechanisms that has demanded new methods for comprehensive and parallel evaluation of putative as well as novel gene targets. Recent advances in DNA microarray technology have enabled the simultaneous evaluation of thousands of genes and the subsequent generation of massive amounts of biological data relevant to CNS injury. This emerging technology can serve to further current knowledge regarding recognized molecular cascades as well as to identify novel molecular mechanisms that occur throughout the post-traumatic period. The elucidation of the complex alterations in gene expression underlying the pathological sequelae following TBI is of central importance in the design of future therapeutic agents.

Targeting of mRNAs to distinct subcellular regions occurs in all polarized cells. The mechanisms by which RNA transport occurs are poorly understood. With the advent of RNA amplification methodologies and expression profiling it is now possible to catalogue the RNAs that are targeted to particular subcellular regions. In particular, neurons are polarized cells in which dendrites receive signals from presynaptic neurons. Upon stimulation (information receipt) the dendrite processes the information such that an immediate dendritic response is generated as well as a longer-term somatic response. The integrated cellular response results in a signal that can be propagated through the axon to the next post-synaptic neuron. Much previous work has shown that mRNAs can be localized in dendrites and that local translation in dendrites can occur. In this chapter the methods for analysis of RNAs that are localized to dendrites are reviewed and a partial list of dendritically localized RNAs is presented. This information may be useful in identifying RNA regulatory regions that are responsible for specifying rate of RNA transport and the dendritic sites at which targeted RNAs dock so that they can be translated.

Individual cells of the same phenotype are commonly viewed as identical functional units of a tissue or organ. However, the deep sequencing of DNA and RNA from single cells suggests a more complex ecology of heterogeneous cell states that together produce emergent system-level function. Continuing development of high-content, real-time, multimodal single-cell measurement technologies will lead to the ultimate goal of understanding the function of an individual cell in the context of its microenvironment.

The neurons of the brain extend axons and dendrites many hundreds of micrometres away from the cell body. The first electron microscope studies of these processes revealed that many of the structures that are found in the cell body are also present in dendrites. For example, particles resembling ribosomes and membrane structures like those of the endoplasmic reticulum (two structures that are important for protein synthesis) were seen in distal regions of dendrites, near synapses. Subsequent studies focused on identifying messenger RNAs in dendrites and providing evidence of dendritic protein synthesis. Transfection technologies have now been used to analyse translation within dendrites in response to pharmacological stimuli. These studies provide us with clues to the physiological role of the dendrite not just as a signal transducer, but also as a modulator of long-term synaptic efficacy.

Thirty years of literature have shown that changes in mRNA abundance provide insight into cellular functioning. Indeed, mRNA abundance measurements have been used to determine how effective particular drugs are in eliciting cellular responses, in monitoring behavioral responses, and in several other

Neuronal processes contain mRNAs and membrane structures, and some forms of synaptic plasticity seem to require protein synthesis in dendrites of hippocampal neurons. To quantitate dendritic protein synthesis, we used multiphoton microscopy of green fluorescent protein synthesized in living isolated dendrites. Transfection of dendrites with mRNA encoding green fluorescent protein resulted in fluorescence that exponentially increased on stimulation with a glutamate receptor agonist; a reaction attenuated by the translation inhibitors anisomycin and emetine. Comparable experiments on whole neurons revealed that (RS)-3,5-dihydroxy-phenylglycine 0.5 H 2 O (DHPG)-stimulated fluorescence was linear in cell bodies relative to the exponential increase seen in dendrites. Detailed spatial analysis of the subdendritic distribution of fluorescence revealed “hotspots,” sites of dendritic translation that were spatially stable. However, detailed temporal analysis of these hotspots revealed heterogeneous rates of translation. A double-label protocol counterstaining for ribosomes indicated that sites of “fastest” translation correlated with increased ribosome density, consistent with ribosome subunit assembly for initiation, the first step of translation. We propose that dendrites have specific sites specialized for fast translation.

The functioning of the neuronal dendrite results from a variety of biological processes including mRNA transport to and protein translation in the dendrite. The complexity of the mRNA population in dendrites suggests that specific biological processes are modulated through the regulation of dendritic biology. There are various classes of mRNAs in dendrites whose translation modulates the ability of the dendrite to receive and integrate presynaptic information. Among these mRNAs are those encoding selective transcription factors that function in the neuronal soma and ionotropic glutamate receptors that function on the neuronal membrane. Conclusive evidence that these mRNAs can be translated is reviewed, and identification of the endogenous sites of translation in living dendrites is presented. These data, as well as those described in the other articles resulting from this colloquium, highlight the complexity of dendritic molecular biology and the exquisitely selective and sensitive modulatory role played by the dendrite in facilitating intracellular and intercellular communication.

The pathogenesis of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) is poorly understood, but changes in the expression of specific messenger RNAs (mRNAs) may reflect mechanisms underlying the formation of NFTs and their consequences in affected neurons. For these reasons, we compared the relative abundance of multiple mRNAs in tangle-bearing versus normal CA1 neurons aspirated from sections of AD and control brains. Amplified antisense RNA expression profiling was performed on individual isolated neurons for analysis of greater than 18,000 expressed sequence tagged complementary DNAs (cDNAs) with cDNA microarrays, and further quantitative analyses were performed by reverse Northern blot analysis on 120 selected mRNAs on custom cDNA arrays. Relative to normal CA1 neurons, those harboring NFTs in AD brains showed significant reductions in several classes of mRNAs that are known to encode proteins implicated in AD neuropathology, including phosphatases/kinases, cytoskeletal proteins, synaptic proteins, glutamate receptors, and dopamine receptors. Because cathepsin D mRNA was upregulated in NFT-bearing CA1 neurons in AD brains, we performed immunohistochemical studies that demonstrated abundant cathepsin D immunoreactivity in the same population of tangle-bearing CA1 neurons. In addition, levels of mRNAs encoding proteins not previously implicated in AD were reduced in CA1 tangle-bearing neurons, suggesting that these proteins (eg, activity-regulated cytoskeleton-associated protein, focal adhesion kinase, glutaredoxin, utrophin) may be novel mediators of NFT formation or degeneration in affected neurons. Thus, the profile of mRNAs differentially expressed by tangle-bearing CA1 neurons may represent a "molecular fingerprint" of these neurons, and we speculate that mRNA expression profiles of diseased neurons in AD may suggest new directions for AD research or identify novel targets for developing more effective AD therapies.