Astrocyte ethanol exposure reveals persistent and defined calcium response subtypes and associated gene signatures.

Astrocytes play a critical role in brain function, but their contribution during ethanol (EtOH) consumption remains largely understudied. In light of recent findings on the heterogeneity of astrocyte physiology and gene expression, an approach with the ability to identify subtypes and capture this heterogeneity is necessary. Here, we combined measurements of calcium signaling and gene expression to define EtOH-induced astrocyte subtypes. In the absence of a demonstrated EtOH receptor, EtOH is believed to have effects on the function of many receptors and downstream biological cascades that underlie calcium responsiveness. This mechanism of EtOH-induced calcium signaling is unknown and this study provides the first step in understanding the characteristics of cells displaying these observed responses. To characterize underlying astrocyte subtypes, we assessed the correlation between calcium signaling and astrocyte gene expression signature in response to EtOH. We found that various EtOH doses increased intracellular calcium levels in a subset of astrocytes, distinguishing three cellular response types and one nonresponsive subtype as categorized by response waveform properties. Furthermore, single-cell RNA-seq analysis of astrocytes from the different response types identified type-enriched discriminatory gene expression signatures. Combining single-cell calcium responses and gene expression analysis identified specific astrocyte subgroups among astrocyte populations defined by their response to EtOH. This result provides a basis for identifying the relationship between astrocyte susceptibility to EtOH and corresponding measurable markers of calcium signaling and gene expression, which will be useful to investigate potential subgroup-specific influences of astrocytes on the physiology and pathology of EtOH exposure in the brain.

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.

Cytoplasmic BK(Ca) channel intron-containing mRNAs contribute to the intrinsic excitability of hippocampal neurons.

High single-channel conductance K+ channels, which respond jointly to membrane depolarization and micromolar concentrations of intracellular Ca2+ ions, arise from extensive cell-specific alternative splicing of pore-forming alpha-subunit mRNAs. Here, we report the discovery of an endogenous BK(Ca) channel alpha-subunit intron-containing mRNA in the cytoplasm of hippocampal neurons. This partially processed mRNA, which comprises approximately 10% of the total BK(Ca) channel alpha-subunit mRNAs, is distributed in a gradient throughout the somatodendritic space. We selectively reduced endogenous cytoplasmic levels of this intron-containing transcript by RNA interference without altering levels of the mature splice forms of the BK(Ca) channel mRNAs. In doing so, we could demonstrate that changes in a unique BK(Ca) channel alpha-subunit intron-containing splice variant mRNA can greatly impact the distribution of the BK(Ca) channel protein to dendritic spines and intrinsic firing properties of hippocampal neurons. These data suggest a new regulatory mechanism for modulating the membrane properties and ion channel gradients of hippocampal neurons.

Astrocytes promote ethanol-induced enhancement of intracellular Ca2+ signals through intercellular communication with neurons.

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.

Subcellular neuropharmacology: the importance of intracellular targeting.

Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.

RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice.

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.

Single neurons as experimental systems in molecular biology.

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.

Identification of RNA cargoes by antibody-positioned RNA amplification.

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.

Quantitative biology of single neurons.

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.

Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons.

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, by 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 a common dendritic targeting element found across multiple RNAs. Retention of intronic sequence is a more general phenomenon than previously thought and plays a functional role in the biology of the neuron, partly mediated by co-opted repetitive sequences.

In vivo identification of ribonucleoprotein-RNA interactions.

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.

Analysis of subcellularly localized mRNAs using in situ hybridization, mRNA amplification, and expression profiling.

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.