Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

Background: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. Results: 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. Conclusion: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

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

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

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

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

CHEX-seq detects single-cell genomic single-stranded DNA with catalytical potential.

Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe "CHEX-seq" which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3'-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.

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.

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

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

Live Cell Genomics: Cell-Specific Transcriptome Capture in Live Single Cells from Complex Tissues.

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.

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

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

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.

Single-Cell Analysis of Long Noncoding RNAs (lncRNAs) in Mouse Brain Cells.

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.

Micro- and Nano-Devices for Studying Subcellular Biology.

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 (<1 µm) heterogeneous organelles and subcellular structures found within each cell. In this review, the emergence of a suite of micro- and nano-technologies for studying intracellular biology on the scale of organelles is described. Devices that use microfluidic and microelectronic components for 1) extracting and isolating subcellular structures from cells and lysate; 2) analyzing the physiology of individual organelles; and 3) recreating subcellular assembly and functions in vitro, are described. The authors envision that the continued development of single organelle technologies and analyses will serve as a foundation for organelle systems biology and will allow new insight into fundamental and clinically relevant biological questions.

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

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

Cell Surface Protein mRNAs Show Differential Transcription in Pyramidal and Fast-Spiking Cells as Revealed by Single-Cell Sequencing.

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.

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

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

Caspase-Activated Oligonucleotide Probe.

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

The BRAIN Initiative and Neuroethics: Enabling and Enhancing Neuroscience Advances for Society.

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 Institutes of Health recently mandated a review of the progress of the BRAIN Initiative progress with the goal of fine-tuning its future directions. The BRAIN Working Group 2 focused its discussion on science while the BRAIN Neuroethics Subgroup focused on neuroethics. The Brain Neuroethics Subgroup deliberated for over a year collecting information and recommendations through in person and video meetings, a public one-day neuroethics symposium and soliciting input from neuroscientists and neuroethicist's. The resulting report entitled "The BRAIN Initiative and Neuroethics: Enabling and Enhancing Neuroscience Advances for Society" was accepted by Director of NIH in October of 2019. The recommendations span many BRAIN research neuroethics concerns including privacy considerations, the use of nonhuman primate model systems, neural modulation and enhancement, subject participation in BRAIN research and equity in neuroscience research. Further the group recommended a transformative project whose goal of detailing the scientific mechanisms and ethical underpinnings of consciousness is one of the most daunting issues that impact our perceptions of ourselves. It is anticipated that the report?s recommendations will provide a foundation or "roadmap" for ensuring that neuroethics and BRAIN research move forward as an integrated effort thereby insuring that BRAIN research is of the highest quality.

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

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

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

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