Jennifer A. Erwin

Mobile DNA elements in the generation of diversity and complexity in the brain

Mobile elements are DNA sequences that can change their position (retrotranspose) within the genome. Although its biological function is largely unappreciated, DNA derived from mobile elements comprises nearly half of the human genome. It has long been thought that neuronal genomes are invariable; however, recent studies have demonstrated that mobile elements actively retrotranspose during neurogenesis, thereby creating genomic diversity between neurons. In addition, mounting data demonstrate that mobile elements are misregulated in certain neurological disorders, including Rett syndrome and schizophrenia.

Nuclear RNA-seq of single neurons reveals molecular signatures of activation

Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.

Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at -80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.

L1-associated genomic regions are deleted in somatic cells of the healthy human brain

The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44–63% of cells of the cells in the healthy brain.

Intersection of diverse neuronal genomes and neuropsychiatric disease: The Brain Somatic Mosaicism Network

Single-cell diversity in the brain The cells that make up an organism may all start from one genome, but somatic mutations mean that somewhere along the line of development, an organisms individual cellular genomes diverge. McConnell et al. review the implications and causes of single-cell genomic diversity for brain function. Somatic mutations caused by mobile genetic elements or errors in DNA repair may underlie certain neuropsychiatric disorders. Science, this issue p. eaal1641 BACKGROUND Elucidating the genetic architecture of neuropsychiatric disorders remains a major scientific and medical challenge. Emerging genomic technologies now permit the analysis of somatic mosaicism in human tissues. The measured frequencies of single-nucleotide variants (SNVs), small insertion/deletion (indel) mutations, structural variants [including copy number variants (CNVs), inversions, translocations, and whole-chromosome gains or losses], and mobile genetic element insertions (MEIs) indicate that each neuron may harbor hundreds of somatic mutations. Given the long life span of neurons and their central role in neural circuits and behavior, somatic mosaicism represents a potential mechanism that may contribute to neuronal diversity and the etiology of numerous neuropsychiatric disorders. ADVANCES Somatic mutations that confer cellular proliferative or cellular survival phenotypes have been identified in patients with cortical malformations. These data have led to the hypothesis that somatic mutations may also confer phenotypes to subsets of neurons, which could increase the risk of developing certain neuropsychiatric disorders. Genomic technologies, including advances in long-read, next-generation DNA sequencing technologies, single-cell genomics, and cutting-edge bioinformatics, can now make it possible to determine the types and frequencies of somatic mutations within the human brain. However, a comprehensive understanding of the contribution of somatic mosaicism to neurotypical brain development and neuropsychiatric disease requires a coordinated, multi-institutional effort. The National Institute of Mental Health (NIMH) has formed a network of 18 investigative teams representing 15 institutions called the Brain Somatic Mosaicism Network (BSMN). Each research team will use an array of genomic technologies to exploit well-curated human tissue repositories in an effort to define the frequency and pattern of somatic mutations in neurotypical individuals and in schizophrenia, autism spectrum disorder, bipolar disorder, Tourette syndrome, and epilepsy patient populations. Collectively, these efforts are estimated to generate a community resource of more than 10,000 DNA-sequencing data sets and will enable a cross-platform integrated analysis with other NIMH initiatives, such as the PsychENCODE project and the CommonMind Consortium. OUTLOOK A fundamental open question in neurodevelopmental genetics is whether and how somatic mosaicism may contribute to neuronal diversity within the neurotypical spectrum and in diseased brains. Healthy individuals may harbor known pathogenic somatic mutations at subclinical frequencies, and the local composition of neural cell types may be altered by mutations conferring prosurvival phenotypes in subsets of neurons. By extension, the neurotypical architecture of somatic mutations may confer circuit-level differences that would not be present if every neuron had an identical genome. Given the apparent abundance of somatic mutations within neurons, an in-depth understanding of how different types of somatic mosaicism affect neural function could yield mechanistic insight into the etiology of neurodevelopmental and neuropsychiatric disorders. The BSMN will examine large collections of postmortem brain tissue from neurotypical individuals and patients with neuropsychiatric disorders. By sequencing brain DNA and single neuronal genomes directly, rather than genomic DNA derived from peripheral blood or other somatic tissues, the BSMN will test the hypothesis that brain somatic variants contribute to neuropsychiatric disease. Notably, it is also possible that some inherited germline variants confer susceptibility to disease, which is later exacerbated by somatic mutations. Confirming such a scenario could increase our understanding of the genetic risk architecture of neuropsychiatric disease and may, in part, explain discordant neuropsychiatric phenotypes between identical twins. Results from these studies may lead to the discovery of biomarkers and genetic targets to improve the treatment of neuropsychiatric disease and may offer hope for improving the lives of patients and their families. Collectively, somatic SNVs, indels, structural variants (e.g., CNVs), and MEIs (e.g., L1 retrotransposition events) shape the genomic landscape of individual neurons. The Brain Somatic Mosaicism Network aims to systematically generate pioneering data on the types and frequencies of brain somatic mutations in both neurotypical individuals and those with neuropsychiatric disease. The resulting data will be shared as a large community resource. Neuropsychiatric disorders have a complex genetic architecture. Human genetic population-based studies have identified numerous heritable sequence and structural genomic variants associated with susceptibility to neuropsychiatric disease. However, these germline variants do not fully account for disease risk. During brain development, progenitor cells undergo billions of cell divisions to generate the ~80 billion neurons in the brain. The failure to accurately repair DNA damage arising during replication, transcription, and cellular metabolism amid this dramatic cellular expansion can lead to somatic mutations. Somatic mutations that alter subsets of neuronal transcriptomes and proteomes can, in turn, affect cell proliferation and survival and lead to neurodevelopmental disorders. The long life span of individual neurons and the direct relationship between neural circuits and behavior suggest that somatic mutations in small populations of neurons can significantly affect individual neurodevelopment. The Brain Somatic Mosaicism Network has been founded to study somatic mosaicism both in neurotypical human brains and in the context of complex neuropsychiatric disorders.

Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology

Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single-human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. Although whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study—GDAP1L1—to isolate highly functional live human neurons in vitro.

Insights into the role of somatic mosaicism in the brain

Somatic mosaicism refers to the fact that cells within an organism have different genomes. It is now clear that somatic mosaicism occurs in all brains and that somatic mutations in a subset of cells can cause various rare neurodevelopmental disorders. However, for most individuals, the extent and consequences of somatic mosaicism are largely unknown. The complexity and unique features of the brain suggest that somatic mosaicism can play an important role in behavior and cognition. Here we review recent manuscripts showing instances of somatic mosaicism in the brain and estimating its extent and possible biological consequences. The consequences of somatic mosaicism span vast dimensions -from a single-locus variant, to genes and gene networks, to cells, to the interactions of the mosaic cells via neural networks affecting behavior and cognition. We highlight how systems biology approaches are particularly well suited for the complex emerging field of brain somatic mosaicism.

Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells.

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently, advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method, referred to as Patch-seq, enables a thorough, multimodal profiling of neurons and permits us to expose the links between functional properties, morphology, and gene expression. Here, we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects, but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders.

Author Correction: L1-associated genomic regions are deleted in somatic cells of the healthy human brain

In the version of this article initially published, NIH grant U01 MH106882 to F.H.G. was missing from the Acknowledgments. The error has been corrected in the HTML and PDF versions of the article.

Corrigendum: Nuclear RNA-seq of single neurons reveals molecular signatures of activation

Nature Communications 7: Article number:1102210.1038/ncomms11022 (2016); Published April192016; Updated June142016