Histone Acetylome-wide Association Study of Autism Spectrum Disorder.

The association of histone modification changes with autism spectrum disorder (ASD) has not been systematically examined. We conducted a histone acetylome-wide association study (HAWAS) by performing H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) on 257 postmortem samples from ASD and matched control brains. Despite etiological heterogeneity, ≥68% of syndromic and idiopathic ASD cases shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex. Similarly, multiple genes associated with rare genetic mutations in ASD showed common "epimutations." Acetylome aberrations in ASD were not attributable to genetic differentiation at cis-SNPs and highlighted genes involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity. By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases. Due to the relative stability of histone modifications postmortem, we anticipate that the HAWAS approach will be applicable to multiple diseases.

Broad transcriptomic dysregulation occurs across the cerebral cortex in ASD.

Neuropsychiatric disorders classically lack defining brain pathologies, but recent work has demonstrated dysregulation at the molecular level, characterized by transcriptomic and epigenetic alterations1-3. In autism spectrum disorder (ASD), this molecular pathology involves the upregulation of microglial, astrocyte and neural-immune genes, the downregulation of synaptic genes, and attenuation of gene-expression gradients in cortex1,2,4-6. However, whether these changes are limited to cortical association regions or are more widespread remains unknown. To address this issue, we performed RNA-sequencing analysis of 725 brain samples spanning 11 cortical areas from 112 post-mortem samples from individuals with ASD and neurotypical controls. We find widespread transcriptomic changes across the cortex in ASD, exhibiting an anterior-to-posterior gradient, with the greatest differences in primary visual cortex, coincident with an attenuation of the typical transcriptomic differences between cortical regions. Single-nucleus RNA-sequencing and methylation profiling demonstrate that this robust molecular signature reflects changes in cell-type-specific gene expression, particularly affecting excitatory neurons and glia. Both rare and common ASD-associated genetic variation converge within a downregulated co-expression module involving synaptic signalling, and common variation alone is enriched within a module of upregulated protein chaperone genes. These results highlight widespread molecular changes across the cerebral cortex in ASD, extending beyond association cortex to broadly involve primary sensory regions.

Transcriptomic Perspectives on Neocortical Structure, Development, Evolution, and Disease.

The cerebral cortex is the source of our most complex cognitive capabilities and a vulnerable target of many neurological and neuropsychiatric disorders. Transcriptomics offers a new approach to understanding the cortex at the level of its underlying genetic code, and rapid technological advances have propelled this field to the high-throughput study of the complete set of transcribed genes at increasingly fine resolution to the level of individual cells. These tools have revealed features of the genetic architecture of adult cortical areas, layers, and cell types, as well as spatiotemporal patterning during development. This has allowed a fresh look at comparative anatomy as well, illustrating surprisingly large differences between mammals while at the same time revealing conservation of some features from avians to mammals. Finally, transcriptomics is fueling progress in understanding the causes of neurodevelopmental diseases such as autism, linking genetic association studies to specific molecular pathways and affected brain regions.

Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Change history: In this Letter, the labels for splicing events A3SS and A5SS were swapped in column D of Supplementary Table 3a and b. This has been corrected online.

Transcription factor combinations that define human astrocyte identity encode significant variation of maturity and function.

Increasing evidence indicates that cellular identity can be reduced to the distinct gene regulatory networks controlled by transcription factors (TFs). However, redundancy exists in these states as different combinations of TFs can induce broadly similar cell types. We previously demonstrated that by overcoming gene silencing, it is possible to deterministically reprogram human pluripotent stem cells directly into cell types of various lineages. In the present study we leverage the consistency and precision of our approach to explore four different TF combinations encoding astrocyte identity, based on previously published reports. Analysis of the resulting induced astrocytes (iAs) demonstrated that all four cassettes generate cells with the typical morphology of in vitro astrocytes, which expressed astrocyte-specific markers. The transcriptional profiles of all four iAs clustered tightly together and displayed similarities with mature human astrocytes, although maturity levels differed between cells. Importantly, we found that the TF cassettes induced iAs with distinct differences with regards to their cytokine response and calcium signaling. In vivo transplantation of selected iAs into immunocompromised rat brains demonstrated long term stability and integration. In conclusion, all four TF combinations were able to induce stable astrocyte-like cells that were morphologically similar but showed subtle differences with respect to their transcriptome. These subtle differences translated into distinct differences with regards to cell function, that could be related to maturation state and/or regional identity of the resulting cells. This insight opens an opportunity to precision-engineer cells to meet functional requirements, for example, in the context of therapeutic cell transplantation.

Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Autism spectrum disorder (ASD) involves substantial genetic contributions. These contributions are profoundly heterogeneous but may converge on common pathways that are not yet well understood. Here, through post-mortem genome-wide transcriptome analysis of the largest cohort of samples analysed so far, to our knowledge, we interrogate the noncoding transcriptome, alternative splicing, and upstream molecular regulators to broaden our understanding of molecular convergence in ASD. Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes. Our data suggest that SOX5, a transcription factor involved in neuron fate specification, contributes to this reduction in regional differences. We further demonstrate that a genetically defined subtype of ASD, chromosome 15q11.2-13.1 duplication syndrome (dup15q), shares the core transcriptomic signature observed in idiopathic ASD. Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression. Our findings illustrate how diverse genetic perturbations can lead to phenotypic convergence at multiple biological levels in a complex neuropsychiatric disorder.

A framework for integrating directed and undirected annotations to build explanatory models of cis-eQTL data.

A longstanding goal of regulatory genetics is to understand how variants in genome sequences lead to changes in gene expression. Here we present a method named Bayesian Annotation Guided eQTL Analysis (BAGEA), a variational Bayes framework to model cis-eQTLs using directed and undirected genomic annotations. We used BAGEA to integrate directed genomic annotations with eQTL summary statistics from tissues of various origins. This analysis revealed epigenetic marks that are relevant for gene expression in different tissues and cell types. We estimated the predictive power of the models that were fitted based on directed genomic annotations. This analysis showed that, depending on the underlying eQTL data used, the directed genomic annotations could predict up to 1.5% of the variance observed in the expression of genes with top nominal eQTL association p-values < 10-7. For genes with estimated effect sizes in the top 25% quantile, up to 5% of the expression variance could be predicted. Based on our results, we recommend the use of BAGEA for the analysis of cis-eQTL data to reveal annotations relevant to expression biology.

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody.

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.

Mouse genomic variation and its effect on phenotypes and gene regulation.

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.

Expression profiling of mouse subplate reveals a dynamic gene network and disease association with autism and schizophrenia.

The subplate zone is a highly dynamic transient sector of the developing cerebral cortex that contains some of the earliest generated neurons and the first functional synapses of the cerebral cortex. Subplate cells have important functions in early establishment and maturation of thalamocortical connections, as well as in the development of inhibitory cortical circuits in sensory areas. So far no role has been identified for cells in the subplate in the mature brain and disease association of the subplate-specific genes has not been analyzed systematically. Here we present gene expression evidence for distinct roles of the mouse subplate across development as well as unique molecular markers to extend the repertoire of subplate labels. Performing systematic comparisons between different ages (embryonic days 15 and 18, postnatal day 8, and adult), we reveal the dynamic and constant features of the markers labeling subplate cells during embryonic and early postnatal development and in the adult. This can be visualized using the online database of subplate gene expression at https://molnar.dpag.ox.ac.uk/subplate/. We also identify embryonic similarities in gene expression between the ventricular zones, intermediate zone, and subplate, and distinct postnatal similarities between subplate, layer 5, and layers 2/3. The genes expressed in a subplate-specific manner at some point during development show a statistically significant enrichment for association with autism spectrum disorders and schizophrenia. Our report emphasizes the importance of the study of transient features of the developing brain to better understand neurodevelopmental disorders.

Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.

The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates.

NSF workshop report: discovering general principles of nervous system organization by comparing brain maps across species.

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene-structure-function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system 'maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of 'reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well-annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross-species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution.

Stratification of acute myocardial and endothelial cell injury, salvage index and final infarct size by systematic microRNA profiling in acute ST-elevation myocardial infarction.

AIM: Acute injury and subsequent remodelling responses to ST-segment elevation myocardial infarction (STEMI) are major determinants of clinical outcome. Current imaging and plasma biomarkers provide delayed readouts of myocardial injury and recovery. Here, we sought to systematically characterize all microRNAs (miRs) released during the acute phase of STEMI and relate miR release to magnetic resonance imaging (MRI) findings to predict acute and late responses to STEMI, from a single early blood sample. METHODS AND RESULTS: miRs were quantified in blood samples obtained from patients after primary PCI (PPCI) for STEMI. Cardiac MRI (cMRI) was performed to quantify myocardial edema, infarct size and salvage index. Regression models were constructed to predict these outcomes measures, which were then tested with a validation cohort. Transcoronary miR release was quantified from paired measurements of coronary artery and coronary sinus samples. A cell culture model was used to identify endothelial cell-derived miRs.A total of 72 patients undergoing PPCI for acute STEMI underwent miR analysis and cMRI. About >200 miRs were detectable in plasma after STEMI, from which 128 miRs were selected for quantification in all patients. Known myocardial miRs demonstrated a linear correlation with troponin release, and these increased across the transcoronary gradient. We identified novel miRs associated with microvascular injury and myocardial salvage. Regression models were constructed using a training cohort, then tested in a validation cohort, and predicted myocardial oedema, infarct size and salvage index. CONCLUSION: Analysis of miR release after STEMI identifies biomarkers that predict both acute and late outcomes after STEMI. A novel miR-based biomarker score enables the estimation of area at risk, late infarct size and salvage index from a single blood sample 6 hours after PPCI, providing a simple and rapid alternative to serial cMRI characterization of STEMI outcome.

Unravelling cell type-specific responses to Parkinson's Disease at single cell resolution.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson's Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase (TH) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets.

Transcribed dark matter: meaning or myth?

Genomic tiling arrays, cDNA sequencing and, more recently, RNA-Seq have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. These methods have led to greatly improved annotations of protein-coding genes, but have also identified transcription outside of annotated exons. One resultant issue that has aroused dispute is the balance of transcription of known exons against transcription outside of known exons. While non-genic 'dark matter' transcription was found by tiling arrays to be pervasive, it was seen to contribute only a small percentage of the polyadenylated transcriptome in some RNA-Seq experiments. This apparent contradiction has been compounded by a lack of clarity about what exactly constitutes a protein-coding gene. It remains unclear, for example, whether or not all transcripts that overlap on either strand within a genomic locus should be assigned to a single gene locus, including those that fail to share promoters, exons and splice junctions. The inability of tiling arrays and RNA-Seq to count transcripts, rather than exons or exon pairs, adds to these difficulties. While there is agreement that thousands of apparently non-coding loci are present outside of protein-coding genes in the human genome, there is vigorous debate of what constitutes evidence for their functionality. These issues will only be resolved upon the demonstration, or otherwise, that organismal or cellular phenotypes frequently result when non-coding RNA loci are disrupted.

Core liver homeostatic co-expression networks are preserved but respond to perturbations in an organism- and disease-specific manner.

Findings about chronic complex diseases are difficult to extrapolate from animal models to humans. We reason that organs may have core network modules that are preserved between species and are predictably altered when homeostasis is disrupted. To test this idea, we perturbed hepatic homeostasis in mice by dietary challenge and compared the liver transcriptome with that in human fatty liver disease and liver cancer. Co-expression module preservation analysis pointed to alterations in immune responses and metabolism (core modules) in both human and mouse datasets. The extent of derailment in core modules was predictive of survival in the cancer genome atlas (TCGA) liver cancer dataset. We identified module eigengene quantitative trait loci (module-eQTL) for these predictive co-expression modules, targeting of which may resolve homeostatic perturbations and improve patient outcomes. The framework presented can be used to understand homeostasis at systems levels in pre-clinical models and in humans. A record of this paper's transparent peer review process is included in the supplemental information.

No Longer Underappreciated: The Emerging Concept of Astrocyte Heterogeneity in Neuroscience.

Astrocytes are ubiquitous in the central nervous system (CNS). These cells possess thousands of individual processes, which extend out into the neuropil, interacting with neurons, other glia and blood vessels. Paralleling the wide diversity of their interactions, astrocytes have been reported to play key roles in supporting CNS structure, metabolism, blood-brain-barrier formation and control of vascular blood flow, axon guidance, synapse formation and modulation of synaptic transmission. Traditionally, astrocytes have been studied as a homogenous group of cells. However, recent studies have uncovered a surprising degree of heterogeneity in their development and function, in both the healthy and diseased brain. A better understanding of astrocyte heterogeneity is urgently needed to understand normal brain function, as well as the role of astrocytes in response to injury and disease.

Identification of region-specific astrocyte subtypes at single cell resolution.

Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain barrier formation, and regulation of blood flow, as well as metabolic support of other brain resident cells. Crucially, emerging evidence shows specific adaptations and astrocyte-encoded functions in regions, such as the spinal cord and cerebellum. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single-cell RNA sequencing. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. The data are available through an online database (https://holt-sc.glialab.org/), providing a resource on which to base explorations of local astrocyte diversity and function in the brain.