25 results found
Yildirim M, Delepine C, Feldman D, et al., 2022, Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome., Elife, Vol: 11
Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.
Breuss MW, Yang X, Schlachetzki JCM, et al., 2022, Somatic mosaicism reveals clonal distributions of neocortical development, NATURE, Vol: 604, Pages: 689-+, ISSN: 0028-0836
Hu D, Abbasova L, Schilder B, et al., 2022, CUT&Tag recovers up to half of ENCODE ChIP-seq peaks, Publisher: Cold Spring Harbor Laboratory
Techniques for genome-wide epigenetic profiling have been undergoing rapid development toward recovery of high quality data from bulk and single cell samples. DNA-protein interactions have traditionally been profiled via chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq), which has become the gold standard for studying histone modifications or transcription factor binding. Cleavage Under Targets & Tagmentation (CUT&Tag) is a promising new technique, which enables profiling of such interactions in situ at high sensitivity and is adaptable to single cell applications. However thorough evaluation and benchmarking against established ChIP-seq datasets are still lacking. Here we comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Across a total of 30 new and 6 published CUT&Tag datasets we found that no experiment recovers more than 50% of known ENCODE peaks, regardless of the histone mark. We tested peak callers MACS2 and SEACR, identifying optimal peak calling parameters. Balancing both precision and recall of known ENCODE peaks, SEACR without retention of duplicates showed the best performance. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.
Yildirim M, Delepine C, Feldman D, et al., 2022, Label-free three-photon imaging of intact human cerebral organoids: tracking early events in brain development and deficits in Rett Syndrome
<jats:title>ABSTRACT</jats:title><jats:p>Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (∼2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.</jats:p>
Murphy KB, Nott A, Marzi SJ, 2021, CHAS, a deconvolution tool, infers cell type-specific signatures in bulk brain histone acetylation studies of brain disorders
<jats:title>Abstract</jats:title><jats:p>Chromatin profiling studies have shown the importance of gene regulation in driving heritability and environmental risk of brain disorders. Acetylation of histone H3 lysine 27 (H3K27ac) has emerged as an informative disease-associated epigenetic mark. However, cell type-specific contributions to epigenetic dysregulation in disease are unclear as studies have often used bulk brain tissue. Therefore, methods for the deconvolution of bulk H3K27ac profiles are critical. Here we developed the Cell type-specific Histone Acetylation Score (CHAS), a computational tool for inferring cell type-specific signatures in bulk brain H3K27ac profiles. CHAS annotates peaks identified in bulk brain studies of H3K27ac to cell type-specific signals in four major brain cell types, and derives cell type-specific histone acetylation scores as a proxy for cell type proportion. Our method was validated in pseudo-bulk samples and applied to three brain disorder epigenome-wide association studies conducted on bulk brain tissue. CHAS exposed shifts in cellular proportions in Alzheimer’s disease (AD), in line with neuropathology, and identified disrupted gene regulatory elements in oligodendrocytes in AD and microglia in autism spectrum disorder (ASD). This contrasts with heritability-based enrichment analyses which indicate genetic risk is associated with microglia in AD and neurons in ASD. Our approach identified cell type specific signalling pathways and putative upstream transcription factors associated with these elements. CHAS enables deconvolution of H3K27ac in bulk brain tissue, yielding cell type-specific biological insights into brain disease-associated regulatory variation.</jats:p>
Nott A, Schlachetzki JCM, Fixsen BR, et al., 2021, Nuclei isolation of multiple brain cell types for omics interrogation, NATURE PROTOCOLS, Vol: 16, Pages: 1629-1646, ISSN: 1754-2189
Morshed N, Ralvenius WT, Nott A, et al., 2020, Phosphoproteomics identifies microglial Siglec-F inflammatory response during neurodegeneration, MOLECULAR SYSTEMS BIOLOGY, Vol: 16, ISSN: 1744-4292
Morshed N, Ralvenius WT, Nott A, et al., 2020, Phosphoproteomics identifies microglial Siglec-F inflammatory response during neurodegeneration., Mol Syst Biol, Vol: 16
Alzheimer's disease (AD) is characterized by the appearance of amyloid-β plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV-2 cell line and human stem cell-derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. Collectively, our results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration.
Breuss MW, Yang X, Antaki D, et al., 2020, Somatic mosaicism in the mature brain reveals clonal cellular distributions during cortical development
<jats:title>Abstract</jats:title><jats:p>The structure of the human neocortex underlies species-specific features and is a reflection of intricate developmental programs. Here we analyzed neocortical cellular lineages through a comprehensive assessment of brain somatic mosaicism—which acts as a neutral recorder of lineage history. We employed deep whole genome and variant sequencing in a single <jats:italic>postmortem</jats:italic> neurotypical human brain across 25 anatomic regions and three distinct modalities: bulk geographies, sorted cell types, and single nuclei. We identified 259 mosaic variants, revealing remarkable differences in localization, clonal abundance, cell type specificity, and clade distribution. We identified a set of hierarchical cellular diffusion barriers, whereby the left-right axis separation of the neocortex occurs prior to anterior-posterior and dorsal-ventral axis separation. We also found that stochastic distribution is a driver of clonal dispersion, and that rules regarding cellular lineages and anatomical boundaries are often ignored. Our data provides a comprehensive analysis of brain somatic mosaicism across the human cerebral cortex, deconvolving clonal distributions and migration patterns in the human embryo.</jats:p><jats:sec><jats:title>One Sentence Summary</jats:title><jats:p>Comprehensive evaluation of brain somatic mosaicism in the adult human identifies rules governing cellular distribution during embryogenesis.</jats:p></jats:sec>
Pao P-C, Patnaik D, Watson LA, et al., 2020, HDAC1 modulates OGG1-initiated oxidative DNA damage repair in the aging brain and Alzheimer's disease, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
Nott A, Holtman IR, Coufal NG, et al., 2019, Brain cell type-specific enhancer-promoter interactome maps and disease-risk association, Science, Vol: 366, Pages: 1134-1139, ISSN: 0036-8075
Nott A, Holtman IR, Coufal NG, et al., 2019, Cell type-specific enhancer-promoter connectivity maps in the human brain and disease risk association
<jats:title>Abstract</jats:title><jats:p>Unique cell type-specific patterns of activated enhancers can be leveraged to interpret non-coding genetic variation associated with complex traits and diseases such as neurological and psychiatric disorders. Here, we have defined active promoters and enhancers for major cell types of the human brain. Whereas psychiatric disorders were primarily associated with regulatory regions in neurons, idiopathic Alzheimer’s disease (AD) variants were largely confined to microglia enhancers. Interactome maps connecting GWAS variants in cell type-specific enhancers to gene promoters revealed an extended microglia gene network in AD. Deletion of a microglia-specific enhancer harboring AD-risk variants ablated <jats:italic>BIN1</jats:italic> expression in microglia but not in neurons or astrocytes. These findings revise and expand the genes likely to be influenced by non-coding variants in AD and suggest the probable brain cell types in which they function.</jats:p><jats:sec><jats:title>One Sentence Summary</jats:title><jats:p>Identification of cell type-specific regulatory elements in the human brain enables interpretation of non-coding GWAS risk variants.</jats:p></jats:sec>
Nott A, Glass CK, 2018, Immune memory in the brain, NATURE, Vol: 556, Pages: 312-313, ISSN: 0028-0836
Durak O, Gao F, Kaeser-Woo YJ, et al., 2016, Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling, Nature Neuroscience, Vol: 19, Pages: 1477-1488, ISSN: 1097-6256
De novo mutations in CHD8 are strongly associated with autism spectrum disorder, but the basic biology of CHD8 remains poorly understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of neural-specific genes by regulating the expression of PRC2 complex components. Furthermore, knockdown of Chd8 disrupts the expression of key transducers of Wnt signaling, and enhancing Wnt signaling rescues the transcriptional and behavioral deficits caused by Chd8 knockdown. We propose that these roles of Chd8 and the dynamics of Chd8 expression during development help negotiate the fine balance between neural progenitor proliferation and differentiation. Together, these observations provide new insights into the neurodevelopmental role of Chd8.
Nott A, Cheng J, Gao F, et al., 2016, Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior, Nature Neuroscience, Vol: 19, Pages: 1497-1505, ISSN: 1097-6256
Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). The RTT missense MECP2R306C mutation prevents MeCP2 from interacting with the NCoR/histone deacetylase 3 (HDAC3) complex; however, the neuronal function of HDAC3 is incompletely understood. We found that neuronal deletion of Hdac3 in mice elicited abnormal locomotor coordination, sociability and cognition. Transcriptional and chromatin profiling revealed that HDAC3 positively regulated a subset of genes and was recruited to active gene promoters via MeCP2. HDAC3-associated promoters were enriched for the FOXO transcription factors, and FOXO acetylation was elevated in Hdac3 knockout (KO) and Mecp2 KO neurons. Human RTT-patient-derived MECP2R306C neural progenitor cells had deficits in HDAC3 and FOXO recruitment and gene expression. Gene editing of MECP2R306C cells to generate isogenic controls rescued HDAC3-FOXO-mediated impairments in gene expression. Our data suggest that HDAC3 interaction with MeCP2 positively regulates a subset of neuronal genes through FOXO deacetylation, and disruption of HDAC3 contributes to cognitive and social impairment.
Nitarska J, Smith JG, Sherlock WT, et al., 2016, A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development, CELL REPORTS, Vol: 17, Pages: 1683-1698, ISSN: 2211-1247
Seneviratne U, Nott A, Bhat VB, et al., 2016, S-nitrosation of proteins relevant to Alzheimer's disease during early stages of neurodegeneration, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 4152-4157, ISSN: 0027-8424
Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein’s function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25–inducible mouse model of Alzheimer’s disease-like neurodegeneration. The approach—SNOTRAP (SNO trapping by triaryl phosphine)—is a direct tagging strategy that uses phosphine-based chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer’s disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.
Seneviratne U, Kodihalli RC, Nott A, et al., 2015, Decoding the S-nitroso proteome in a mouse model of Alzheimer's by SNOTRAP and mass spectrometry - clues for altered signaling pathways, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Madabhushi R, Gao F, Pfenning AR, et al., 2015, Activity-induced DNA breaks govern the expression of neuronal early-response genes, Cell, Vol: 161, Pages: 1592-1605, ISSN: 0092-8674
Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIβ (Topo IIβ), and knockdown of Topo IIβ attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.
Nott A, Cho S, Seo J, et al., 2015, HDAC2 expression in parvalbumin interneurons regulates synaptic plasticity in the mouse visual cortex., Neuroepigenetics, Vol: 1, Pages: 34-40, ISSN: 2214-7845
An experience-dependent postnatal increase in GABAergic inhibition in the visual cortex is important for the closure of a critical period of enhanced synaptic plasticity. Although maturation of the subclass of Parvalbumin (Pv)-expressing GABAergic interneurons is known to contribute to critical period closure, the role of epigenetics on cortical inhibition and synaptic plasticity has not been explored. The transcription regulator, histone deacetylase 2 (HDAC2), has been shown to modulate synaptic plasticity and learning processes in hippocampal excitatory neurons. We found that genetic deletion of HDAC2 specifically from Pv-interneurons reduces inhibitory input in the visual cortex of adult mice, and coincides with enhanced long-term depression (LTD) that is more typical of young mice. These findings show that HDAC2 loss in Pv-interneurons leads to a delayed closure of the critical period in the visual cortex and supports the hypothesis that HDAC2 is a key negative regulator of synaptic plasticity in the adult brain.
Nott A, Tsai L-H, 2013, The Top3 beta way to untangle RNA, NATURE NEUROSCIENCE, Vol: 16, Pages: 1163-1164, ISSN: 1097-6256
Nott A, Nitarska J, Veenvliet JV, et al., 2013, S-nitrosylation of HDAC2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration., Proc Natl Acad Sci U S A, Vol: 110, Pages: 3113-3118
Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.
Nott A, Riccio A, 2009, Nitric oxide-mediated epigenetic mechanisms in developing neurons, CELL CYCLE, Vol: 8, Pages: 725-730, ISSN: 1538-4101
Nott A, Watson PM, Robinson JD, et al., 2008, S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons, NATURE, Vol: 455, Pages: 411-U67, ISSN: 0028-0836
Nott A, Levin ED, 2006, Dorsal hippocampal alpha 7 and alpha 4 beta 2 nicotinic receptors and memory, BRAIN RESEARCH, Vol: 1081, Pages: 72-78, ISSN: 0006-8993
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