ABSTRACT
Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.
Subject(s)
Cell Lineage , Cerebral Cortex/cytology , Neurogenesis , Cerebral Cortex/growth & development , Cerebral Cortex/metabolism , High-Throughput Nucleotide Sequencing , Humans , Mutation Accumulation , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Sequence Analysis, DNA , Single-Cell AnalysisABSTRACT
Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Axonal Transport/radiation effects , Axons/radiation effects , Gene Expression Regulation/radiation effects , Neurons/cytology , Ultraviolet Rays , Amyloid beta-Protein Precursor/deficiency , Animals , Axons/drug effects , Axons/metabolism , Axons/ultrastructure , Cells, Cultured , Embryo, Mammalian , Hippocampus/cytology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuroblastoma/pathology , Neurons/radiation effects , Presenilin-1/deficiency , Presenilin-2/deficiency , TransfectionABSTRACT
Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.
Subject(s)
Agrin/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Presenilin-1/metabolism , Presenilin-2/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Cells, Cultured , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Biological , Presenilin-1/deficiency , Presenilin-1/genetics , Presenilin-2/deficiency , Presenilin-2/genetics , Promoter Regions, Genetic , Signal TransductionABSTRACT
BACKGROUND: Mosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation. METHODS: To overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples. RESULTS: The Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions. CONCLUSIONS: MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.
Subject(s)
INDEL Mutation , Humans , Neoplasms , SoftwareABSTRACT
Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution.
Subject(s)
Cell Lineage , Gastrulation , Mutation , Neural Stem Cells/cytology , Prosencephalon/cytology , Adolescent , Adult , Cell Division , Clone Cells/cytology , Embryonic Development/genetics , Female , Gastrula/cytology , Genetic Variation , Germ Layers/cytology , Humans , Male , Neurons/cytology , Organogenesis , Polymorphism, Single Nucleotide , Prosencephalon/embryology , Single-Cell Analysis , Whole Genome SequencingABSTRACT
We characterize the landscape of somatic mutations-mutations occurring after fertilization-in the human brain using ultra-deep (~250×) whole-genome sequencing of prefrontal cortex from 59 donors with autism spectrum disorder (ASD) and 15 control donors. We observe a mean of 26 somatic single-nucleotide variants per brain present in ≥4% of cells, with enrichment of mutations in coding and putative regulatory regions. Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2-3 mutations in subsequent generations. This suggests that a typical individual possesses ~80 somatic single-nucleotide variants present in ≥2% of cells-comparable to the number of de novo germline mutations per generation-with about half of individuals having at least one potentially function-altering somatic mutation somewhere in the cortex. ASD brains show an excess of somatic mutations in neural enhancer sequences compared with controls, suggesting that mosaic enhancer mutations may contribute to ASD risk.
Subject(s)
Autism Spectrum Disorder/pathology , Prefrontal Cortex/pathology , Cell Division/genetics , Chromatin/genetics , Embryonic Development/genetics , Epigenesis, Genetic , Exons , Female , Gene Regulatory Networks/genetics , Genetic Predisposition to Disease , Genome, Human/genetics , Germ-Line Mutation/genetics , High-Throughput Nucleotide Sequencing , Humans , Polymorphism, Single Nucleotide , Pregnancy , Whole Genome SequencingABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
We systematically analyzed postzygotic mutations (PZMs) in whole-exome sequences from the largest collection of trios (5,947) with autism spectrum disorder (ASD) available, including 282 unpublished trios, and performed resequencing using multiple independent technologies. We identified 7.5% of de novo mutations as PZMs, 83.3% of which were not described in previous studies. Damaging, nonsynonymous PZMs within critical exons of prenatally expressed genes were more common in ASD probands than controls (P < 1 × 10-6), and genes carrying these PZMs were enriched for expression in the amygdala (P = 5.4 × 10-3). Two genes (KLF16 and MSANTD2) were significantly enriched for PZMs genome-wide, and other PZMs involved genes (SCN2A, HNRNPU and SMARCA4) whose mutation is known to cause ASD or other neurodevelopmental disorders. PZMs constitute a significant proportion of de novo mutations and contribute importantly to ASD risk.