Somatic genomic changes in single Alzheimer's disease neurons.

Dementia in Alzheimer's disease progresses alongside neurodegeneration1-4, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations5 at rates similar to those of dividing cells6,7 which suggests that genetic factors, environmental exposures or disease states might influence this accumulation5. Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer's disease and neurotypical control individuals. We found that somatic DNA alterations increase in individuals with Alzheimer's disease, with distinct molecular patterns. Normal neurons accumulate mutations primarily in an age-related pattern (signature A), which closely resembles 'clock-like' mutational signatures that have been previously described in healthy and cancerous cells6-10. In neurons affected by Alzheimer's disease, additional DNA alterations are driven by distinct processes (signature C) that highlight C>A and other specific nucleotide changes. These changes potentially implicate nucleotide oxidation4,11, which we show is increased in Alzheimer's-disease-affected neurons in situ. Expressed genes exhibit signature-specific damage, and mutations show a transcriptional strand bias, which suggests that transcription-coupled nucleotide excision repair has a role in the generation of mutations. The alterations in Alzheimer's disease affect coding exons and are predicted to create dysfunctional genetic knockout cells and proteostatic stress. Our results suggest that known pathogenic mechanisms in Alzheimer's disease may lead to genomic damage to neurons that can progressively impair function. The aberrant accumulation of DNA alterations in neurodegeneration provides insight into the cascade of molecular and cellular events that occurs in the development of Alzheimer's disease.

Persistent DNA damage associated with ATM kinase deficiency promotes microglial dysfunction.

The autosomal recessive genome instability disorder Ataxia-telangiectasia, caused by mutations in ATM kinase, is characterized by the progressive loss of cerebellar neurons. We find that DNA damage associated with ATM loss results in dysfunctional behaviour of human microglia, immune cells of the central nervous system. Microglial dysfunction is mediated by the pro-inflammatory RELB/p52 non-canonical NF-κB transcriptional pathway and leads to excessive phagocytic clearance of neuronal material. Activation of the RELB/p52 pathway in ATM-deficient microglia is driven by persistent DNA damage and is dependent on the NIK kinase. Activation of non-canonical NF-κB signalling is also observed in cerebellar microglia of individuals with Ataxia-telangiectasia. These results provide insights into the underlying mechanisms of aberrant microglial behaviour in ATM deficiency, potentially contributing to neurodegeneration in Ataxia-telangiectasia.

H2AZ is enriched at polycomb complex target genes in ES cells and is necessary for lineage commitment.

Elucidating how chromatin influences gene expression patterns and ultimately cell fate is fundamental to understanding development and disease. The histone variant H2AZ has emerged as a key regulator of chromatin function and plays an essential but unknown role during mammalian development. Here, genome-wide analysis reveals that H2AZ occupies the promoters of developmentally important genes in a manner that is remarkably similar to that of the Polycomb group (PcG) protein Suz12. By using RNAi, we demonstrate a role for H2AZ in regulating target gene expression, find that H2AZ and PcG protein occupancy is interdependent at promoters, and further show that H2AZ is necessary for ES cell differentiation. Notably, H2AZ occupies a different subset of genes in lineage-committed cells, suggesting that its dynamic redistribution is necessary for cell fate transitions. Thus, H2AZ, together with PcG proteins, may establish specialized chromatin states in ES cells necessary for the proper execution of developmental gene expression programs.

Genome aging: somatic mutation in the brain links age-related decline with disease and nominates pathogenic mechanisms.

Aging is a mysterious process, not only controlled genetically but also subject to random damage that can accumulate over time. While DNA damage and subsequent mutation in somatic cells were first proposed as drivers of aging more than 60 years ago, whether and to what degree these processes shape the neuronal genome in the human brain could not be tested until recent technological breakthroughs related to single-cell whole-genome sequencing. Indeed, somatic single-nucleotide variants (SNVs) increase with age in the human brain, in a somewhat stochastic process that may nonetheless be controlled by underlying genetic programs. Evidence from the literature suggests that in addition to demonstrated increases in somatic SNVs during aging in normal brains, somatic mutation may also play a role in late-onset, sporadic neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. In this review, we will discuss somatic mutation in the human brain, mechanisms by which somatic mutations occur and can be controlled, and how this process can impact human health.

PaSD-qc: quality control for single cell whole-genome sequencing data using power spectral density estimation.

Single cell whole-genome sequencing (scWGS) is providing novel insights into the nature of genetic heterogeneity in normal and diseased cells. However, the whole-genome amplification process required for scWGS introduces biases into the resulting sequencing that can confound downstream analysis. Here, we present a statistical method, with an accompanying package PaSD-qc (Power Spectral Density-qc), that evaluates the properties and quality of single cell libraries. It uses a modified power spectral density to assess amplification uniformity, amplicon size distribution, autocovariance and inter-sample consistency as well as to identify chromosomes with aberrant read-density profiles due either to copy alterations or poor amplification. These metrics provide a standard way to compare the quality of single cell samples as well as yield information necessary to improve variant calling strategies. We demonstrate the usefulness of this tool in comparing the properties of scWGS protocols, identifying potential chromosomal copy number variation, determining chromosomal and subchromosomal regions of poor amplification, and selecting high-quality libraries from low-coverage data for deep sequencing. The software is available free and open-source at https://github.com/parklab/PaSDqc.

SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state.

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors.

ATM-deficiency-induced microglial activation promotes neurodegeneration in ataxia-telangiectasia.

While ATM loss of function has long been identified as the genetic cause of ataxia-telangiectasia (A-T), how it leads to selective and progressive degeneration of cerebellar Purkinje and granule neurons remains unclear. ATM expression is enriched in microglia throughout cerebellar development and adulthood. Here, we find evidence of microglial inflammation in the cerebellum of patients with A-T using single-nucleus RNA sequencing. Pseudotime analysis revealed that activation of A-T microglia preceded upregulation of apoptosis-related genes in granule and Purkinje neurons and that microglia exhibited increased neurotoxic cytokine signaling to granule and Purkinje neurons in A-T. To confirm these findings experimentally, we performed transcriptomic profiling of A-T induced pluripotent stem cell (iPSC)-derived microglia, which revealed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, A-T microglia co-culture with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may promote neurodegeneration in A-T.

Histone H3K27ac separates active from poised enhancers and predicts developmental state.

Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.

Single-cell transcriptomic and genomic changes in the aging human brain.

Aging brings dysregulation of various processes across organs and tissues, often stemming from stochastic damage to individual cells over time. Here, we used a combination of single-nucleus RNA-sequencing and single-cell whole-genome sequencing to identify transcriptomic and genomic changes in the prefrontal cortex of the human brain across life span, from infancy to centenarian. We identified infant-specific cell clusters enriched for the expression of neurodevelopmental genes, and a common down-regulation of cell-essential homeostatic genes that function in ribosomes, transport, and metabolism during aging across cell types. Conversely, expression of neuron-specific genes generally remains stable throughout life. We observed a decrease in specific DNA repair genes in aging, including genes implicated in generating brain somatic mutations as indicated by mutation signature analysis. Furthermore, we detected gene-length-specific somatic mutation rates that shape the transcriptomic landscape of the aged human brain. These findings elucidate critical aspects of human brain aging, shedding light on transcriptomic and genomics dynamics.

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex.

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

Reprogramming of postnatal neurons into induced pluripotent stem cells by defined factors.

Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression.

The two faces of DNA oxidation in genomic and functional mosaicism during aging in human neurons.

Maintaining genomic integrity in post-mitotic neurons in the human brain is paramount because these cells must survive for an individual's entire lifespan. Due to life-long synaptic plasticity and electrochemical transmission between cells, the brain engages in an exceptionally high level of mitochondrial metabolic activity. This activity results in the generation of reactive oxygen species with 8-oxo-7,8-dihydroguanine (8-oxoG) being one of the most prevalent oxidation products in the cell. 8-oxoG is important for the maintenance and transfer of genetic information into proper gene expression: a low basal level of 8-oxoG plays an important role in epigenetic modulation of neurodevelopment and synaptic plasticity, while a dysregulated increase in 8-oxoG damages the genome leading to somatic mutations and transcription errors. The slow yet persistent accumulation of DNA damage in the background of increasing cellular 8-oxoG is associated with normal aging as well as neurological disorders such as Alzheimer's disease and Parkinson's disease. This review explores the current understanding of how 8-oxoG plays a role in brain function and genomic instability, highlighting new methods being used to advance pathological hallmarks that differentiate normal healthy aging and neurodegenerative disease.

Prevalence and mechanisms of somatic deletions in single human neurons during normal aging and in DNA repair disorders.

Replication errors and various genotoxins cause DNA double-strand breaks (DSBs) where error-prone repair creates genomic mutations, most frequently focal deletions, and defective repair may lead to neurodegeneration. Despite its pathophysiological importance, the extent to which faulty DSB repair alters the genome, and the mechanisms by which mutations arise, have not been systematically examined reflecting ineffective methods. Here, we develop PhaseDel, a computational method to detect focal deletions and characterize underlying mechanisms in single-cell whole genome sequences (scWGS). We analyzed high-coverage scWGS of 107 single neurons from 18 neurotypical individuals of various ages, and found that somatic deletions increased with age and in highly expressed genes in human brain. Our analysis of 50 single neurons from DNA repair-deficient diseases with progressive neurodegeneration (Cockayne syndrome, Xeroderma pigmentosum, and Ataxia telangiectasia) reveals elevated somatic deletions compared to age-matched controls. Distinctive mechanistic signatures and transcriptional associations suggest roles for somatic deletions in neurodegeneration.

Somatic mutations in single human cardiomyocytes reveal age-associated DNA damage and widespread oxidative genotoxicity.

The accumulation of somatic DNA mutations over time is a hallmark of aging in many dividing and nondividing cells but has not been studied in postmitotic human cardiomyocytes. Using single-cell whole-genome sequencing, we identified and characterized the landscape of somatic single-nucleotide variants (sSNVs) in 56 single cardiomyocytes from 12 individuals (aged from 0.4 to 82 years). Cardiomyocyte sSNVs accumulate with age at rates that are faster than in many dividing cell types and nondividing neurons. Cardiomyocyte sSNVs show distinctive mutational signatures that implicate failed nucleotide excision repair and base excision repair of oxidative DNA damage, and defective mismatch repair. Since age-accumulated sSNVs create many damaging mutations that disrupt gene functions, polyploidization in cardiomyocytes may provide a mechanism of genetic compensation to minimize the complete knockout of essential genes during aging. Age-related accumulation of cardiac mutations provides a paradigm to understand the influence of aging on cardiac dysfunction.

Single-cell genome sequencing of human neurons identifies somatic point mutation and indel enrichment in regulatory elements.

Accurate somatic mutation detection from single-cell DNA sequencing is challenging due to amplification-related artifacts. To reduce this artifact burden, an improved amplification technique, primary template-directed amplification (PTA), was recently introduced. We analyzed whole-genome sequencing data from 52 PTA-amplified single neurons using SCAN2, a new genotyper we developed to leverage mutation signatures and allele balance in identifying somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) in PTA data. Our analysis confirms an increase in nonclonal somatic mutation in single neurons with age, but revises the estimated rate of this accumulation to 16 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels increase by at least three per year per neuron and are enriched in functional regions of the genome such as enhancers and promoters. Our data suggest that indels in gene-regulatory elements have a considerable effect on genome integrity in human neurons.

Linked-read analysis identifies mutations in single-cell DNA-sequencing data.

Whole-genome sequencing of DNA from single cells has the potential to reshape our understanding of mutational heterogeneity in normal and diseased tissues. However, a major difficulty is distinguishing amplification artifacts from biologically derived somatic mutations. Here, we describe linked-read analysis (LiRA), a method that accurately identifies somatic single-nucleotide variants (sSNVs) by using read-level phasing with nearby germline heterozygous polymorphisms, thereby enabling the characterization of mutational signatures and estimation of somatic mutation rates in single cells.

Aging and neurodegeneration are associated with increased mutations in single human neurons.

It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of 15 normal individuals (aged 4 months to 82 years), as well as 9 individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age-which we term genosenium-shows age-related, region-related, and disease-related molecular signatures and may be important in other human age-associated conditions.