Lai J, Kim J, Jeffries AM, Tolles A, Chittenden TW, Buckley PG, Yu T, Lodato MA**, Lee EA**. Single-nucleus transcriptomic analyses reveal microglial activation underlying cerebellar degeneration in Ataxia Telangiectasia. bioRxiv Submitted;
While ATM loss-of-function has long been identified as the genetic cause of Ataxia Telangiectasia (AT), how this genetic mutation leads to selective and progressive cerebellar degeneration of Purkinje and granule cells remains unknown. We performed single-nucleus RNA-sequencing of the human cerebellum and prefrontal cortex from individuals with AT and matched unaffected controls to identify AT-associated transcriptomic changes in a cell-type- and brain-region-specific manner. We provide the largest single-nucleus transcriptomic atlas of the adult human cerebellum to-date (126,356 nuclei), identify upregulation of apoptotic and ER stress pathways in Purkinje and granule neurons, and uncover strong downregulation of calcium ion homeostasis genes in Purkinje neurons. Our analysis reveals prominent inflammation of microglia in AT cerebellum with transcriptional signatures similar to aging and neurodegenerative microglia, and suggests that microglia activation precedes Purkinje and granule neuron death in disease progression. Our data implicates a novel role of microglial activation underlying cerebellar degeneration in AT.


Miller MB*, Huang AY*, Kim, J, Zhou, Z, Kirkham SL, Maury EA, Ziegenfuss JS, Reed HC, Neil JE, Rento, L, Ryu SC, Ma CC, Luquette LJ, Ames HM, Oakley DH, Frosch, M.P, Hyman BT, Lodato MA**, Lee EA**, Walsh CA**. Somatic genomic changes in single Alzheimer’s disease neurons. Nature 2022;604(7907):714-722.
Dementia in Alzheimer’s disease progresses alongside neurodegeneration, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations at rates similar to those of dividing cells which suggests that genetic factors, environmental exposures or disease states might influence this accumulation. 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 cells. 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 oxidation, 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.
Choudhury, S*, Huang AY*, Kim, J, Zhou, Z, Morillo, K, Maury EA, Tsai JW, Miller MB, Lodato MA, Araten, S, Hilal N, Lee EA**, Chen MH**, Walsh CA**. Somatic mutations in single human cardiomyocytes reveal age-associated DNA damage and widespread genotoxicity. Nature Aging 2022;
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.
Bourseguin J, Cheng W, Talbot E, Hardy L, Lai J, Jeffries A, Lodato M, Lee EA, Khoronenkova S. Persistent DNA damage associated with ATM kinase deficiency promotes microglial dysfunction. Nucleic Acids Res 2022;50(5):2700-2718.
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.
Huang AY, Lee EA. Identification of somatic mutations from bulk and single-cell sequencing data. Frontiers in Aging (mini review) 2022;2:800380

Somatic mutations are DNA variants that occur after the fertilization of zygotes and accumulate during the developmental and aging processes in the human lifespan. Somatic mutations have long been known to cause cancer, and more recently have been implicated in a variety of non-cancer diseases. The patterns of somatic mutations, or mutational signatures, also shed light on the underlying mechanisms of the mutational process. Advances in next-generation sequencing over the decades have enabled genome-wide profiling of DNA variants in a high-throughput manner; however, unlike germline mutations, somatic mutations are carried only by a subset of the cell population. Thus, sensitive bioinformatic methods are required to distinguish mutant alleles from sequencing and base calling errors in bulk tissue samples. An alternative way to study somatic mutations, especially those present in an extremely small number of cells or even in a single cell, is to sequence single-cell genomes after whole-genome amplification (WGA); however, it is critical and technically challenging to exclude numerous technical artifacts arising during error-prone and uneven genome amplification in current WGA methods. To address these challenges, multiple bioinformatic tools have been developed. In this review, we summarize the latest progress in methods for identification of somatic mutations and the challenges that remain to be addressed in the future.