Recurrent patterns of widespread neuronal genomic damage shared by major neurodegenerative disorders

Abstract

Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) are common neurodegenerative disorders for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 429 neurons from three C9ORF72 ALS, six C9ORF72 FTD, seven AD, and twenty-three neurotypical control brains revealed significantly increased burdens in somatic single nucleotide variant (sSNV) and insertion/deletion (sIndel) in all three disease conditions. Mutational signature analysis identified a disease-associated sSNV signature suggestive of oxidative damage and an sIndel process, affecting 28% of ALS, 79% of FTD, and 65% of AD neurons but only 5% of control neurons (diseased vs. control: OR=31.20, p = 2.35×10^-10). Disease-associated sIndels were primarily two-basepair deletions resembling signature ID4, which was previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Duplex sequencing confirmed the presence of sIndels and identified similar single-strand events as potential precursor lesions. TOP1-associated sIndel mutagenesis and resulting genome instability may thus represent a common mechanism of neurodegeneration.

Fig. 1.. Experimental strategy and characterization of isolated neuronal nuclei from C9ORF72 ALS and FTD brains.

(A) Overview of the experimental design. Neuronal nuclei with and without nuclear TDP-43 depletion were isolated from 3 C9ORF72 ALS and 6 C9ORF72 FTD brains, then subjected to genome sequencing and analysis. These were compared with neuronal nuclei from neurotypical brains, including newly generated data and previously published datasets. Previously generated scWGS data from AD brains were included. (B) Representative FACS plots showing neuronal nuclei from a C9ORF72 ALS brain and a neurotypical control brain co-stained with NeuN and TDP-43 antibodies. The P5 gate (right, circle) indicates neuronal nuclei with depletion of nuclear TDP-43. (C) UMAP clustering of snRNA-seq data from neuronal nuclei, colored by major cell types. TDP-43− neuronal nuclei come exclusively from C9ORF72 ALS and FTD brains, while TDP-43+ neuronal nuclei come from both diseased and neurotypical brains; control indicates neurotypical brains. IT: intratelencephalic neurons. CBT: corticobulbar tract neurons. VEN: Von Economo neurons. CT: corticothalamic neurons. PV: parvalbumin. IN: inhibitory neurons. Ch IN: cholinergic inhibitory neurons. OL: oligodendrocytes. OPC: oligodendrocyte precursor cells. Red dashed circles indicate reduced inhibitory neurons under TDP-43− condition (D) Heatmap of cryptic exonization showing the proportion of transcripts with cryptic exons in neurons across different conditions. (E) IGV Genome browser tracks of STMN2 showing snRNA-seq read coverage and splice junctions. TDP-43− neurons (red) contain a marked increase in cryptic exon inclusion (highlighted region), which is absent or low in TDP-43+ (orange) and control (black) neurons. Numbers next to splice junctions are read counts supporting the junction. PFC: prefrontal cortex. preMC: premotor cortex.

Fig. 2.. Increased somatic mutation burdens in neurons from C9ORF72 ALS, C9ORF72 FTD and AD.

(A) PFC (prefrontal cortex) and preMC (premotor cortex) neurons from neurotypical brains show comparable burdens of sSNVs and sIndels. Each point represents the burden of one neuron, compared against the expected burden for its age (Methods). (B) Extrapolated genome-wide sSNV and sIndel burdens for neurons from C9ORF72 ALS, C9ORF72 FTD and AD brains compared to those for neurons from neurotypical brains as a function of age. Identical control neuron points and regression (mixed-effects linear regression, Methods) are shown in gray in each panel for comparison. (C) Mutation burdens corrected for age (Methods); preMC and PFC neurons are combined to form the control set. (D) TDP-43+ and TDP-43− neurons from C9ORF72 ALS and FTD brains show similar levels of sSNVs and sIndels. All P-values in this figure represent Wilcoxon rank-sum tests.

Fig. 3.. Neurons from C9ORF72 ALS, C9ORF72 FTD and AD brains share an indel mutational signature.

(A) Distribution of sIndel lengths across conditions. Diseased neurons have higher levels of 2-bp deletions. (B) Fraction of 2 bp deletions in neurons and oligodendrocytes across conditions. All P-values in this figure represent Wilcoxon rank-sum tests. (C) Both de novo sIndel signatures identified from joint analysis of diseased and neurotypical control neurons amplified by PTA, as well as previously published neurons and oligodendrocytes amplified using an older amplification technology (MDA). COSMIC ID4 is a signature previously identified in cancer and normal aging neurons. Top part illustrates the disease-associated 2-bp deletions in ID-A signature. (D) Burdens of ID-A and ID-B sIndels; P-values are Wilcoxon rank-sum tests. (E) Fraction of cells with high levels of ID-A sIndels in neurons and oligodendrocytes across conditions, determined by a classifier (Methods); P-values represent Fisher tests of independence between disease status and ID-A-high classifications. (F) sIndel spectra of neurons and oligodendrocytes stratified by ID-A-high and -normal classifications. Oligo: oligodendrocyte.

Fig. 4.. ID-A-like deletions reflect characteristics of TOP1-mediated ribonucleotide excision.

(A) Model of TOP1-mediated ribonucleotide excision repair. (B) Rates of 2-bp deletions by the deleted dinucleotide. (C) DNA motifs of 2-bp deletion sites; positions 5 and 6 are the deleted nucleotides. (D) Enrichment of sIndels in relation to local gene expression levels from previously published snRNA-seq data of excitatory neurons. Enrichment tests divide the genome into 10 roughly equally-sized, non-contiguous regions ranked by gene expression. I.e., decile 10 represents the subset of the genome containing the top 10% of expressed genes. Solid lines: enrichment analysis of ID-A-like sIndels (see Fig. 3A), dotted lines: all other sIndels. P-values are enrichment tests (Methods) indicating that the observed/expected ratio significantly differs from 1. (E) Agarose gel electrophoresis of denatured gDNA from prefrontal cortex tissues, ordered by age. Smears on the gel indicate single-strand breaks. Red rectangles indicate cases with high ID-4 like sIndels (Methods). (F) Abundance of single-strand breaks in prefrontal cortex tissues. Y-axis values are quantifications of the smears in (E). Red circles indicate brains for which most scWGS neurons were classified as ID-A-high. (G) Indel spectra of excess (subtracting sIndels of age-matched controls) double-strand and single-strand sIndels in neurons from C9ORF72 FTD cases compared to neurons from neurotypical controls. Mini-bulk samples (50 neurons per case) were sequenced using META-CS. (H) Fraction of 2-bp deletions among single-strand lesions and double-strand sIndels (numbers of 2-bp deletions divided by total numbers of Indels). P-values are two-tailed unpaired t-test.

Fig. 5.. Potential mechanisms underlying the dysregulation of ribonucleotide excision repair.

(A) Model of TOP1-mediated mutagenesis in neurons from neurodegenerative conditions. RNR: ribonucleotide reductase. (B) Expression levels of RNASEH2B and RRM2B in neurons measured by qPCR. P-values are two-tailed unpaired t-test. (C) Correlation between burdens of de novo signatures SBS-B sSNVs and ID-A sIndels. SBS-B resembles the COSMIC SBS30 signature, which is associated with a deficiency in NTHL1 and base excision repair of oxidative DNA damage repair. P-values indicate regression t-tests on slope.