Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration [BLISS]

Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's Disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human post-mortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases. Overall design: BLISS was performed as previously described in Yan et al., 2017. Sorted neurons were attached to a MatTek 35 mm Dish | No. 1.0 Coverslip | 14 mm Glass Diameter | Poly-D-Lysine Coated (P35GC-1.0-14-C) by adding sorted cells to the center coverslip in 4% formaldehyde in PBS and spinning at 2000xg for 2 minutes. The attached nuclei were then end-repaired and A-tailed. Next BLISS adaptors were ligated overnight at 16°C. Excess BLISS adaptors were washed off and genomic DNA was isolated. Next DNA was sonicated to an average size of 500 bp and in-vitro transcribed. Transcribed RNA was isolated, and reverse transcribed to cDNA to prepare Illumina sequencing libraries. Custom BLISS adaptors containing RA3 and RA5 sequences were amplified using RP1 and selected RPI Illumina primers as described in the Illumina TruSeq Small RNA Library Preparation Kit. Illumina libraries were sequenced in 75 bp single-end mode. FASTQ files were demultiplexed based on sample barcodes. PCR duplicates were removed based on the UMI. Reads after removing PCR duplicates were mapped to mm10 genome build using Bowtie2. Mapped sam files were converted to bam files using samtools view and to bed files using bedtools bamtobed.