Deletion of Topoisomerase 1 in excitatory neurons causes genomic instability and early onset neurodegeneration

Abstract

Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD +) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.

NuMorph: tools for cellular phenotyping in tissue cleared whole brain images

“…A previous study demonstrated that deletion of Top1 in postmitotic excitatory neurons within the cortex and hippocampus results in massive neurodegeneration in these structures by postnatal day 15 (P15) (Fragola et al, 2020). Interestingly, while all cortical layers were affected by Top1 deletion, the lower cortical layers (Layers 5-6) showed a noticeably greater reduction in thickness and cell count compared to the upper cortical layers (Layers 2-4) (Fragola et al, 2020). These observations however were limited to the somatosensory cortex, which itself is a large structure that can be further decomposed into multiple functional regions.…”

“…We processed one brain hemisphere from four wild-type (WT) and four Top1 cKO mice at P15 -when the Top1 cKO had displayed large, bilateral deficits in brain structure (Fragola et al, 2020). We labeled layer-specific cell-types using antibodies for Cux1 (upper layer neuron marker) and Ctip2 (lower layer neuron marker) in addition to staining all cell nuclei with TO-PRO-3 (TP3) during iDISCO+ processing.…”

“…Top1 conditional knockout mice ( Top1 fl/fl ;Neurod6 -Cre, Top1 cKO) were bred by crossing Top1 fl/fl mice (Mabb et al, 2016) with the Neurod6 -Cre mouse line (Jackson Laboratory) (Goebbels et al, 2006) as described previously (Fragola et al, 2020). Cre-negative mice ( Top1 fl/fl ) were used as controls (WT).…”

“…The mean and standard deviation of all correlation coefficients in each bin (<100kb or >100kb) was used to compare correlation coefficients between bins (Welch’s t-test). A list of differentially expressed genes in Top1 cKO cortex as measured by scRNA-seq was acquired from (Fragola et al, 2020) for additional comparisons.…”

“…To address these issues, we developed a group of image analysis tools called NuMorph (Nuclear-Based Morphometry) (available here: https://bitbucket.org/steinlabunc/numorph/) for end-to-end processing to perform cell-type quantification within the mouse cortex after tissue clearing and imaging by a conventional light-sheet microscope. To demonstrate the effectiveness of the tool, we first applied and evaluated NuMorph to quantify structural changes in a mouse model with large differences in cortical structure, a topoisomerase I ( Top1 ) conditional knockout ( Top1 cKO) mouse model that exhibits clear reductions in both cortical size and specific cell types (Fragola et al, 2020). We then apply NuMorph to investigate a neurofibromin I ( Nf1 ) conditional knockout ( Nf1 cKO) model, a gene harboring mutations in individuals with Neurofibromitosis type I.…”