The chemotherapeutic agent doxorubicin induces brain senescence, with modulation by APOE genotype.

Many cancer patients experience serious cognitive problems related to their treatment, which can greatly affect their quality of life. The molecular mechanisms of this cancer chemotherapy-induced cognitive impairment (CICI) are unknown, thus slowing the development of preventative approaches. We hypothesized that cancer chemotherapies could induce cellular senescence in the brain, creating a pro-inflammatory environment and damaging normal brain communication. We tested this hypothesis using the common chemotherapeutic agent doxorubicin in two independent mouse models. In the first model, we used mice that express tdTomato under the pdkn2a (p16) promoter; p16 is a regulator of cellular senescence, and its upregulation is denoted by the presence of fluorescently tagged cells. Two weeks after exposure to three doses of 5 mg/kg doxorubicin, the number of tdTomato positive cells were increased nearly three-fold in both the cerebral cortex and the hippocampus. tdTomato staining co-localized with neurons, microglia, oligodendrocyte precursor cells, and endothelial cells, but not astrocytes. In the second model, we used APOE knock-in mice, since the APOE4 allele is a risk factor for CICI in humans and mouse models. We isolated RNA from the cerebral cortex of APOE3 and APOE4 mice from one to 21 days after a single dose of 10 mg/kg doxorubicin. Using NanoString analysis of over 700 genes related to neuroinflammation and RT-qPCR analysis of cerebral cortex transcripts, we found two-fold induction of four senescence-related genes at three weeks in the APOE4 mice compared to the APOE3 control mice: p21(cdkn1a), p16, Gadd45a, and Egr1. We conclude that doxorubicin promotes cellular senescence pathways in the brain, supporting the hypothesis that drugs to eliminate senescent cells could be useful in preventing CICI.

A mouse model of chemotherapy-related cognitive impairments integrating the risk factors of aging and APOE4 genotype.

Some cancer survivors experience marked cognitive impairment, referred to as cancer-related cognitive impairment (CRCI). CRCI has been linked to the genetic factor APOE4, the strongest genetic risk factor for Alzheimer's disease (AD). We used APOE knock-in mice to test whether the relationship between APOE4 and CRCI can be demonstrated in a mouse model, to identify associations of chemotherapy with behavioural and structural correlates of cognition, and to test whether chemotherapy affects markers of AD. Twelve-month old C57BL/6 J female APOE3 (n = 30) and APOE4 (n = 31) knock-in mice were randomized to treatment with either doxorubicin (10 mg/kg) or saline. Behavioural assays at 2-21 weeks-post exposure included open field maze, elevated zero maze, pre-pulse inhibition, Barnes maze, and fear conditioning. Ex-vivo magnetic resonance imaging was used to determine regional volume differences at 31-35 weeks-post exposure, and tissue sections were analyzed for markers of AD pathogenesis. Minimal toxicities were observed in the aged mice after doxorubicin exposure. In the Barnes maze assay, APOE3 mice did not exhibit impairment in spatial learning after doxorubicin treatment, but APOE4 mice demonstrated significant impairments in both the initial identification of the escape hole and the latency to full escape at 6 weeks post-exposure. Both APOE3 and APOE4 mice treated with doxorubicin showed impairment of spatial memory. Grey matter volume in the frontal cortex decreased in APOE4 mice treated with doxorubicin vs. APOE3 mice. This study demonstrates cognitive impairments in aged APOE4 knock-in mice after doxorubicin treatment and establishes this system as a novel and powerful model of CRCI.

Development of a Human APOE Knock-in Mouse Model for Study of Cognitive Function After Cancer Chemotherapy.

Cancer-related cognitive impairment in breast cancer patients exposed to multi-agent chemotherapy regimens is associated with the apolipoprotein E4 (APOE4) allele. However, it is difficult to determine the effects of specific agents on cognitive impairment in human studies. We describe the development of a human APOE knock-in congenic C57BL/6J mouse model to study cancer-related cognitive impairment. Female APOE3 and APOE4 homozygous mice were either left untreated or treated with the most commonly used breast cancer therapeutic agent, doxorubicin. APOE3 and APOE4 mice had similar behaviors in exploratory and anxiety assays, which were affected transiently by doxorubicin treatment. Spatial learning and memory were measured in a Barnes maze: after 4 days of training, control APOE3 and APOE4 mice were able to escape with similar latencies. In contrast, doxorubicin-treated APOE4 mice had markedly impaired learning compared to doxorubicin-treated APOE3 mice at all time points. Voxel-based morphometry of magnetic resonance images revealed that doxorubicin treatment caused significant changes in the cortex and hippocampus of in both APOE3 and APOE4 mouse brains, but the differences were significantly greater in the APOE4 brains. The results indicate that doxorubicin-exposed APOE4 mice recapitulate key aspects of human cancer-related cognitive impairment. These data support the usefulness of this novel preclinical model for future elucidation of the genetic and molecular interactions of APOE genotype with chemotherapy; this model can also allow extension to prospective studies of older mice to study these interactions in the context of aging.