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

Abstract

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.

Keywords: Alzheimer’s disease; Apolipoprotein E; Cancer; Chemotherapy; Cognitive impairment; Mouse behaviour.

Figure 1:. Doxorubicin treatment does not alter locomotive and anxiety behaviours.

A. Timeline of doxorubicin or control exposure, behavioural assessments, and euthanasia. B-E, Results of Open Field and Elevated Zero behavioural assessments; “pre-treatment” time point two weeks prior to doxorubicin or control exposure, “post 1” two weeks following completion of treatment, “post 2” 15 weeks following completion of treatment. B. Distance traveled in a 5 minute Open Field exploration task. C. Time spent in the Inner Zone of the Open Field. Distance traveled in a 5 minute Elevated Zero exploration task. D. Distance traveled in a 5 minute Elevated Zero exploration task. E. Time spent in the Open Zone of the Elevated Zero apparatus. Three-way ANOVA with Tukey’s multiple comparisons test to assess differences between time points followed by two-way ANOVA with Sidak’s multiple comparisons test to assess interactions of time point and genotype, * p <.05, ** p<.01.

Figure 2:. Doxorubicin treatment does not alter Pre-Pulse Inhibition (PPI).

A. Prior to treatment, percent inhibition of startle response to a short tone following a pre-pulse of 3, 6, 9, or 12 decibels (dB) above background noise (PP3 – PP12) compared to startle with no pre-pulse. B. Following treatment, percent inhibition of startle response to a tone following a pre-pulse 3 decibels above background noise compared to startle with no pre-pulse. One way ANOVA with Tukey’s multiple comparisons test **** p<.0001

Figure 3:. Doxorubicin treatment impairs spatial learning in APOE4 but not APOE3 mice.

A. Average speed of locomotion on apparatus during Barnes Maze habituation task. B. Latency to full entry to escape hole by day, average of four trials per day for each of four consecutive days. C. Latency to first approach to escape hole by day, average of four trials per day for each of four consecutive days. D. Delay to complete escape after first approach to escape hole, average of four trials per day for each of four consecutive days (excludes all mice that did not make an approach to the escape hole during the trial). Three-way ANOVA with Tukey’s multiple comparisons test: &, APOE3 Ctrl vs. APOE3 Doxo; +, APOE3 Ctrl vs. APOE4 Ctrl; #, APOE3 Ctrl vs. APOE4 Doxo; $, APOE3 Doxo vs. APOE4 Ctrl; %, APOE3 Doxo vs. APOE4 Doxo; *, APOE4 Ctrl vs. APOE4 Doxo; @, All APOE3 vs. all APOE4. * p<.05, ** p<.01, ***p<.001, ****p<.0001.

Figure 4:. Doxorubicin impairs spatial memory.

A. Time until total escape from Barnes Maze, single trial conducted 72 hours following the final training trial. ROUT removal of outliers Q = 1% followed by two-way ANOVA. *** p<.001.

Figure 5:. Doxorubicin does not impair conditioned or cued memory in APOE3 or APOE4 mice.

A. Percent time spent freezing during the second of two training shocks, during tone cue in cue test, and in the conditioned environment. B. Percent time spent freezing during the five minute cue test, comparison of pre-tone, tone, and post-tone periods. Mixed-effects analysis with Tukey’s multiple comparisons test to evaluate freezing behaviour in different tasks. Three-way ANOVA with Tukey’s multiple comparisons test to assess differences between time points followed by two-way ANOVA with Sidak’s multiple comparisons test to assess genotype effects in cue test. ** p<.01, **** p<.0001.

Figure 6:. Long-term effects of doxorubicin on the brain structure of APOE mice are modest.

Nine months after treatment, 3-D MRI and VMB were performed on control and doxorubicin-treated APOE3 and APOE4 mice. A and C show maximum intensity projections (MIP) and the design of the matrix for the study. B and D depict color overlays of the t-test values on the co-registered template image and the location of significant clusters in this comparison. The color bar measures t test values of the statistical analyses. A & B. VBM analysis comparing the grey matter of untreated versus doxorubicin-treated APOE4 mice revealed a mild atrophy predominantly in the frontal cortex (colored areas). APOE3 mice did not show any long-term brain regional differences in response to doxorubicin (data not shown). C & D. The comparison of all (both treated and untreated) APOE3 mice versus all APOE4 mice, detected regions of grey matter that were significantly larger in APOE3 mice than in APOE4 mice, regardless of the treatment.

Figure 7:. Immunohistochemical staining for pathogenic markers of AD.

Coronal sections across the anterior/posterior brain regions were stained from 3–5 brains of APOE3 and APOE4 mice treated with control (con) or doxorubicin (dox). Images show staining of frontal cortex (ctx) and hippocampus (hip): Hematoxylin and eosin staining (H&E), IBA-1, GFAP, phosphorylated tau. Images were taken using brightfield microscopy at 20× magnification.