Genotype Load Modulates Amyloid Burden and Anxiety-Like Patterns in Male 3xTg-AD Survivors despite Similar Neuro-Immunoendocrine, Synaptic and Cognitive Impairments

Abstract

The wide heterogeneity and complexity of Alzheimer's disease (AD) patients' clinical profiles and increased mortality highlight the relevance of personalized-based interventions and the need for end-of-life/survival predictors. At the translational level, studying genetic and age interactions in a context of different levels of expression of AD-genetic-load can help to understand this heterogeneity better. In the present report, a singular cohort of long-lived (19-month-old survivors) heterozygous and homozygous male 3xTg-AD mice were studied to determine whether their AD-genotype load can modulate the brain and peripheral pathological burden, behavioral phenotypes, and neuro-immunoendocrine status, compared to age-matched non-transgenic controls. The results indicated increased amyloid precursor protein (APP) levels in a genetic-load-dependent manner but convergent synaptophysin and choline acetyltransferase brain levels. Cognitive impairment and HPA-axis hyperactivation were salient traits in both 3xTg-AD survivor groups. In contrast, genetic load elicited different anxiety-like profiles, with hypoactive homozygous, while heterozygous resembled controls in some traits and risk assessment. Complex neuro-immunoendocrine crosstalk was also observed. Bodyweight loss and splenic, renal, and hepatic histopathological injury scores provided evidence of the systemic features of AD, despite similar peripheral organs' oxidative stress. The present study provides an interesting translational scenario to study further genetic-load and age-dependent vulnerability/compensatory mechanisms in Alzheimer's disease.

Keywords: Alzheimer’s disease; BPSD; NPS; end-of-life; frailty; genetic load; heterogeneity; neuro-immunoendocrine crosstalk; survival.

Figure 1

Graphical abstract. Genotype load modulates amyloid burden and anxiety-like patterns in male 3xTg-AD survivors despite similar neuro-immunoendocrine and cognitive impairments. Experimental design and main findings. White square: NTg mice, yellow square: heterozygous 3xTg-AD mice, blue square: homozygous 3xTg-AD mice; Green square: 3xTg-AD mice (both 3xTg-AD genotypes, since no genotype-load differences were found).

Figure 2

Survival, HPA axis endocrine status and physical health. (A) Survival; (B) Corticosterone levels; (C) Body weight and (D) spleen weight in 19-month-old mice. Results are expressed as the mean ± SEM. Male NTg, n = 8 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; Blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; Green bar, mean value of both 3xTg-AD genotypes, since no genotype-load differences were found). Statistics: two-tailed unpaired Student’s t-test (above the line) for Genotype differences (G): * p < 0.05; one-way analysis of variance (ANOVA) for comparisons between all the groups of mice followed by Bonferroni’s post-hoc test. * p < 0.05 vs. homozygous 3xTg-AD-group; # p < 0.05 vs. heterozygous 3xTg-AD-group.

Figure 3

Neuropathology and Synaptic Function: Brain biochemical analysis of APP (A), synaptophysin (B) and ChAT (C) in the prefrontal cortex, hippocampus, and entorhinal cortex. (D) Western blot images. Results are expressed as the relative levels as fold change ± SEM. Male NTg, n = 3–4 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; yellow and blue bars, mean value for heterozygous and homozygous 3xTg-AD genotypes, respectively, since genotype-load differences were found). Statistics: two-tailed unpaired Student’s t-test (above the line) for Genotype differences (G): ** p < 0.01; one-way analysis of variance (ANOVA) for comparisons between all the groups of mice followed by Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. homozygous 3xTg-AD-group; # p < 0.05 vs. heterozygous 3xTg-AD-group.

Figure 4

Mental Health: Neuropsychiatric-like phenotype in 2-day Open-field test. (A) Exploratory activity; (B) Ethogram day 2; (C) Time in periphery day 2 and (D) Heat maps representation of how much time animals spends in different parts of the apparatus during a test, with blue as the shortest time and red as the longest. Results are expressed as the mean ± SEM. Male NTg, n = 8 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; green bar, mean value of both 3xTg-AD genotypes, since no genotype-load differences were found; yellow and blue bars, mean value for heterozygous and homozygous 3xTg-AD genotypes, respectively, since genotype-load differences were found). Statistics: one-way analysis of variance (ANOVA) for comparisons between all the groups of mice followed by Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. homozygous 3xTg-AD-group.

Figure 5

Mental Health: Neuropsychiatric-like phenotype and cognitive impairment in Dark and light box test (A) and T-Maze test (B). Results are expressed as the mean ± SEM. Male NTg, n = 8 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; yellow and blue bars, mean value for heterozygous and homozygous 3xTg-AD genotypes, respectively, since genotype-load differences were found). Statistics: one-way analysis of variance (ANOVA) for comparisons between all the groups of mice followed by Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, vs. homozygous 3xTg-AD-group.

Figure 6

Mental Health: Cognitive impairment in Morris Water Maze (A) Long-term learning and memory; (B) Short- and long-term memory; (C) Short-term memory in Removal: Time in target quadrant; (D) Heat maps representation in removal. (E) Short-term memory in Removal: Latency to reach the platform; (F) Time immobile in removal; (G) Immobile episodes in removal. Results are expressed as the mean ± SEM. Male NTg, n = 8 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; green bar, mean value of both 3xTg-AD genotypes, since no genotype-load differences were found; yellow and blue bars, mean value for heterozygous and homozygous 3xTg-AD genotypes, respectively, since genotype-load differences were found). Heat maps representation of how much time animals spends in different parts of the apparatus during a test, with blue as the shortest time and red as the longest. Statistics: two-tailed unpaired Student’s t-test (above line) for Genotype differences (G): * p < 0.05, ** p < 0.01.

Figure 7

Pathological Status of Peripheral Organs in 19-month-old male 3xTg-AD mice and NTg counterparts: (A) Representative images of histopathological evaluation of peripheral organs (kidney, spleen, and liver), Black arrow: amyloidosis damage, *: proteinuria; (B) Incidence and degree of tissue damage; (C) General systemic total score. Results are expressed as the mean ± SEM. Male NTg, n = 8 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; Blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; Green bar, mean value of both 3xTg-AD genotypes, since no genotype-load differences were found). Statistics: two-tailed unpaired Student’s t-test (above line) for Genotype differences (G): * p < 0.05.

Figure 8

Pathological Status of Peripheral Organs: Oxidative stress parameters in peripheral organs: (A) Total Glutathione (GSH); (B) Glutathione Peroxidase (GPx); (C) Glutathione Reductase (GR) in 19-month-old male 3xTg-AD mice and NTg counterparts. Results are expressed as the mean ± SEM. Male NTg, n = 8 (White circles for individual values; white bar, mean value); male 3xTg-AD n = 9 (Yellow circles, individual values of heterozygous 3xTg-AD +/− mice, n = 5; Blue circles, individual values for homozygous 3xTg-AD +/+ mice, n = 4; Green bar, mean value of both 3xTg-AD genotypes, since no genotype-load differences were found). Statistics: n.s., no significative, p > 0.05.