Adolescent Binge Alcohol Enhances Early Alzheimer's Disease Pathology in Adulthood Through Proinflammatory Neuroimmune Activation

Abstract

Epidemiological studies suggest that heavy alcohol use early in life is associated with increased risk for Alzheimer's disease (AD). However, mechanisms connecting AD with alcohol use have not been identified. Both heavy alcohol use and AD feature increased proinflammatory signaling. Therefore, we hypothesized that adolescent binge ethanol would increase AD molecular and behavioral pathology in adulthood through proinflammatory signaling. The 3xTg-AD mouse model (APPSwe, tauP301, Psen1^tm1Mpm) which features amyloid (Aβ) and tau pathology beginning at 6-12 months underwent adolescent intermittent ethanol (AIE, 5 g/kg/d, i.g., P25-55) with assessment of AD pathologic mediators at P200. A second group of mice received AIE +/- minocycline (30 mg/kg/d, IP) followed by behavioral testing in adulthood. Behavioral testing and age of testing included: locomotor activity and exploration (27-28 weeks), novel object recognition (NORT, 28-30 weeks), 3-chamber sociability and social memory (29-31 weeks), prepulse inhibition (PPI, 30-32 weeks), Morris Water Maze with reversal (MWM, 31-35 weeks), and Piezo sleep monitoring (35-37 weeks). We found that AIE increased levels of neurotoxic Aβ_1-42 in adult female hippocampus as well as intraneuronal Aβ_1-42 in amygdala and entorhinal cortex. Phosphorylated tau at residue Thr181 (p-tau-181) was also increased in female hippocampus by AIE. Several proinflammatory genes were persistently increased by AIE in the female hippocampus, including IL-1β, MCP-1, IL-6, and IFNα. Expression of these genes was strongly correlated with the levels of Aβ_1-42 and p-tau-181 in hippocampus. AIE caused persistent decreases in locomotor activity (open-field and NORT habituation) and increased anxiety-like behavior (thigmotaxis) while reducing memory retention. Treatment with the anti-inflammatory compound minocycline during AIE blocked persistent increases in Aβ_1-42 in amygdala and p-tau-181 in hippocampus, and prevented AIE-induced thigmotaxis and memory loss. Together, these data find that adolescent binge ethanol enhances AD molecular and behavioral pathology in adulthood through proinflammatory signaling. Blockade of proinflammatory signaling during ethanol exposure prevents ethanol-induced effects on pathologic accumulation of AD-associated proteins and persistent behavior changes relevant to human AD.

Keywords: Alzheiemer’s disease; addiction; adolescence; alcohol; amyloid; neuroinflammation.

FIGURE 1

Experimental design and tissue allocation for both experiments. (A, B) Experiment 1. (A) Adolescent 3xTg male and female mice received either water gavage (i.g.) or AIE (5 g/kg/d, i.g.) from P25-55, in a 2-days on 2 days off pattern. Mice were then left without intervention until P200. (B) 16 mice were sacrificed by cardiac perfusion with 4% PFA for immunohistochemistry (Females: N = 5 control, 4 ethanol; Males: N = 3 control, 4 ethanol), with remaining mice perfused with PBS (Females: N = 6 control, 8 ethanol; Males: N = 6 control, 8 ethanol) and the cortex and hippocampus dissected and frozen in liquid nitrogen. One half of the cortex and hippocampus were prepared for western blot, while the other halves were prepared for RT-PCR. (C, D) Experiment 2. (C) Female mice received either water gavage + vehicle (i.p.) (N = 5), minocycline (Min, 30 mg/kg, i.p., N = 5), AIE (N = 9), or AIE + Min (N = 8) in the same schedule as Experiment 1, with behavioral testing beginning at P200. Mice were sacrificed at the end of the behavioral testing on P270. (D) Mice were sacrificed by cardiac perfusion with PBS and one hemisphere was drop-fixed in PFA for IHC and the other hemisphere dissected for cortex and hippocampus. Dissected cortex and hippocampus from one hemisphere were divided equally for western blot and RT-PCR analyses. (E) Body weights of male subjects across treatment and maturation into adulthood. (F) Body weights of female subjects across treatment and maturation into adulthood.

FIGURE 2

AIE increases amyloid pathology in adult female 3xTg-AD. 3xTg-AD mice received either AIE (5 g/kg/day, i.g., 2-days on 2-days off, P25-55) or water gavage and were assessed for amyloid pathology at P200. (A) RT-PCR for human amyloid precursor protein (APP) transgene at P200 found AIE did not significantly alter APP gene expression (p = 0.14). N = 6 control, 7 ethanol (B) Western blot found AIE caused a 42% increase in neurotoxic Aβ_1–42 protein in female hippocampus (*p < 0.05, Sidak’s post-test). (C) Representative image of immunoreactive (+IR) intraneuronal Aβ_1–42 staining in the subiculum of control and AIE-treated subjects at P200. (D) Quantification of intraneuronal Aβ_1–42 staining in subiculum revealed a 4.8-fold increase after AIE. **p < 0.01, t-test. N = 5 control, 4 ethanol (E) Representative image of intraneuronal Aβ_1–42 staining in the entorhinal cortex (ENT Cx) (F) Quantification of intraneuronal Aβ_1–42 staining in ENT cortex found a 56% increase after AIE. **p < 0.01 Sidak’s post-test. N = 5 control, 4 ethanol (G) Representative image of intraneuronal Aβ_1–42 staining in the amygdala (AMG) of control and AIE-treated subjects at P200, white dashed outline. (H) Quantification of intraneuronal Aβ_1–42 staining in AMG found a 92% (**p < 0.01) increase after AIE. *p < 0.05, **p < 0.01. N = 5 control, 4 ethanol.

FIGURE 3

AIE enhances tau pathology in adult female 3xTg-AD mice. 3xTg-AD mice received either AIE (5 g/kg/day, i.g., 2-days on 2-days off, P25-55) or water gavage and were assessed for tau pathology at P200. (A) Western blot of adult hippocampus found an 80% increase in phosphorylated tau at Thr181 (p-tau-181) after AIE. **p < 0.01, Sidak’s multiple comparisons test. N = 6 control, 6 ethanol (B) Representative high magnification image of p-tau-181 staining in CA1 of control and AIE-treated subjects at P200. (C) Quantification of p-tau-181 staining in female CA1 found a 2-fold, increase after AIE. **p < 0.01, t-test N = 4 control, 4 ethanol (D) Western blot of adult whole cortex showed no significant change in p-Tau-181 (p = 0.21, Sidak’s multiple comparisons test). N = 7 control, 6 ethanol. (E) Representative high magnification image of p-tau-181 staining in ENT Cx region of control and AIE-treated subjects at P200. (F) Quantification of p-tau-181 staining in the ENT Cx found a 51%, (**p < 0.01, Sidak’s post-test N = 5 control, 4 ethanol) increase by AIE. *p < 0.05, **p < 0.01.

FIGURE 4

AIE persistently induces inflammation in adult (P200) female brain that correlate with levels of Aβ and tau pathology. 3xTg-AD mice underwent either AIE (5 g/kg/day, i.g., 2-days on 2-days off, P25-55) or water gavage and hippocampus at P200 was assessed for proinflammatory and microglial genes by RT-PCR. (A) AIE persistently increased proinflammatory gene expression in IFNα, IL-6, IL-1β, MCP-1, TNFα, and TLR4 in the female hippocampus. 2-way ANOVA with Sidak’s post-test and FDR p-value correction, ***q < 0.001, ****q < 0.0001. N = 6 control, 7 ethanol. (B) AIE significantly altered the expression of microglial DAM genes Cst7, C3, Tmem119, and B2M in the female hippocampus at P200 relative to control. Mann-Whitney t-tests with FDR correction, **q < 0.01. N = 6 control, 7 ethanol. (C) Proinflammatory gene expression was positively correlated with levels of neurotoxic soluble Aβ_1–42 protein (western blot) in female hippocampus with Pearson R² values between 0.86 and 0.99. (D) Proinflammatory gene expression was positively correlated with p-tau-181 protein levels (western blot) in female hippocampus with Pearson R² values between R² = 0.69 and R² = 0.87 with p values **< 0.01, ***< 0.001, ****< 0.0001.

FIGURE 5

AIE worsens AD-associated activity and memory deficits in adult female 3xTg-AD mice (P200) with prevention by minocycline. (A, B) Locomotor activity AIE caused a reduction in locomotor activity in adulthood as measured by (A) 1 h in the open field that was blunted by minocycline (63% reduction, 1-way ANOVA treatment effect F_3,24 = 3.353, p = 0.036, *p < 0.05, Sidak’s multiple comparison test), and (B) 10 min in the open field during habituation for the novel object recognition test (NORT) that was prevented by minocycline; 60% reduction, F_3,24 = 7.366, p = 0.001, **p < 0.01, ***p < 0.001 Sidak’s post-test. (C) Center time in the open field. AIE reduced time spent in the center of an open field with prevention by minocycline as measured by 10 min in the open field during habituation for the NORT; 82% reduction, F_3,24 = 3.9, p = 0.02, *p < 0.01 Sidak’s test. (D, E) Novel Object Recognition Test (NORT). (D) A trend toward an increase in time sniffing the novel object was seen in the AIE treatment group, p = 0.07. (E) No significant differences between groups were observed in time sniffing the familiar object during the NORT. (F, H) Morris Water Maze (MWM) learning and retention (F) AIE had no effect on spatial learning in the MWM. AIE, however, caused an impaired memory retention that was prevented by minocycline as measured during the probe trial with (G) less time spent in the correct quadrant; 33% reduction, F_3,23 = 4.7, p = 0.01, *p < 0.05 Sidak’s test, and (H) fewer swim path crosses over the target location. 1-way ANOVA treatment effect *p < 0.05, **p < 0.01. N = 6 Control, 5 Mino, 8 AIE, 8 AIE + Mino.

FIGURE 6

Minocycline reduces AD pathology caused by AIE. 3xTg-AD female mice received either AIE (5 g/kg/day, i.g., 2-days on 2-days off, P25-55) +/− minocycline (30 mg/kg/d) or water gavage. Mice were assessed for amyloid and tau pathology after behavioral testing in adulthood (P270) by IHC. (A) Representative images of intraneuronal Aβ_1–42 staining in the amygdala (AMG, white dashed outline). (B) Quantification of intraneuronal Aβ_1–42 in AMG found a 67% increase by AIE and that was blocked by minocycline. ANOVA F_3,15 = 15.68, p < 0.0001, Sidak’s multiple comparison test. N = 5 control, N = 3 control + Mino, N = 6 AIE, N = 7 AIE + mino (C) Representative images of p-tau-181 in the CA1 region of hippocampus. AIE increased staining in cell bodies and dendrites of the CA1. (D) Quantification of a significant main effect of treatment (F_3,18 = 31.60, p < 0.0001, Sidak’s post-test, N = 5 control, N = 3 control + Mino, N = 8 AIE, N = 6 AIE + mino) with a ∼2-fold increase in p-tau-181 by AIE was blocked by minocycline. (E) A negative correlation between memory retention and hippocampal p-tau IHC was found. R = −0.43, *p < 0.05. (F) A negative correlation between target path crosses in the MWM probe trial and hippocampal p-tau-181 immunoreactivity was found. R = −0.46, *p < 0.04.*p < 0.05, **p < 0.01 ***p < 0.001, ***p < 0.0001.