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. 2026 Mar 23;44(2):14. doi: 10.1007/s12640-026-00794-2

Decreased Length of Locus Coeruleus Norepinephrine Axons and Increased Amyloid Beta Pathology in Male APP/PS1 Mice During Protracted Abstinence From Alcohol

Ivy J Z Garland 1, Shaydel Engel 1, Matthew Scalf 1, Nichole R Payne 1, Anna M Lee 1, Steven M Graves 1,✉
PMCID: PMC13009128  PMID: 41870711

Abstract

Alzheimer’s disease (AD) is the leading cause of dementia and evidence suggests that alcohol, the most commonly used addictive substance, may increase AD risk. Locus coeruleus (LC) neurons are the primary source of norepinephrine in the brain and these neurons degenerate early in AD. In rodent models, lesioning the LC increases amyloid beta (Aβ) pathology suggesting that LC integrity and norepinephrine signaling obstruct Aβ pathogenesis. We recently reported a decrease in the number of LC norepinephrine neurons and increased Aβ pathology when measured after protracted abstinence from chronic intermittent alcohol consumption in female APP/PS1 mice. Clinically, female subjects are at a higher risk for AD; additionally, female mice consume more alcohol than male mice making it unclear as to whether alcohol consumption would produce similar adverse outcomes in male subjects. To address this gap, male APP/PS1 and non-transgenic mice underwent chronic intermittent access (IA) to alcohol followed by protracted abstinence with water drinking controls run in parallel, consistent with our prior study. In contrast to our previous results with female mice, the number of LC norepinephrine neurons was unchanged in male APP/PS1 mice that had IA to alcohol; however, the length of LC axons was decreased and Aβ pathology was increased in male APP/PS1 mice that consumed alcohol. These data demonstrate that alcohol consumption during early adulthood results in negative consequences in male APP/PS1 mice, although the effect may not be as severe as previously observed in female mice.

Keywords: Alcohol, Amyloid beta, Male, Locus coeruleus, Motor cortex

Introduction

The leading cause of dementia and the most common neurodegenerative disease is Alzheimer’s disease (AD) (Alzheimer's disease 2024; Collaborators 2024; Gustavsson et al. 2023) which is characterized by pathological hallmarks including aggregates of amyloid beta (Aβ) and neurofibrillary tangles (DeTure and Dickson 2019; Tiwari et al. 2019). In the classic trajectory of AD, pathology begins to develop during a prodromal phase during which Aβ accumulates resulting in extracellular plaques, followed by the formation of intracellular neurofibrillary tangles, and the eventual emergence of clinical symptoms (Long and Holtzman 2019). Locus coeruleus (LC) neurons are the primary source of norepinephrine in the brain and LC neurodegeneration becomes evident during early stages of AD (Grudzien et al. 2007; Kelly et al. 2017). Loss of LC neurons and norepinephrine signaling appears to be detrimental in AD progression. Evidence suggests that norepinephrine signaling may be neuroprotective by preventing amyloid toxicity (Counts and Mufson 2010; Liu et al. 2015), whereas lesioning the LC in AD mouse models increases Aβ pathology and neuroinflammation (Chalermpalanupap et al. 2018; Heneka et al. 2002, 2010, 2006; Jardanhazi-Kurutz et al. 2010, 2011; Kalinin et al. 2007).

The most widely used substance of abuse is alcohol (Substance Abuse and Mental Health Services Administration 2024) which can also have deleterious effects on LC neurons in humans (Arango et al. 1994) and rats (Jaatinen et al. 2003; Kjellstrom et al. 1993; Lu et al. 1997; Rintala et al. 1998). We recently demonstrated LC degeneration after protracted abstinence from chronic intermittent alcohol consumption in female APP/PS1 mice (Engel et al. 2025), a transgenic mouse model of AD that develops progressive Aβ pathology and associated neurodegeneration (Holcomb et al. 1998; Jankowsky et al. 2004, 2001; Liu et al. 2008). The degeneration of LC neurons observed in female APP/PS1 mice was also associated with an increase in Aβ pathology (Engel et al. 2025). These data provide evidence suggesting that alcohol consumption during early adulthood increases the vulnerability of LC neurons to degeneration which may contribute to exacerbated Aβ, at least in female APP/PS1 mice. There is a difference of AD prevalence between sexes with women being at a higher risk than men (Beam et al. 2018; Gong et al. 2023; Mosconi et al. 2017; O'Neal 2023; Snyder et al. 2016). Similarly, female APP/PS1 mice have elevated Aβ (Jiao et al. 2016; Li et al. 2016; Mifflin et al. 2021; Wang et al. 2003) as well as more pronounced neurodegeneration (Jiao et al. 2016) compared to male APP/PS1 mice. Female mice also consume greater amounts of alcohol than male mice (DeBaker et al. 2020; Downs et al. 2023; Hwa et al. 2011; O'Rourke et al. 2016; Sneddon et al. 2019). In light of these sex differences relating to AD and alcohol, it is unclear whether alcohol consumption during early adulthood similarly impacts male subjects.

The goal of the present study was to determine the impact of chronic intermittent alcohol consumption during early adulthood on LC neurodegeneration and Aβ pathology in male mice. To do so, transgenic APP/PS1 and non-transgenic (non-Tg) male mice underwent 8 weeks of intermittent access (IA) to alcohol during early adulthood followed by 23 weeks of abstinence with water drinking control mice run in parallel, as in our prior study (Engel et al. 2025). Stereological analyses were used to quantify the axon length of LC norepinephrine neurons, the total number of LC norepinephrine neurons, and amount of Aβ pathology. In our prior study with female APP/PS1 mice, we present evidence of both axonal and somatic loss of LC norepinephrine neurons and a near doubling of Aβ pathology (Engel et al. 2025). In contrast, the current study provides evidence of axonal but not somatic loss in male APP/PS1 mice that had consumed alcohol and a more modest (~ 30%) increase in Aβ pathology. These outcomes suggest that alcohol consumption during early adulthood does have adverse long-term consequences relating to AD in male mice, however, the impact does not appear to be as severe as reported in female APP/PS1 mice (Engel et al. 2025).

Materials and Methods

Experimental Subjects

Experimental protocols and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the University of Minnesota Institutional Animal Care and Use Committee. Subjects were provided free access to food (Teklad Irradiated Global 18% Protein Rodent Diet (#2918)) and water and maintained on a 12-h light/dark cycle. Transgenic APP/PS1 mice, which develop monoaminergic degeneration (Liu et al. 2008) and progressive amyloid beta (Aβ) pathology (Holcomb et al. 1998; Jankowsky et al. 2004, 2001), and APP/PS1 negative non-transgenic (non-Tg) littermates were used throughout the study. The transgenic APP/PS1 breeding colony in the Graves lab was originally established using mice generously provided by Dr. Michael K. Lee at the University of Minnesota. The breeding colony was supplemented with B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax (RRID:MMRRC_034829-JAX) obtained from Mutant Mouse Resource and Research Center (MMRRC) to maintain the colony and mice were backcrossed for at least 10 generations onto a C57BL/6 J background (RRID:IMSR_JAX:000664) before being incorporated into breeding or experimental groups. Genotyping was performed on tail biopsies by Transnetyx (Cordova, TN) using real time PCR.

Experiments in the current study were performed using male mice exclusively; the impact of chronic intermittent access (IA) to alcohol on locus coeruleus (LC) noradrenergic neurons and Aβ pathology in female mice is reported in our prior study (Engel et al. 2025). Studies examining the impact of alcohol consumption in APP/PS1 mice during early adulthood were conducted as separate and independent studies based on known sex differences in the development of pathology and neurodegeneration in APP/PS1 mice (Jiao et al. 2016; Li et al. 2016; Mifflin et al. 2021; Wang et al. 2003) as well as known sex differences in the amount of alcohol consumed between male and female mice (DeBaker et al. 2020; Downs et al. 2023; Hwa et al. 2011; O'Rourke et al. 2016; Sneddon et al. 2019).

Two-Bottle Intermittent Access Paradigm

Male APP/PS1 and non-Tg mice (approximately 8 weeks of age) were individually housed for a minimum of 3 days to acclimate after which mice underwent an eight week IA paradigm as in our prior study (Engel et al. 2025); a timeline illustrating the experimental workflow is provided in Fig. 1. Mice assigned to the alcohol group were given 24-h access to alcohol on Mondays, Wednesdays, and Fridays; a second bottle containing water was always present. The first week of the paradigm consisted of a ramping period during which mice were provided access to a bottle of 3% (v/v) alcohol in tap water on Monday for 24 h, 10% alcohol on Wednesday for 24 h, and 20% alcohol on Friday for 24 h; 20% alcohol was provided on each test day for the remaining seven weeks of the paradigm (weeks 2–8). Bottles containing alcohol and water were weighed before and after each IA session. To account for side preference, the positions of the bottles were alternated each session. On all other days, animals received two bottles of tap water. Water drinking control subjects were provided with two bottles of tap water every day for eight weeks. All experimental subjects had free access to food and water at all times and body weight was measured weekly. After eight weeks of the IA paradigm, mice remained singly housed with free access to food and water for an additional 23 weeks of abstinence, thereby aging mice to approximately nine months of age, after which they were sacrificed, brains fixed and extracted for immunostaining for stereological analyses. Alcohol is the most misused in young adults 18–25 years of age (Substance Abuse and Mental Health Services Administration 2024); given that our paradigm provided IA to alcohol during early adulthood after which mice were aged to approximately 9 months of age, our abstinence timepoint aligns with long-term sobriety stretching into middle-age and perhaps early stages of late adulthood in humans.

Fig. 1.

Fig. 1

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Timeline of experimental workflow. At approximately 8 weeks of age, male APP/PS1 and non-Tg mice were singly housed for at least 3 days prior to starting the 8-week intermittent access (IA) paradigm with water drinking control mice run in parallel. After completing 8 weeks of IA, all subjects remained individually housed for a 23-week period of abstinence after which mice were sacrificed and brains fixed with 4% paraformaldehyde (PFA). Fixed brain tissue was subsequently sectioned, stained using immunofluorescence, and stereologically quantified

Alcohol consumption was quantified by multiplying the weight of fluid consumed by the percent concentration of alcohol and alcohol density, divided by the animal’s weight. To calculate preference, the weight of alcohol consumed was divided by the weight of total fluid (water and alcohol) consumed. Blood ethanol concentration (BEC) was not examined in the current study. However, C57BL/6 J male mice at age 8–10 weeks undergoing an IA paradigm are reported to have BEC levels of approximately 79.58–166.93 mg/dL after 2 h (Hwa et al. 2011). Chronic IA exposure is also capable of inducing signs of alcohol dependence as handling-induced convulsions (Hwa et al. 2011) during withdrawal as well as alcohol-induced hyperalgesia and liver perturbations (Peng et al. 2024).

Immunofluorescence

Mice were injected intraperitoneally with ketamine (50 mg/kg)/xylazine (4.5 mg/kg) 23 weeks after the last IA session and after verifying a lack of toe pinch response, were transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted, post-fixed in 4% PFA overnight, and subsequently placed in 30% sucrose in PBS for cryoprotection. As in prior studies (Du et al. 2022; Engel et al. 2025; Pilski and Graves 2023), 40 µm coronal sections spanning the entirety of the locus coeruleus (LC) and M1 motor cortex were collected. LC norepinephrine neurons were labeled by staining for tyrosine hydroxylase (TH) with every third section being stained. Axons of LC norepinephrine neurons were stained for the norepinephrine transporter (NET) in the M1 motor cortex with every sixth section stained. Similarly, an additional series of coronal sections entailing the M1 motor cortex were stained for amyloid beta (Aβ) with every sixth section stained. Sections were incubated in 10% (for Aβ staining) or 20% (for TH and NET staining) formic acid, then blocked with blocking buffer containing 5% normal donkey serum (Sigma Aldrich, Cat No. S30-100 mL) and 0.3% (Aβ staining) or 3% (NET and TH staining) Triton X-100, followed by treatment in AffiniPure® Fab fragment donkey anti-mouse IgG (H + L) (Jackson ImmunoResearch, 715–007-003, 1:200). The LC was stained for TH (primary antibody: rabbit anti-TH polyclonal, AB152, Millipore, 1:2000; secondary antibody: Alexa 555 donkey anti-rabbit, A31572, Invitrogen, 1:200). To examine axons, the M1 motor cortex was stained for NET (primary antibody: mouse IgG1 anti-NET monoclonal [CL3063], MA5-24,647, ThermoFisher, 1:1000; secondary antibody: Alexa 555 donkey anti-mouse, A31570, Invitrogen, 1:200). Aβ pathology was assessed by staining for Aβ (primary antibody: mouse purified anti-β-amyloid, 1–16 monoclonal [6E10], 803,001, BioLegend, 1:1000; secondary antibody: Alexa 555 donkey anti-mouse A31570, Invitrogen, 1:200). All blocking and antibody solutions were made in PBS with 0.05% Tween-20, and washes were completed after incubation steps using PBS-T. ProLong Diagmond Antifade Mountant (Invitrogen) was used to mount LC and M1 motor cortex stained brain sections onto glass slides (Fig. 1; Electron Microscopy Services); slides with slices stained for immunofluorescence were cover slipped (Globe Scientific) and stored at -20 ºC.

Stereological Analyses

Experimenters were blinded to genotype and treatment history for all stereological analyses. Parameters were quantified using a Zeiss microscope with a motorized stage and digital camera controlled by StereoInvestigator software (version 2025.1.2, MBF Bioscience) using methods consistent with our prior investigations (Du et al. 2022; Engel et al. 2025; Pilski and Graves 2023). In brief, anatomical boundaries were contoured under an objective lens of 2.5X/0.085NA for all probes. The optical fractionator probe (Deniz et al. 2018) was used to quantify the number of TH+ cells in the LC under a 63X/1.4NA objective lens, with a counting frame of 150 μm × 150 μm, grid size of 275 μm × 175 μm, and a 3 μm guard zone as in prior studies (Du et al. 2022; Engel et al. 2025; Pilski and Graves 2023). The Gunderson coefficient of error (m = 1) ranged from 0.05–0.06 for TH+ cells. The Spaceballs probe (West 2018) was used to determine the length of NET+ axons in the M1 motor cortex under a 63X/1.4NA objective lens. For axon length quantification, a hemisphere of 20 μm, grid size of 250 μm × 250 μm, and guard zone of 3 μm were used as in our prior studies (Du et al. 2022; Engel et al. 2025; Pilski and Graves 2023), resulting in a Gunderson coefficient of error (m = 1) ranging from 0.04–0.05. To quantify Aβ pathology in the M1 motor cortex, the area fraction fractionator probe was used (Howard 1998; Warille et al. 2023) with an objective lens of 20X/0.5NA, a counting frame of 600 μm × 600 μm, and a grid size of 650 μm × 650 μm (Engel et al. 2025); the Gunderson coefficient of error (m = 1) was 0.015–0.03.

Statistical Analyses

There were 13 non-Tg male mice assigned to the water group, 12 non-Tg mice assigned to IA alcohol, 12 APP/PS1 mice assigned to water, and 13 APP/PS1 mice assigned to IA alcohol. Tissue from a subset of these animals was collected and used for stereological analyses. For somatic and axonal loss, n = 8 non-Tg water, n = 8 non-Tg alcohol, n = 8 APP/PS1 water, n = 8 APP/PS1 alcohol animals were assessed; for Aβ pathology n = 7 and n = 8 for APP/PS1 water and APP/PS1 alcohol groups, respectively.

Survival curves were evaluated using a Mantel-Cox log-rank test while body weight, preference, alcohol consumption, and total fluid consumption were assessed using a mixed effects model with repeated measures for time with multiple comparisons performed using Sidak’s post-hoc test; four outliers in the body weight, one outlier in the preference, and seven outliers in the total fluid consumption datasets were detected using Grubb’s test and excluded from analyses. Survival data are presented as survival curves with the non-Tg water and alcohol treatment subjects being pooled as one group. Body weight, alcohol preference, alcohol consumption, and total fluid consumption are presented as mean ± SEM across weeks. Statistical outliers were identified via ROUT 1% which resulted in one subject being excluded from TH+ LC cell counts and one subject being excluded from NET+ axon length quantification. After the removal of outliers, all stereological datasets passed Shapiro–Wilk normality testing and were analyzed using unpaired t-tests or two-way ANOVAs with Tukey’s post-hoc test. Stereological data are presented as mean ± SEM with Aβ quantification normalized to water control subjects and expressed as percent water control. All data analyses were completed using GraphPad Prism software (version 10.5.0); α = 0.05.

Results

Alcohol Consumption had no Impact on Survival or Body Weight

Individually housed male APP/PS1 and non-Tg littermates underwent an 8-week chronic intermittent access (IA) paradigm for voluntary alcohol consumption with water drinking control subjects run in parallel beginning at approximately 8 weeks of age. After eight weeks of IA, mice remained individually housed for an additional 23 weeks with food and water provided ad libitum. Non-Tg alcohol and water control subjects were pooled for survival analyses as there were no mortalities in these groups and compared to APP/PS1 mice that had IA to alcohol and APP/PS1 water control mice. Comparing non-Tg subjects, APP/PS1 water controls, and APP/PS1 mice that had IA to alcohol showed a difference in survival (Fig. 2A, Log-rank Mantel-Cox test, χ2 = 6.750, p = 0.0342). Regardless of the treatment, the mortality rates of APP/PS1 mice were higher than that of non-Tg animals (non-Tg vs. APP/PS1 water control, p = 0.0089; non-Tg vs. APP/PS1 IA, p = 0.0123). Additionally, there was no difference between the survival rate of APP/PS1 animals that consumed alcohol or water (p = 0.8724) indicating that the higher incidence of mortality in the current study was driven by genotype and not alcohol.

Fig. 2.

Fig. 2

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Eight weeks of voluntary intermittent alcohol consumption during early adulthood had no impact on mortality or weight. (A) Mice had intermittent access (IA) to alcohol for 8 weeks with water control subjects run in parallel. Eight weeks of IA was followed by 23 weeks of protracted abstinence. There was no mortality in non-Tg mice consuming water or alcohol and groups were therefore pooled. APP/PS1 mice consuming water or alcohol had decreased probability of survival relative to non-Tg mice (Log-rank Mantel-Cox test, χ2 = 6.750, p = 0.0342). APP/PS1 IA (p = 0.0123) and APP/PS1 water control mice (p = 0.0090) had an increased mortality compared to non-Tg mice (n = 25 non-Tg water + IA, n = 12 APP/PS1 water, n = 13 APP/PS1 IA); *p < 0.05. (B) When comparing genotypes and treatment groups, there were no differences in body weight throughout the eight-week IA period; n = 13 non-Tg water, n = 12 non-Tg IA, n = 12 APP/PS1 water, n = 13 APP/PS1 IA

Animals were weighed weekly during the 8-week IA paradigm. Over time, all groups exhibited an increase of body weight (Ftime (2.926,124.1) = 94.22, p < 0.0001), but there was no difference in body weights between the treatment groups (Ftreatment (3,45) = 1.362, p = 0.2665). However, there was an interaction of time X treatment (Ftime x treatment (8.778,124.1) = 2.098, p = 0.0358); Sidak’s multiple comparisons did not indicate any differences between groups at specific time points (Fig. 2B).

Male APP/PS1 and Non-Transgenic Control Mice Exhibited Similar Drinking Behavior

There was no effect of time (Ftime (3.480,71.54) = 1.206, p = 0.3154), genotype (Fgenotype (1,23) = 0.08178, p = 0.7775), or time X genotype interaction (Ftime x genotype (3.480,71.54) = 1.300, p = 0.2800) on preference for alcohol (Fig. 3A). Overall, alcohol consumption increased across weeks of the IA paradigm in both non-Tg and APP/PS1 mice (Fig. 3B; Ftime (3.621,74.83) = 14.31, p < 0.0001). Similarly, there was no difference in alcohol consumption between non-Tg and APP/PS1 groups (Fgenotype (1,23) = 0.3045, p = 0.5864), nor was there a time X genotype interaction (Ftime x genotype (3.621,74.83) = 1.109, p = 0.3561). Total fluid consumption also increased across weeks (Fig. 3C; Ftime (3.495,69.51) = 4.454, p = 0.0043) with no genotype (Fgenotype (1,23) = 0.01825, p = 0.8937) or time X genotype interaction (Ftime x genotype (3.495,69.51) = 1.407, p = 0.2447). Altogether these data indicate that drinking behavior was similar between male non-Tg and APP/PS1 subjects.

Fig. 3.

Fig. 3

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There were no differences in drinking behavior between male APP/PS1 and non-Tg mice. (A) There was no difference in alcohol preference between groups; n = 13 non-Tg water, n = 12 non-Tg IA, n = 12 APP/PS1 water, n = 13 APP/PS1 IA. (B) IA alcohol consumption was not different between groups; n = 13 non-Tg water, n = 12 non-Tg IA, n = 12 APP/PS1 water, n = 13 APP/PS1 IA. (C) Total fluid consumption did not differ between groups; n = 13 non-Tg water, n = 12 non-Tg IA, n = 12 APP/PS1 water, n = 13 APP/PS1 IA

The Length of Axons Stained for the Norepinephrine Transporter in the M1 Motor Cortex Was Decreased in Male APP/PS1, but not in Non-Transgenic Mice With a History of Intermittent Access to Alcohol

In female APP/PS1 mice, voluntary alcohol consumption using the same 8-week IA paradigm with a 23-week abstinence period resulted in LC axon loss in the M1 motor cortex (Engel et al. 2025), we therefore examined whether a history of alcohol similarly impacts LC axon length in male mice. A significant genotype (Fgenotype (1,27) = 25.64, p < 0.0001) and genotype X treatment interaction (Fgenotype x treatment (1,27) = 7.964, p = 0.0088) was detected with no treatment effect (Ftreatment (1,27) = 2.692, p = 0.1125). Tukey’s post-hoc analysis indicated that in non-Tg mice, there was no effect of alcohol on NET+ axon length in the M1 motor cortex (p = 0.8300), nor was there a significant difference in axon length between non-Tg and APP/PS1 water control mice (p = 0.3883) or APP/PS1 water controls compared to non-Tg IA subjects (p = 0.0890). However, NET+ axon length was decreased in APP/PS1 mice that had IA to alcohol compared to non-Tg water control mice (p = 0.0004), non-Tg IA mice (p < 0.0001), and APP/PS1 water control mice (p = 0.0218) (Fig. 4). Compared to APP/PS1 water control mice, mean NET+ axon length in male APP/PS1 mice that had IA to alcohol was decreased by 15.18%.

Fig. 4.

Fig. 4

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Axon length of LC norepinephrine neurons in the M1 motor cortex was decreased in male APP/PS1 but not non-transgenic mice that had consumed alcohol during early adulthood and undergone protracted abstinence. Representative z-stack projection images showing norepinephrine transporter (NET+, red) stained axons in the M1 motor cortex of a male (A non-Tg water, (B) non-Tg IA, (C) APP/PS1 water, and (D) APP/PS1 IA mouse; scale bars denote 20 µm. (E) In the M1 motor cortex, NET+ axon length was decreased in APP/PS1 IA mice compared to APP/PS1 water control (p = 0.0218), non-Tg IA (p < 0.0001), and non-Tg water control mice (p = 0.0004); n = 7 APP/PS1 IA, n = 8 APP/PS1 water, n = 8 non-Tg IA, n = 8 non-Tg water; * p < 0.05, *** p < 0.001, **** p < 0.0001

A History of Intermittent Access to Alcohol had no Impact in the Number of Tyrosine Hydroxylase-Stained LC Neurons in Male APP/PS1 and Non-Transgenic Mice

In female APP/PS1 mice, the deleterious effects measured after abstinence from chronic IA to alcohol was not restricted to axons, but also resulted in a loss of LC norepinephrine neurons (Engel et al. 2025). To determine whether there was evidence of LC norepinephrine cell loss in male mice, the total number of TH+ neurons in the LC was stereologically quantified. There was no evidence of LC norepinephrine cell loss in non-Tg or APP/PS1 male mice after abstinence from IA to alcohol (Fig. 5; Ftreatment (1,27) = 0.01230, p = 0.9125; Fgenotype (1,27) = 1.384, p = 0.2497; Fgenotype x treatment (1,27) = 0.004974, p = 0.9443). These data suggest that although a history of alcohol consumption does result in deficits, i.e. decreased LC axon length, in male APP/PS1 mice (Fig. 4), it is insufficient to result in neurodegeneration of LC norepinephrine neurons (Fig. 5) at the timepoint under investigation.

Fig. 5.

Fig. 5

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The number of tyrosine hydroxylase-stained neurons in the locus coeruleus (LC) was unaffected by a history of alcohol consumption during early adulthood in male non-transgenic and APP/PS1 mice. Representative images of LC norepinephrine neurons stained for tyrosine hydroxylase (TH) in red from a male (A) non-Tg water, (B) non-Tg IA, (C) APP/PS1 water, and (E) APP/PS1 IA mouse; scale bars indicate 200 µm. (E) Stereological quantification of the number of TH+ neurons in the LC indicates no difference across genotypes or treatment; n = 7 APP/PS1 IA, n = 8 APP/PS1 water, n = 8 non-Tg IA, n = 8 non-Tg water

Amyloid Beta Pathology in the M1 Motor Cortex was Increased in Male APP/PS1 Mice that had a History of Intermittent Access to Alcohol

To determine whether the observed axonal deficits (Fig. 4) were associated with exacerbated Aβ pathology in the M1 motor cortex, Aβ pathology was stereologically quantified, and data normalized to APP/PS1 water control male mice. In male APP/PS1 mice that had IA to alcohol, mean Aβ pathology was increased in the M1 motor cortex by 32.63% compared to APP/PS1 water control subjects (Fig. 6; t (13) = 2.869, p = 0.0132, two-tailed) providing evidence that alcohol consumption during early adulthood may exacerbate disease severity.

Fig. 6.

Fig. 6

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Amyloid beta (Aβ) pathology in the M1 motor cortex was increased in male APP/PS1 mice that had undergone abstinence from intermittent access (IA) to alcohol. A Representative images of the M1 motor cortex stained with Aβ (red) depicting Aβ pathology in a male APP/PS1 water (left) and APP/PS1 IA (right) mouse; scale bars denote 200 μm. B Stereological quantification of Aβ pathology in the M1 motor cortex indicates increased Aβ pathology in male APP/PS1 mice that had IA to alcohol during early adulthood followed by 23 weeks of abstinence relative to male APP/PS1 mice that had consumed water (p = 0.0132). Quantification of Aβ pathology was normalized to the APP/PS1 water control group and is presented as % water; n = 8 APP/PS1 IA, n = 7 APP/PS1 water; * p < 0.05

Discussion

The goal of the current study was to examine the impact of alcohol consumption during early adulthood on potential LC degeneration and associated Aβ pathology measured after a period of protracted abstinence in male mice. While alcohol consumption had no impact on overall survival rates, there was increased mortality in APP/PS1 mice compared to non-Tg mice. After protracted abstinence from IA to alcohol, there was no change in the number of norepinephrine neurons in the LC; however, LC axon length in the M1 motor cortex was decreased in APP/PS1 mice that had IA to alcohol during early adulthood, but not in non-Tg mice. This axonal deficit was also associated with increased Aβ pathology in the M1 motor cortex of male APP/PS1 mice.

The reason for increased mortality in male APP/PS1 mice is unclear, although we previously found a similar increase in mortality in female APP/PS1 (Engel et al. 2025). The premature mortality rate in the current study peaked at approximately 3 months of age, where the survival rate was approximately 85% (Fig. 2A); previous studies have found similar survival trajectories and peak mortality rates occurring at approximately 3–5 months of age in APP/PS1 mice (Engel et al. 2025; Hegnet et al. 2025; Huang and Lemke 2022; Minkeviciene et al. 2009; Tzeng et al. 2018), a timepoint which slightly precedes significant Aβ pathology reported at approximately 6 months of age (Garcia-Alloza et al. 2006; Jankowsky et al. 2004; van Groen et al. 2006). These premature deaths may be in part attributed to neuronal hyperexcitability, particularly observed in young to middle-aged mice (Jin et al. 2018; Minkeviciene et al. 2009). Taken together, the predisposition of potential seizure activity due to neuronal hyperexcitability in young animals may contribute to the premature mortality observed in APP/PS1 mice.

In the current study, we found that a history of alcohol consumption resulted in axonal deficits with no somatic loss of LC norepinephrine neurons and a moderate increase in Aβ pathology in male subjects. In contrast, our prior study using female subjects found decreased axon length as well as a 21.9% decrease in the number of norepinephrine neurons in the LC which was associated with an approximately two-fold increase in Aβ pathology (Engel et al. 2025) suggesting that that the deleterious effects of alcohol in the context of AD are more severe in females than males. The simplest explanation for the differential outcomes in male and female subjects relates to the differences in alcohol consumed. We previously observed that female mice consumed approximately 22 g/kg of alcohol per day (Engel et al. 2025) while in the current study, male mice consumed approximately 12 g/kg of alcohol per day. Sex differences in alcohol consumption with females consuming more than males are reported across multiple studies (DeBaker et al. 2019; Downs et al. 2023; Hwa et al. 2011; O'Rourke et al. 2016; Sneddon et al. 2019) and may be a contributing factor to observed outcomes between our prior study in female mice (Engel et al. 2025) and the current report. However, one limitation of our work is that the current study in male mice and our previous investigation using female mice are independent studies and were not designed to directly test for sex differences. Future studies will be necessary to examine potential dose–response relationships with lifetime intake of alcohol and implications for AD in both sexes.

Differences in neuroinflammation, specifically microglia, may also contribute to sex differences. Exacerbated Aβ plaques and neuroinflammation has been found in female mouse models of AD (Yang et al. 2018). In AD, microglia from female mice develop disease-associated and senescent phenotypes to a greater extent than males (Ocanas et al. 2023), and alterations of phagocytosis of Aβ by microglia is decreased in female APP/PS1 mice compared to males despite the co-localization of phagolysosomes in microglia (Guillot-Sestier et al. 2021). Similar to AD, female rats have a greater number of microglia in the brain compared to males following binge drinking of alcohol (Barton et al. 2017). This evidence suggests that there are sex-dependent neuroinflammatory responses in alcohol and AD that may contribute to the differences observed in our female mice (Engel et al. 2025) compared to this study in male mice.

The LC is vulnerable to degeneration in AD mouse models (Liu et al. 2013) and in patients (Kelly et al. 2017). Aβ pathology and neuroinflammation is worsened by lesioning the LC (Chalermpalanupap et al. 2018; Heneka et al. 2002, 2010, 2006; Jardanhazi-Kurutz et al. 2010, 2011; Kalinin et al. 2007) or blocking of norepinephrine receptors (Branca et al. 2014; Evans et al. 2020), thereby suggesting that Aβ pathogenesis is obstructed by norepinephrine signaling from LC neurons. Following chronic alcohol consumption, damage to LC neurons was noted in male rats (Kjellstrom et al. 1993; Lu et al. 1997; Rintala et al. 1998) and in studies with both male and female rats (Lu et al. 1997; Rintala et al. 1998). In the brains of alcoholic men and women, fewer LC neurons compared to non-alcoholic controls were also found (Arango et al. 1994), however, sex differences were not analyzed. This collection of evidence suggests that the LC may be a point of intersection between AD and alcohol. One possible mechanism of LC degeneration is from the metabolic pathway of norepinephrine via monoamine oxidase (MAO) enzymes. In axons of substantia nigra pars compacta (SNc) dopamine neurons, disrupting vesicular packaging using the psychostimulant methamphetamine (meth) increases MAO-dependent mitochondrial oxidative stress (Du et al. 2021; Graves et al. 2020). Meth similarly induces MAO-dependent mitochondrial oxidative stress in axons of LC norepinephrine neurons (Du et al. 2022); chronic in vivo administration of meth results in MAO-dependent degeneration of both SNc dopamine and LC norepinephrine neurons in male mice (Du et al. 2022, 2021; Graves et al. 2021; Pilski and Graves 2023). These studies collectively indicate that MAO metabolism of substrate increases mitochondrial oxidative stress which can contribute to neurodegeneration including axonal loss which preceded somatic loss (Pilski and Graves 2023). Both MAO-B (Adolfsson et al. 1980; Emilsson et al. 2002; Schedin-Weiss et al. 2017) and MAO-A (Burke et al. 1999; Emilsson et al. 2002; Syed et al. 2023) activity is heightened in male and female patients with AD, and chronic alcohol consumption in men and women is associated with escalated MAO-A activity levels (Matthews et al. 2014). Together, these data suggest that MAO activity may contribute to the observed axonal loss in the current study. If this is the case, MAO inhibition could potentially be an intervention strategy to help mitigate the impact of past alcohol use on AD risk.

The inherent neurophysiology of LC norepinephrine may also be a contributing factor to the observed axonal loss in the current study as well as the axonal and somatic loss reported in our prior study in female mice (Engel et al. 2025). L-type calcium channel activity contributes to mitochondrial oxidative stress in LC neurons in both somatic and axonal compartments (Du et al. 2022; Sanchez-Padilla et al. 2014). Alcohol consumption increases the excitability of LC neurons (Downs et al. 2023) which could lead to increased overall calcium load and by extension mitochondrial oxidative stress experienced by LC norepinephrine neurons. Presumably this proposed increase in calcium influx leading to mitochondrial oxidative stress could also increase the vulnerability of LC norepinephrine neurons to degeneration. Consistent with this, we’ve also shown that administration of isradipine, an L-type calcium channel inhibitor, prevents chronic meth induced degeneration of LC norepinephrine neurons (Du et al. 2022). L-type calcium channels may therefore be an additional druggable target which may provide neuroprotection.

Metabolism of alcohol itself may also be a contributing factor. The liver is the main organ responsible for the metabolism of alcohol, although, other tissues in the body, including the brain can metabolize alcohol (Leung and Nieto 2013; Zakhari 2006). Cytochrome P450 2E1 (CYP2E1) is one of the enzymes involved in metabolizing alcohol into acetaldehyde in both the liver and brain (Leung and Nieto 2013; Zakhari 2006). One consequence of alcohol metabolism by CYP2E1 is the production of reactive oxygen species (ROS; Leung and Nieto 2013; Zakhari 2006). The production of ROS via alcohol metabolism in the brain could also contribute to oxidative stress and damage. Furthermore, alcohol-induced oxidative stress increases activity of β-secretase (BACE1), an enzyme that cleaves APP to generate Aβ, and APP expression (Kim et al. 2011) whereas inhibiting BACE1 decreases Aβ1-40 and Aβ1-42 levels (Ulku et al. 2024). Thus, Aβ1-40 and Aβ1-42 content could be increased following alcohol exposure due to oxidative stress. In addition, γ-secretase cleaves APP into Aβ peptides of varying lengths, where γ-secretase activity is elevated following alcohol due to oxidative stress (Gong et al. 2021). The impacts of alcohol-induced oxidative stress on BACE1 and γ-secretase indicate potential mechanisms by which alcohol may exacerbate Aβ pathogenesis.

Conclusions

Results from the current study demonstrate that protracted abstinence from IA to alcohol results in axonal but not somatic loss of LC norepinephrine neurons in male APP/PS1 mice and that this axonal deficit is associated with exacerbated Aβ pathology. In contrast, our previous study demonstrated that in female APP/PS1 mice, there was both somatic and axonal loss of LC norepinephrine neurons after protracted abstinence from IA to alcohol and a seemingly more robust increase in Aβ pathology (Engel et al. 2025). Taken together, our two complementary studies raise significant concerns about the impact of alcohol consumption during early adulthood on AD. Further studies are needed to identify mechanisms driving the alcohol induced increased vulnerability in LC neurons and associated increase in Aβ pathology as well as efforts to determine whether the relationship between LC deficits and increased Aβ is causal.

Acknowledgements

We would like to acknowledge Sarah M. Mulloy, Ketki Pawaskar, Ruth Dobbelmann, Heba Khattab, and Madison Dillerud for their assistance in investigation.

Author Contributions

Credit authorship statement: Steven M. Graves: conceptualization, formal analysis, funding acquisition, investigation, methodology, writing – original draft, review and editing. Anna M. Lee: conceptualization, formal analysis, funding acquisition, investigation, methodology, review and editing. Nichole R. Payne: investigation. Matthew Scalf: investigation. Shaydel Engel: investigation. Ivy J.Z. Garland: formal analysis, investigation, writing – original draft, review and editing. All authors reviewed the manuscript.

Funding

This work was supported by National Institutes of Health (DA051450 and AG070962 to SMG).

Data Availability

Data generated in the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data generated in the current study are available from the corresponding author upon reasonable request.

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