Background
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder [
1]. Autosomal dominant mutations account for less than 5% of all Alzheimer’s cases [
2], thus, most AD cases are likely due to a combination of environmental and genetic risk factors [
3‐
5]. A gene-environment interaction (GXE) between genetic risk factors and environmental exposures may perturb hippocampus-dependent learning and memory, accelerate cognitive decline, and contribute to neurodegenerative diseases, including AD. However, there is a paucity of information supporting this GXE hypothesis.
Apolipoprotein E4 allele (ApoE4) is the strongest genetic risk factor for late-onset AD [
6]; it increases the frequency and decreases the age at onset of AD in a gene dose-dependent manner [
6]. ApoE4 carriers also have a worse prognosis following traumatic brain injury, a higher prevalence of mild cognitive impairment, and accelerated cognitive decline [
7‐
11].
ApoE4 impairs hippocampus-dependent learning and memory in an age- and sex-dependent manner in transgenic knock-in (KI) mouse models [
12‐
14]. In contrast to aging ApoE3-KI mice or male ApoE4-KI mice, female transgenic ApoE4-KI mice exhibit spatial learning and memory deficits starting at 16 months of age [
12,
14]. Interestingly, female human ApoE4 carriers have an increased risk of AD compared to males, and female ApoE4 carriers with mild cognitive impairment experience greater hippocampal volume loss and reduced memory performance [
15,
16]. Due to the negative effects of ApoE4 on cognition, ApoE4 carriers, particularly female carriers, may be more susceptible to toxicant-induced cognitive impairment compared to ApoE4 non-carriers.
Lead is a ubiquitous environmental contaminant and a significant public health concern in the U.S. and globally [
17‐
19]. This is exemplified by the recent water crisis in Flint, Michigan, where the water supply was contaminated with lead due to lead leaching from old water pipes. The fact that many U.S. cities still have lead service pipes for water distribution underscores the continuing risk of lead exposure in the U.S. [
20].
Both animal and epidemiological studies have reported an association between lead exposure and accelerated cognitive decline and/or AD-associated neuropathology in adults [
21‐
25]. Interestingly, among workers occupationally exposed to lead, those with at least one ApoE4 allele experienced accelerated cognitive decline relative to ApoE4 non-carriers [
26]. This epidemiological finding suggests that an interaction between lead and ApoE4 may accelerate cognitive decline. However, an experimental model is necessary in order to directly assess for a GXE between ApoE4 and lead regarding cognitive impairment. In this study, we used homozygous male and female ApoE3-KI and ApoE4-KI mice as an experimental model in order to assess the effect of lead exposure and ApoE4 on cognitive behavior and to examine any potential sex differences in susceptibility. We used deficits in hippocampus-dependent learning and memory as a measure of cognitive decline and AD-related memory impairment because the hippocampus is one of the earliest brain regions affected in AD patients and deficits in hippocampus-dependent spatial learning and memory may develop prior to the onset of a clinical diagnosis [
27‐
29].
Interestingly, perturbation of adult hippocampal neurogenesis may cause cognitive behavior deficits, accelerate cognitive decline, and increase AD risk [
30]. During adult hippocampal neurogenesis, adult neural precursor cells in the dentate gyrus (DG) of the hippocampus continuously generate adult-born neurons [
31] which contribute to hippocampus-dependent learning and memory [
32‐
37]. Various factors have been shown to modulate adult neurogenesis [
31,
33,
34,
37,
38], however, little is known about whether toxicants or GXE may impair adult hippocampal neurogenesis. ApoE is expressed in adult neural precursor cells in the DG [
6,
31,
39], and in vivo studies using ApoE4-KI mice found that ApoE4 alters adult-born neuron survival and maturation in an age- and sex-dependent fashion [
12‐
14,
40]. Furthermore, we previously reported that lead can impair the proliferation, survival, and differentiation of adult neural precursor cells from the DG of the hippocampus in vitro [
41]. No study to date has assessed the effect of adult-only lead exposure on adult hippocampal neurogenesis and cognitive behavior. Thus, we also investigated whether impaired adult hippocampal neurogenesis may contribute to this GXE between ApoE4 and lead exposure regarding cognitive impairment.
Methods
Mice
Humanized Apolipoprotein E3 and E4 knock-in (ApoE3-KI and ApoE4-KI) mice were generated as previously described [
42] and provided by Dr. Nobuyo Maeda at the University of North Carolina, Chapel Hill. The human E3 or E4 allele in the ApoE3-KI or ApoE4-KI mice is expressed at physiological levels under control of the endogenous mouse ApoE promoter [
13]. ApoE3-KI and ApoE4-KI animals were maintained as homozygous lines and all animals were housed in standard conditions (12 h light/dark cycle) with food and water provided ad libitum.
Reagents
Animal drinking water with 0.2% lead (II) acetate (Cat. 316512, Sigma-Aldrich, St. Louis, MO) was prepared from a stock lead acetate solution and replaced weekly. The preparation, use, and disposal of hazardous agents was carried out according to the Environmental Health and Safety Office at the University of Washington.
Lead exposure
Male and female ApoE3-KI and ApoE4-KI animals were weaned at 28 days and littermates of the same sex were randomly mixed into groups of 3–5 animals per cage. At 8 weeks of age, the mice were either switched to drinking water with 0.2% lead acetate (lead-treated) or kept on normal animal drinking water (control). Body weight was recorded every 1–2 weeks throughout the exposure. Water consumption was monitored for the first week of the exposure period. Lead-treated animals were exposed to lead drinking water for 12 weeks, at which point all the animals were switched to normal drinking water for the remaining duration of the study for the behavior cohort. We used separate cohorts of animals, one for behavior and one for cellular studies, to assess cognitive behavior and adult hippocampal neurogenesis, respectively. The behavior cohorts had n = 8–13 animals per genotype/treatment/sex and the cellular cohorts had n = 6–10 animals per genotype/treatment/sex.
Open field test
The open field test was conducted before and after the lead exposure to assess for the development of any lead-induced locomotor deficits or anxiety. Briefly, each animal was placed into a TruScan Photo Beam Tracking arena (Colbourn Instruments, Whitehall, PA) with Plexiglas walls (10″ X 10″ X 16″) and their movement was monitored with two sets of infrared breams. The animal was allowed to freely explore the arena for 20 min and the data was collected by TruScan 2.0 software (Colbourn Instruments).
Elevated plus maze
We conducted the elevated plus maze test in order to assess the effect of lead exposure on anxiety. The maze (26″ x 26″ x 15.25″; San Diego Instruments, San Diego, CA) consisted of four plastic arms with 7″ walls (two enclosed and two open), and it was placed in the center of the behavior room. Each animal was placed into the center of the maze facing an open arm and allowed to freely explore the maze for 5 min. The open and closed arm ends were defined as the distal 1/3 of the arms. ANYmaze software (San Diego Instruments) was used to collect data on the animal’s movement during the test.
Novel object location test
In order to assess hippocampus-dependent, short-term spatial working memory, we used the 1 h novel object location (NOL) test. This test was performed as previously described with a few modifications [
34]. Briefly, each animal was placed into an open field arena (Colbourn Instruments) with two identical objects in two different locations. During training, the animal was allowed to freely explore the two objects for 5 min and then returned to its home cage. After 1 h, the animal was returned to the arena with the same two objects; one object remained in its original location and one object had been moved to a novel location. The time the animal spent actively investigating each object during the training and testing was quantified. Each training and testing session was recorded and scored offline by an experimenter blinded to the animal’s genotype and treatment. We calculated the discrimination index by dividing the difference in exploration time between the novel (C) and familiar (A) locations by the total exploration time.
Cued-contextual fear-conditioning
Contextual fear conditioning is another form of hippocampus-dependent learning and memory [
33,
37,
43]. We tested 5–6-month-old animals and used a weak foot shock conditioning paradigm (3 x 0.3 mA, 2 s shocks with 2 min inter-trial intervals) as previously described [
33]. For conditioning, the mouse was placed into the foot shock context (10″ x 10″ x 16″ arena with grid shock floor (Colbourn Instruments) and star-shaped wallpaper) and allowed to freely explore the arena for 2 min before the presentation of a 90 dB, 30 s tone (conditioned stimulus, CS). During the last 2 s of the tone presentation, a 0.3 mA foot shock (unconditioned stimulus, US) was delivered via the grid shock floor. This cycle was repeated two more times for a total of three cycles before the animal was returned to its home cage. The CS and US were automated and delivered by TruScan software (Colbourn Instruments). The contextual fear memory test was conducted 24 h after conditioning. The mouse was placed back into the foot shock context for 2 min in the absence of any foot shock. For the cued test (performed 2 h after context test), the animal was placed into a different context (new room; hexagonal Plexiglas arena; cartoon wallpaper) and allowed to freely explore for 2 min followed by the presentation of the CS for 2 min. For the novel context test (performed 2 h after the cued test), the mouse was placed into a novel context (new room; rat cage; striped wallpaper) and allowed to freely explore without any CS or US presentation. In all three tests, persistent freezing behavior (four paws on the ground, no head or body movement besides breathing) was recorded manually every 5 s during the 2 min scoring periods by an experimenter blinded to animal genotype and treatment.
T-maze continuous alternation task
We assessed spontaneous alternation in 12–13- month-old animals using a continuous alternation T-maze protocol with slight modifications [
44]. Briefly, the black, plastic T-maze had two goal arms and a start arm (12.2″ x 4.5″ x 8.26″), and was placed on a platform (22.5″) in the center of a room. The test consisted of a first-forced trial followed by 14 free-choice trials. For the first-forced trial, one of the goal arms was (randomly) blocked with a plastic guillotine door. The animal was sequestered in the distal one-third of the start arm for 5 s before the guillotine door was raised and the animal was allowed to enter the unblocked goal arm. Once the animal returned to the start arm, it was sequestered in the start arm for 5 s before the start of the 14 free-choice trials. For each free-choice trial, the mouse was allowed to enter either of the unblocked goal arms; once it entered a goal arm, the other goal arm was blocked with a guillotine door. The animal eventually returned to the start arm and was sequestered for 5 s while all of the goal arms were unblocked. This was repeated for a total of 14 free-choice trials. The alternation percentage was calculated by dividing the number of times the animal entered alternating arms by 14 (free-choice trials). We defined arm entry as the animal’s tail tip entering the arm and defined repetitive arm entries as an animal re-entering the same arm three times in a row (e.g., 5 sequential entries into the same arm is 3 repetitive entries). An experimenter blinded to animal genotype and treatment scored each test.
Brain and blood lead analysis
We collected blood and brain tissue for lead analysis at sacrifice after the cessation of the lead exposure from a subset of the mice in the cellular cohort (20 weeks old; n = 3–5 per genotype/sex/treatment). The Environmental Health Laboratory at the University of Washington measured blood lead levels using inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500ce, Agilent Technologies, Santa Clara, CA). The spatial distribution and semi-quantitative measurement of lead and zinc levels (ppm) in 30 μm brain hemisphere sections from 20-week-old ApoE4-KI control or lead-treated females was determined by laser ablation ICP-MS (LA-ICP-MS) using an NWR213 laser ablation system (ESI, Portland, OR) connected to an Agilent 7500ce ICP-MS (Agilent Technologies, Santa Clara, CA). Quantification was achieved using spiked protein matrix as external standards.
TUNEL staining
TUNEL staining was performed using the Trevigen TACS 2 TdT-fluor In situ Apoptosis Detection Kit (Cat. 4812-30-K, Sigma-Aldrich, St. Louis, MO) on 30 μm fresh frozen coronal brain sections according to the manufacturer’s instructions. The cells were permeabilized using Trevigen’s CytoninTM permeabilization buffer and an experimental positive control was included (treated with TACS-NucleaseTM).
Quantification of dentate gyrus volume
The volume of the dentate gyrus was quantified using coronal images (20X magnification) from 15-month-old female ApoE3-KI and ApoE4-KI mice from the behavior cohorts as previously described [
45].
BrdU administration
5-bromo-2′-deoxyuridine (BrdU) was from Sigma (Cat. B9285, Sigma-Aldrich, St. Louis, MO) and stored as a 20 mg/ml stock in saline with 0.007% NaOH at -20 °C. After 9 weeks of lead exposure, the mice in one of the cellular cohorts were dosed with 100 mg/kg BrdU by intraperitoneal injection 5 times in 1 day (every 2 h) and sacrificed 3 weeks later (at the end of the 12 week lead exposure) in order to assess the maturation of BrdU-labeled adult-born cells. A second cellular cohort was dosed with BrdU 1 X 100 mg/kg 2 h prior to sacrifice (at the end of the 12 week lead exposure period) in order to assess for proliferative BrdU-labeled cells.
Immunohistochemistry
The primary antibodies and dilutions used in immunohistochemistry were: rat monoclonal anti-BrdU (1:500, Bio-Rad Laboratories AbD Serotec, Raleigh, NC), mouse monoclonal anti-NeuN (1:1000, Millipore, Billerica, MA), mouse monoclonal anti-GAD67 (1:2000, Millipore, Billerica, MA), and donkey polyclonal anti-DCX (1:200, Santa Cruz Biotechnology, Dallas, TX), Goat anti-rat, goat anti-mouse, and donkey anti-rat Alexa Fluor-conjugated secondary antibodies as well as Hoechst 33342 (2.5 μg/ml) were from Invitrogen (Carlsbad, CA). All of the primary and secondary antibodies were diluted into the appropriate blocking buffer (10% donkey or goat serum and 1% BSA).
Mice were anesthetized with ketamine/xylazine and then euthanized by decapitation. One brain hemisphere was freshly frozen for brain lead analysis or other biochemical analysis, while the other hemisphere was post-fixed in 10% neutral-buffered formalin for 3–4 days followed by 30% (w/v) sucrose in phosphate-buffered saline (PBS, pH 7.4) at 4 °C for 3–4 days until saturated. Each post-fixed hemisphere was then stored in cryoprotectant media (30% glycerol; 30% ethylene glycol; 40% PBS) at -80 °C until sectioning. Coronal brain sections (30 μm thick) were used for immunohistochemistry (IHC) using the free-floating antibody staining method as previously described [
38].
Quantification and imaging of immunostained cells
Immunostained cells were quantified as previously described [
34,
38]. Every eighth serial section (30 μm) was immunostained for each IHC marker (or combination of markers). An experimenter blinded to treatment and genotype quantified marker
+ cells in the subgranular zone and granule cell layer of the DG. This number was multiplied by 8 in order to estimate the total number of marker
+ cells per DG. Marker colocalization (double-positive cell) was defined as overlapping fluorescent signals in a single cell using a Z-series stack. All the marker
+ cells from at least 9 coronal sections per mouse (
n = 3–5 mice per genotype/sex/treatment) were quantified for each immuno marker or marker combination. All images were captured with an Olympus Fluoview-1000 laser scanning confocal microscope with the following lenses: numerical aperture (NA) 0.75 10X, NA 0.75 20X, NA1.3 40X (oil), or NA 1.35 60X (oil). Optical Z-sections (1 μm thick) were collected and processed using ImageJ software (NIH, Bethesda, MD). Images were uniformly adjusted for color, brightness, and contrast with Adobe Photoshop CS4 (Adobe Systems Inc., San Jose, CA).
Quantification of dendritic morphology
The dendritic morphology of DCX
+ cells in control and lead-treated ApoE3-KI and ApoE4-KI mice (at least 13 individual neurons per genotype/sex/treatment) was assessed by Sholl analysis as previously described [
34].
Histology
Kidney and liver sections from control and lead-treated ApoE3-KI and ApoE4-KI males and females (
n = 3–4 per genotype/treatment) were fixed in 10% neutral buffered formalin, paraffin embedded, cut into 4- to 5-μm sections, routinely stained with hematoxylin and eosin (H&E), and evaluated by a board-certified veterinary pathologist. Slides were coded to remove information regarding genotype and treatment. Kidneys were scored semi-quantitatively using a 0–4 scale, in which “0” was normal; “1” indicated minimal expansion of the glomerular mesangial matrix and minimal multifocal tubular protein; “2” indicated mild changes of the glomerulus with mild interstitial fibrosis, interstitial nephritis, and tubular protein; “3” indicated moderate multifocal to coalescing changes characterized by more pronounced glomerular, tubular and interstitial changes; and “4” indicated end stage disease with obsolescent glomeruli and severe multifocal to coalescing interstitial nephritis and fibrosis with or without dilation of the renal pelvis (hydronephrosis). Intranuclear inclusion bodies within the proximal renal tubules were noted when present. Livers were assessed using modified criteria from a previous study [
46] and scored semi-quantitatively for changes including necrotic foci, cytoplasmic vacuolation, and mononuclear cell infiltration on a 0–4 system in which “0” was normal; “1” indicated minimal changes; “2” indicated mild changes; “3” indicated moderate changes; and “4” indicated severe changes. Images of representative lesions were acquired using NIS-Elements BR 3.2 64-bit and plated in Adobe Photoshop Elements. Image brightness and contrast was adjusted using Auto Smart Fix and Auto White Balance manipulations applied to the entire image.
Statistical analysis
Statistical analyses were conducted using GraphPad Prism software (version 6.0 h for Mac, GraphPad Software Inc., San Diego, CA, USA) and Stata (version 12.0 for Mac, StataCorp LP, College Station, TX, USA). Two-way analysis of variance (ANOVA) with the Holm-Sidak post-test (α = 0.05) was used to analyze the open field, elevated plus maze, contextual fear, and T-maze data in order to account for the main effect of genotype (ApoE3-KI vs. ApoE4-KI), treatment (control vs. lead), or time. Two-tailed t-test (α = 0.05) was used for within-genotype comparisons for the NOL data. Multilevel mixed-effects linear regression (α = 0.05) was used for longitudinal analysis of within-genotype NOL discrimination index data. Two-way analysis of variance (ANOVA) with Fisher’s LSD post-test (α = 0.05) was used to analyze all of the cellular data. All data represent mean ± SEM, n.s. not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.
Discussion
AD currently affects nearly 35 million people and is projected to affect 117 million people worldwide by 2050 [
57]. AD and cognitive decline are associated with significant societal, financial, and health care costs. Yet, there is a paucity of research on the role of environmental exposures and GXE on neurodegenerative disease risk and AD pathogenesis. Thus, additional investigation into how environmental factors and GXE may influence AD risk or accelerate cognitive decline is of immense public health importance.
Functional impairment in cognition is often used as a proxy to estimate the presence and degree of AD-associated neuropathology [
58,
59]. Here, we used humanized transgenic knock-in mice as an animal model of human ApoE4 carriers to assess whether there is a GXE between lead and ApoE4 on cognitive behavior, and potentially on AD risk. ApoE3-KI and ApoE4-KI female mice develop spontaneous learning and memory deficits starting at 15–18 months [
12‐
14]. Consequently, we assessed learning and memory prior to 15 months of age to determine if lead can accelerate the onset of cognitive behavior deficits. We defined a GXE as a different effect of lead exposure on cognitive behavior in mice expressing the human ApoE4 allele from those expressing the human ApoE3 allele. Based on this definition, we concluded that lead interacts with ApoE4 because we observed statistically significant deficits in contextual fear memory and spontaneous alternation in lead-treated ApoE4-KI female mice but not in other animals. In addition, lead causes an earlier onset of spatial working memory deficits in the NOL test in lead-treated ApoE4-KI mice compared to ApoE3-KI mice, for both males and females. Together, these data suggest that the effect of lead exposure on cognitive behavior is different depending on ApoE genotype. More specifically, a GXE between lead and ApoE4 may accelerate cognitive decline, contribute to a more rapid progression from cognitive aging to mild cognitive impairment, and potentially contribute to AD. Furthermore, females may be more susceptible to this GXE.
We found that lead exposure decreased spontaneous alternation in the T-maze in female ApoE4-KI animals. While alternation is not strictly hippocampus-dependent, alternation performance is very sensitive to hippocampal lesions [
44,
54,
60]. More importantly, impaired alternation and/or repetitive arm entry has been reported in several different AD-mouse models, including models with presenilin and amyloid precursor protein mutations [
61‐
64].
Lead exposure also impaired hippocampus-dependent spatial memory in the NOL test in all lead-treated animals. Interestingly, lead-exposed ApoE3-KI male mice developed memory loss in the NOL test at 6 months post-lead exposure. Moreover, the memory loss in all lead-exposed animals was persistent through 10 months post-lead. These data suggest that sub-chronic adult lead exposure alone is sufficient to cause long lasting or irreversible memory impairment, and that this behavior phenotype can occur long after exposure has ended. These findings are pertinent to human health because irreversible loss of memory formation is characteristic of AD, senile dementia, and up to 50% of Parkinson’s disease cases [
65]. Importantly, the aging population in the U.S. experienced high lead exposure in early life [
66]. Thus, it is possible that lead exposure may lead to cognitive impairment later in life and contribute to cognitive decline associated with aging and neurodegeneration, such as late-onset AD.
Epidemiological reports suggest an increased risk of late-onset AD and increased cognitive decline with mild cognitive impairment in women [
15,
16]. We observed that lead impaired contextual fear memory and working memory in the T-maze in female but not male ApoE4-KI mice. Furthermore, lead-induced spatial memory impairment in the NOL test manifested earlier in females than males for both ApoE3-KI and ApoE4-KI mice. These data provide strong evidence for sex differences in susceptibility to lead-induced cognitive decline in an animal model and suggest that women may be more susceptible to cognitive impairment and/or AD following exposure to toxicants such as lead.
We report average peak blood lead levels of 44.4–63.4 μg/dL, similar to previous animal studies and comparable to some occupational exposure settings in the 20th century [
49,
67‐
69]. Elevated blood lead levels (≥10 μg/dL for adults) can still be encountered among U.S. workers, and Occupational Safety & Health Administration regulations do not require U.S. workers to be removed from their job sites until they have a blood lead level ≥ 50 μg/dL. [
70]. Importantly, high levels of lead exposure from environmental sources and in occupational settings are still a major concern in developing countries [
18,
71]. Therefore, 0.2% lead acetate serves as an experimental model for occupational exposures globally, for environmental exposure in developing countries, and for the US population who were exposed to high levels of environmental lead prior to the ban of leaded gasoline and paint.
Brain and blood lead levels were highest in female ApoE4-KI mice. There were no differences in water consumption during the first week but water consumption thereafter was not monitored. Although we cannot formally exclude the possibility that ApoE4-KI females drank more water than other animals after the first week of lead exposure, there was no statistically significant difference between blood lead levels of female ApoE3-KI and ApoE4-KI mice. Thus, it seems unlikely that higher water consumption alone led to more lead deposition in the brain of female ApoE4-KI.
It is possible that the ApoE4-KI enhances lead deposition in the brain and differences in brain lead disposition may contribute to the GXE between ApoE4 and lead. Interestingly, ApoE4-KI mice exhibit decreased basement membrane thickness, reduced collagen and tight junction protein expression, as well as increased matrix metalloproteinase 9 activity in the blood-brain barrier, suggesting that more lead may accumulate in the brain of ApoE4-KI mice due to reduced blood-brain barrier integrity [
72‐
74]. Furthermore, human ApoE4 carriers with AD also exhibit a thinner basement membrane, increased fibrin extravasion, and pericyte loss in the blood-brain barrier compared to ApoE3-KI carriers with AD or non-AD controls [
75,
76]. Thus, effects of E4 on the blood-brain barrier may increase the accumulation of lead in the brain in ApoE4-KI mice or maybe even in humans, and contribute to more severe or earlier onset of learning and memory impairments.
We previously found that lead can impair the survival, proliferation, and differentiation of adult neural precursor cells in vitro [
41]. Thus, we sought to determine whether lead can interact with ApoE4 to impair adult hippocampal neurogenesis in vivo and whether this could be an underlying mechanism for the observed cognitive deficits. We exposed mice to lead starting at 8 weeks of age in order to avoid the cumulative effects of lead on both the developing and adult brain [
77]. Unlike previous reports using the ApoE3-KI and ApoE4-KI models, we did not see a significant effect of ApoE4 alone on adult-born cell proliferation and maturation. This may be due to the fact that the effects of ApoE4 are age-dependent and we analyzed adult neurogenesis 1.5–2.5 months earlier (in 4.6-month-old vs. 6–7-month-old mice) than previous studies [
12,
14,
40]. Data presented here demonstrate that adult lead exposure impairs adult-born cell proliferation in all lead-treated animals; this mechanism may contribute to lead impairment of spatial memory in the novel object location test that eventually develop in all lead-treated mice. Furthermore, lead exposure significantly impaired adult-born neuron differentiation and decreased dendritic complexity specifically in ApoE4-KI females. These cellular changes correlate with the fact that lead impairment of cognition is most pronounced in ApoE4-KI females. There was also a statistically significant reduction in the number of adult-born mature neurons (BrdU
+NeuN
+ cells) in male ApoE4-KI mice, although the reduction is smaller than that in females. This cellular change occurs at the end of the 12-week lead exposure and correlates with the onset of NOL deficits in the lead-treated ApoE4-KI males in the behavior cohort. Together, these data suggest that lead and ApoE4 may interact to perturb neuronal differentiation and maturation during adult hippocampal neurogenesis, and that this mechanism may contribute to the lead and ApoE4 interaction on accelerated cognitive impairment in ApoE4 mice and the heightened sensitivity of ApoE4 females.
The survival and integration of adult-born neurons is activity-dependent and occurs at approximately three weeks after neuronal birth [
78,
79]. Several different cell types and inputs facilitate the survival and maturation of adult-born cells during this window, including GABAergic interneurons that may exert trophic effects on adult-born immature neuron survival and maturation [
56,
80,
81]. Previous studies found that there are basal sex differences in the number of GAD67
+ interneurons in ApoE3-KI and ApoE4-KI mice, with female mice having significantly more GAD67
+ interneurons, and that female ApoE4-KI mice have significantly fewer GAD67
+ cells than ApoE3-KI mice starting at 6–7 months of age [
40]. We found that lead significantly decreased the total number of GAD67
+ cells in 20-week-old, lead-treated ApoE4-KI females. This cellular effect correlates closely with our observation of significantly reduced adult-born neuron maturation and dendritic complexity in ApoE4-KI females, suggesting that an interaction between lead and ApoE4 may contribute to earlier impairment of GABAergic input and contribute to the observed impairment in immature neuron maturation in ApoE4-KI females.
Adult-born, immature neurons play a critical role in synaptic plasticity within the DG [
79,
82,
83]. Notably, the increased responsiveness and excitability of newborn granule cells may give these adult-born cells a specific and significant role in learning and memory [
55,
84]. In rats, a greater proportion of young, adult-born neurons are activated during spatial exploration compared to existing granule cells [
85,
86]. Similarly, the selective loss of immature neurons impairs long-term spatial memory retention and contextual fear extinction [
36]. Thus, the disruption of adult-born immature neuron maturation in lead-treated ApoE4-KI females may be associated with the impaired hippocampus-dependent learning and memory in these mice.
Although lead was deposited in the mouse brain broadly including the hippocampus and no regional specific deposition was found, we did not observe overt motor impairment. In humans, high levels of lead exposure can cause fine and gross motor impairments. However, lead exposure during development, which interferes with the proper development of the nervous system, likely plays a significant role in these cases. In the present study, mice were exposed to lead beginning at 8 weeks of age, eliminating the effect of lead on the developing nervous system. We only measured gross motor function (open field test), so we cannot rule out if lead exposure caused fine motor dysfunction. We did not observe a significant increase in TUNEL+ cells in the hippocampus or cortex of lead-treated animals. However, lead impaired adult neurogenesis in the hippocampus, with the greatest effect seen in female ApoE4-KI mice. Thus, newborn cells and neurons in the hippocampus, especially those in mice expressing human ApoE4 allele, may be more vulnerable than neurons in other regions of the brain to lead toxicity in the adult mice.
Similar to other studies that have reported basal sex differences in adult neurogenesis in these mice [
14], we report baseline differences in the total number of adult-born cells (total BrdU
+ cells), and the total number of GAD67
+ cells in the DG between control males and females. However, we do not think these differences alter the conclusions of this study. For example, we found that all control mice performed equally well in tests for contextual fear memory, spontaneous alternation in the T-maze, and memory for novel object location (NOL). In addition, while ApoE3-KI and ApoE4-KI male mice had a lower number of BrdU
+ and GAD67
+ cells, they did not show statistically significant memory deficits after lead exposure when tested for contextual fear memory or spontaneous alternation in the T-maze. They also exhibited a later onset of deficits in the NOL test than their respective females of the same genotype. These data suggest that the lower basal adult neurogenesis in males does not affect their basal shot-term spatial and contextual memory, nor lead to higher sensitivity to lead impairment of cognition.