The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Long-term administration of excess zinc impairs learning and memory in aged mice
Kaoru YoshidaMin GiMasaki FujiokaIsao TeramotoHideki Wanibuchi
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2019 Volume 44 Issue 10 Pages 681-691

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Abstract

Zinc (Zn) is an essential element, but excess amounts are known to cause neurotoxic effects. The risk of excessive Zn intake is increased by supplementing food intake with dietary supplements. Ageing affects many cellular processes that predispose individuals to neurodegeneration. Indeed, the prevalence of senile dementia such as Alzheimer’s disease, Parkinson’s disease, and vascular-type dementia increases with age. As such, we investigated the effects of long-term exposure to excess Zn on learning and memory in aged mice. ICR-JCL female mice (aged 26 weeks) were administered 0, 200, or 500 ppm Zn as zinc chloride in drinking water for 30 weeks. After 30-week administration, aged female animals were subjected to Y-maze, novel object recognition, and step-through passive avoidance tests. Chronic exposure to Zn did not inhibit learning and memory in the Y-maze test, but dose-dependently inhibited learning and memory in novel object recognition and step-through passive avoidance tests. These results indicate the potential for chronic Zn exposure to dose-dependently inhibit both long-term and novel object recognition memory. Results of microarray analysis revealed significant changes in gene expression of transthyretin and many olfactory receptors in the hippocampus of Zn-treated mice.

INTRODUCTION

Many trace elements, such as iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn), are both nutrients and neurotoxicants. As such, a deficiency or excess of trace elements may lead to different metabolic disorders (Takeda, 2004; Hambidge, 2000). In the brain, abnormalities in Zn, Fe, Cu, and Mn metabolism have been reported in a variety of neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (Barnham and Bush, 2008; Schrag et al., 2011). In addition, ageing affects many cellular processes that can predispose individuals to neurodegeneration. Accordingly, the prevalence of senile dementia including AD, PD, and vascular-type dementia (VD) increases with age (Nussbaum and Ellis, 2003; Hindle, 2010).

Zn is an essential element that plays an important role in various physiological activities such as immune system function and protein synthesis, and also serves as a co-factor for many enzymes (Frassinetti et al., 2006). Zn, which exists mostly in testes, muscle, liver, and brain tissue, is the second most abundant trace element in brain after Fe; indeed, its concentration in the brain is higher than Cu and Mn. In the adult brain, Zn is especially enriched in the hippocampus and cerebral cortex (Frederickson et al., 2000). Zn homeostasis in the brain is tightly controlled by the blood-brain barrier (BBB), which ensures proper neuronal function and protects the brain from injury and disease (Weiss et al., 2009). However, BBB breakdown is an early event in the ageing brain (Montagne et al., 2015) that may enable Zn to enter the hippocampus during long-term Zn administration.

Although Zn deficiency is known to adversely affect neurodevelopment, effects of excess Zn on neurological disorders are essentially unknown. It is believed that an excess of free Zn is detrimental and can lead to neuronal death, as excess Zn in vivo can cause focal neuronal pathology. There are three main hypotheses for how excess Zn elicits neurotoxicity: excitotoxicity, induction of oxidative stress, and impairment of cellular energy generation (Gower-Winter and Levenson, 2012). An influx of excess Zn into neurons has been shown to result in excitotoxic changes to post-synaptic neurons (Manzerra et al., 2001). In addition, excess Zn has been implicated in oxidative stress by increasing free radical and reactive oxygen species (ROS) production, which leads to neuronal death. Indeed, Zn is a strong inducer of oxidative stress because it promotes both mitochondrial and extra-mitochondrial production of ROS (Frazzini et al., 2006). Thus, it is possible that excess Zn induces mitochondrial dysfunction. Floriańczyk also suggested that the main cause of neuronal death is low energy production by mitochondria. However, the detailed mechanism by which Zn exerts toxic activity is unknown.

Zn concentrations increase with age in the hippocampus, cortical matter and basal ganglia (Serpa et al., 2006; Hebbrecht et al., 1999), and increased Zn concentrations have been reported for all brain areas in individuals with AD or PD (Boruchowska et al., 2001). AD is a polygenic neurodegenerative disorder involving abnormal accumulation and deposition of a Zn–Cu metalloprotein amyloid beta (Aβ). Aggregation of Aβ is mediated by interaction with metals, in particular Zn, Cu and Fe, which are concentrated in and around amyloid plaques in AD brains (Opazo et al., 2002; Dong et al., 2003; Maynard et al., 2005). High levels of Zn have also been reported in amyloid plaques of the Tg2576 (APPsw) mouse model of AD (Maynard et al., 2005). A recent study reported a role for Zn metabolism in AD as a trigger for Aβ aggregation and neuronal plaque formation (Kawahara et al., 2014). Excess Zn is also a common finding in other neurodegenerative diseases (Cai et al., 2005; Cuajungco and Fagét, 2003).

We hypothesised that excess Zn may cause neuronal damage in the hippocampus and lead to impaired learning and memory in aged mice. Therefore, in the present study, we investigated the effects of chronic Zn exposure on step-through passive avoidance, Y-maze, and novel object recognition tests in aged mice. In addition, we performed microarray analysis to investigate changes in neurodevelopment- and function-related gene expression in the hippocampus during learning memory impairments elicited by long-term Zn administration.

MATERIALS AND METHODS

Chemicals

Zinc chloride (ZnCl2) was obtained from Kanto Chemical Industry Co., Ltd., Tokyo, Japan.

Animals and experimental design

Female Jcl:ICR mice were purchased at 7 weeks of age from Clea Japan, Inc., Tokyo, Japan. Animals were housed in polycarbonate cages (five mice per cage) in experimental animal rooms with temperature maintained at 22 ± 3°C, relative humidity of 55% ± 5%, and a 12:12 hr light:dark cycle. Mice were fed standard CE2 pellet (Clea Japan) and given tap water ad libitum until the beginning of Zn administration. Zn drinking water (200 and 500 ppm) was prepared by dissolving ZnCl2 in tap water. Twenty-six-week-old mice were given 0 (n=10), 200 (n = 15), or 500 ppm (n = 15) Zn in drinking water for 33 weeks. Body weights and water intake were monitored on a weekly basis until the day of dissection.

After 30-week administration of Zn to aged mice, effects of long-term Zn exposure on learning and memory were investigated by behavioural tests including Y-maze, novel object recognition, and step-through passive avoidance tests. All behavioural tests were performed within 3 weeks prior to sacrifice in a special room with constant temperature (22 ± 3°C). Before testing, mice were allowed to adapt to the testing room for 30 min. The same animals were used for all three behavioural tests. The order of behavioural tests was as follows: Y-maze, novel object recognition, and step-through passive avoidance. At the end of behavioural experiments, mice were euthanised by inhalation of an overdose of isoflurane (Abbott Japan Co., Ltd., Tokyo, Japan) using a Small Animal Anesthetizer (MK-A110D, Muromachi Kikai Co., Ltd., Tokyo, Japan) coupled with an Anesthetic Gas Scavenging System (MK-T 100E, Muromachi Kikai). At necropsy, brains were rapidly excised, cut into five coronal sections, fixed in 10% phosphate-buffered formalin, embedded in paraffin, and processed for hematoxylin/eosin and immunohistochemical staining.

Animal experimental protocols were approved by The Laboratory Animal Center of Osaka City University Graduate School of Medicine (Osaka, Japan), which is accredited by the Center for the Accreditation of Laboratory Animal Care and Use, Japan Health Sciences Foundation.

Y-maze test

The Y-maze test was used to evaluate short-term memory by monitoring spontaneous alternation behaviours. The Y-maze apparatus (Muromachi Kikai) consisted of three identical arms (40 × 4 × 10 cm) made of grey opaque polyvinyl chloride with equal angles between each arm. Each mouse was placed alone into one arm and allowed to explore the maze for 8 min. The mouse’s performance was monitored with a DV-Track Video Tracking System (Muromachi Kikai). Alternation behaviour (%) was calculated as the ratio of actual to possible alternations using the formula: (total number of arm entries − 2) × 100.

Novel object recognition test

The novel object recognition test is based on the tendency of mice to explore a novel object longer than a familiar one. The novel object recognition test can evaluate recognition memory, object recognition memory, and long-term memory. For testing, each mouse was first habituated to a grey open field (60-cm diameter × 30-cm height) for 10 min. During the training phase (24 hr after open field exploration), two identical objects (A) were placed in the field equidistantly from each other and the wall. Each mouse was allowed to explore the objects for 10 min in the apparatus. In the test phase (3 hr after the training phase), one of the identical objects was replaced by a novel object; the position at which the new object was placed was defined as the novel object position. Mice were allowed to explore the open field for 5 min in the presence of one familiar (A) and one novel (B) object. Object recognition in the test phase was investigated after 3 hr because the object recognition memory of control mice was significantly decreased after 24 hr. Time spent exploring each object during the training and test phases was recorded using a video-assisted tracking system (Muromachi Kikai). The open field arena and objects were thoroughly cleaned with 70% ethanol solution or water and dried between trials. Exploratory preference and ratios of time spent exploring either of the two identical objects (A) during the training phase or the familiar (A) and novel (B) object during the test phase over the total time spent exploring both objects were used to measure object recognition memory. Discrimination between the two objects was also calculated using a discrimination index (DI) as follows: DI = (novel object position exploration time during test phase / total exploration time) − (novel object position exploration time during training phase / total exploration time) × 100. This equation takes into account individual differences in total exploration time and the palatability of the position where each object was placed.

Step-through passive avoidance test

The step-through passive avoidance test can evaluate non-spatial long-term memory. The experimental apparatus (O’Hara & Co. Ltd, Tokyo, Japan) consisted of two compartments: one illuminated and one dark. Illumination was provided by a 13 W lamp above the apparatus. The two compartments were separated with a guillotine door. During acquisition trials, each mouse was placed in the illuminated compartment. Ten seconds later, the door to the dark compartment was opened. When the mouse entered the dark compartment, the guillotine door was closed, and the latency to enter was recorded. Five seconds after the door was closed, a footshock (0.3 mA, 5- sec duration) was delivered. After confirming that the mouse reacted to the electric shock, such as vociferation or jumping, the mouse was removed and returned to the home cage. In test trials (1 day after acquisition trials), the mouse was returned to the illuminated compartment without the guillotine door between illuminated and dark compartments. When the mouse entered the dark compartment with all four paws, the latency to enter the dark compartment was recorded. Latency to refrain from entering into the dark compartment served as an index of the ability to avoid, thus allowing memory to be assessed. If a mouse did not enter the dark compartment within 300 sec, the latency was recorded as the cutoff time of 300 sec. After each trial, all chambers were cleaned with 70% ethanol solution or water to prevent bias based on olfactory cues.

Clinical chemistry parameters

After collecting blood by cardiac blood collection, serum was separated from the collected blood. Serum biochemical analysis of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, amylase, total protein, albumin, albumin/globulin ratio, urea nitrogen, and creatinine were conducted by LSI Medience Corporation, Tokyo, Japan.

Histopathological analysis

Serial sections (4-µm thickness) cut from paraffin-embedded brain specimens were examined for expression of Aβ peptides and ionised calcium binding adaptor molecule 1 (Iba1) by immunohistochemical staining using an avidin–biotin–peroxidase complex (ABC) method. Antigen retrieval was performed for Aβ staining by microwaving sections at 98°C for 20 min in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% H2O2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, sections were incubated with rabbit polyclonal anti-β amyloid antibody (ab2539, Abcam, Cambridge, UK) at a dilution of 1:20, or rabbit polyclonal anti-Iba1 antibody (090-19741, Wako, Tokyo, Japan) at a dilution of 1:500 overnight at 4°C. Immunoreactivity was detected using a VECSTAIN Elite ABC Kit (PK-6101, Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine hydrochloride (Sigma Chemical Co., St Louis, MO, USA). Omission of the primary antibody served as the negative control, which was included with each staining procedure.

Microarray analysis

Hippocampal tissues from three mice administered 500 ppm Zn and three control mice were processed for microarray analysis. Ten serial brain sections (10-µm thickness) were cut from paraffin-embedded brain specimens. The first and last sections from each brain sample were stained with haematoxylin and eosin to identify the hippocampus area for needle microdissection. After deparaffinization, hippocampal tissues were collected using sterile toothpicks under a light microscope and transferred immediately to Eppendorf tubes containing lysis buffer from the ReliaPrep™ FFPE Total RNA Miniprep System (Promega, Madison, WI, USA), which was used to extract total RNA according to the manufacturer’s instructions. A total of 2.1 µg of mRNA from three mice administered 500 ppm Zn (700 ng from each mouse) and 2.1 µg of mRNA from three control mice (700 ng from each mouse) were used for microarray analysis.

Microarray analysis using a GeneChip® Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) was performed by Cell Innovator Inc., Fukuoka, Japan. Briefly, the quality of extracted RNA was analysed using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). cRNA was amplified and labelled using a GeneChip® WT Pico Kit, and then hybridised to a GeneChip® Mouse Gene 2.0 ST Array. Raw signal intensities of all samples were normalised by quantile algorithm with Affymetrix® Power Tool version 1.15.0 software. To identify upregulated or downregulated genes, Z-scores and ratios (non-log scaled fold-change) were calculated from the normalised signal intensities of each probe for comparison between control and treatment samples. Upregulated genes were defined as Z-score ≥ 2.0 and ratio ≥ 2-fold, while downregulated genes were defined as Z-score ≤ −2.0 and ratio ≤ 2-fold. Functional annotation and pathway analysis of differentially upregulated genes and downregulated genes were performed using Ingenuity Pathway Analysis (Qiagen, Redwood City, CA, USA).

TaqMan real-time quantitative PCR

mRNA expression levels of transthyretin (TTR) in the hippocampus were evaluated in three mice from each group by TaqMan real-time quantitative PCR. cDNA synthesis was performed with 200 ng of RNA using an Advantage RT-for-PCR kit (Takara Bio, Shiga, Japan). PCR reagents, sequence-specific primers, and probes for TTR (Taqman Gene Expression Assay) were purchased from Applied Biosystems (Foster City, CA, USA). mRNA expression assays were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems). Relative expression levels of mRNA were calculated using the 2-ΔΔCt method and normalized to the internal control 18S.

Statistical Analysis

GraphPad Prism 5 statistical software (GraphPad Software, San Diego, CA, USA) was used to compare differences between multiple groups by one-way ANOVA followed by post hoc testing with Tukey’s test, while the Mann–Whitney U test was used to perform comparisons of two groups. Values of p < 0.05 were considered significant. Data are expressed as mean ± standard error.

RESULTS

Body weight and daily Zn intake

Water intake of each cage and the body weight of each mouse were recorded weekly. Mice were given drinking water containing specific amounts of ZnCl2 (200 or 500 ppm Zn) for periods of up to 33 weeks. Amounts of daily water and Zn intake for mice, and body weights are shown in Fig. 1. The 500-ppm treatment group exhibited lower water intake compared with other groups. However, there was no significant difference in final body weights among groups.

Fig. 1

Amounts of water intake (a) and Zn intake (b), and body weights (c) of mice exposed to 0, 200, or 500 ppm Zn. Data are expressed as mean ± SE.

Effect of long-term Zn administration on learning and memory in aged mice

The Y-maze test was used to evaluate the effects of long-term Zn administration short-term memory by monitoring spontaneous alternation behaviour in aged mice. There were no differences among the three groups with regard to spontaneous alternation behaviour, which yielded values of 58.9% ± 3.2%, 60.4% ± 2.6%, and 60.4% ± 5.6% for mice administered 0, 200, and 500 ppm Zn, respectively. Zn did not have any marked effects on percent alternation or total arm entries.

Loss of long-term memory is a particularly devastating feature of a variety of cognitive disorders, diseases, and injuries. The simplest and most commonly used tests for long-term memory in mice are novel object recognition and step-through passive avoidance tests. Novel object recognition, which can also evaluate object recognition memory, is based on the tendency of mice to spend more time exploring a novel object compared with a familiar one. We assumed that mice spent equal amounts of time exploring the two identical objects during the training phase; however, exploratory preference percentages were not equal for mice exposed to 0 and 500 ppm Zn, and were significantly different between the two identical objects for mice exposed to 0 ppm Zn (Fig. 2a). These results indicated that each mouse has a different location preference. During the test phase (3 hr after the training phase), there were significant differences between the exploratory preferences of familiar and novel objects in the three groups, but no dose-dependent difference was observed (Fig. 2b). Comparing the exploratory preference of training and test phases in terms of novel object position (where the novel object was placed in the apparatus) revealed a dose-dependent decrease in exploratory preference between training and test phases (Fig. 3a). These results indicated that exploratory preference for a novel object after long-term Zn exposure decreased in a dose-dependent manner.

Fig. 2

Effect of 30-week administration of 0, 200, or 500 ppm Zn on exploratory preference of aged female mice in a novel recognition test during (a) training phase and (b) test phase. *p < 0.05, ** p < 0.01.

Fig. 3

Effects of 0, 200, or 500 ppm Zn on (a) exploration time (%) of aged female mice to a novel object position in the training phase (0 hr) and test phase (3 hr), and (b) DI after 30 week-administration in an object recognition test. *p < 0.05, ** p < 0.01.

To clarify the effect of Zn exposure on object recognition memory, we calculated discrimination between novel and familiar objects using a DI equation. DI was previously calculated by taking into account only individual differences in the total amount of exploration as follows: DI = (novel object exploration time / total exploration time) − (familial object exploration time / total exploration time) × 100 (Antunes and Biala, 2012; Stefanko et al., 2009). However, as exploration time differed for the identical object in the training phase, as shown in Fig. 2a, it is necessary to consider that each mouse has a location preference with regard to where the object was placed in the apparatus. In the present study, we calculated discrimination between novel and familiar objects using a new DI equation that takes into consideration not only individual differences in the total amount of exploration time, but also individual differences in location preference by accounting for where the object was placed: DI = (novel object position exploration time during test phase / total exploration time) − (novel object position exploration time during training phase / total exploration time) × 100. Using the new formula to calculate DI revealed a dose-dependent reduction of novel object recognition in Zn-exposed mice (Fig. 3b). This result indicates that Zn exposure reduced the ability of mice to discriminate novel from familiar objects in a dose-dependent manner, suggesting that chronic Zn exposure inhibits long-term memory and object recognition memory in a dose-dependent manner.

We investigated the effects of long-term Zn administration on passive avoidance responses using the step-through passive avoidance task. As shown in Fig. 4, Zn administration dose-dependently shortened the latency of aged mice tested after 31-week Zn administration. These results indicated that long-term exposure to Zn attenuated the impairment of non-spatial long-term memory in aged mice.

Fig. 4

Effects of 31-week administration of 0, 200, or 500 ppm Zn to aged female mice on step through latency (a) and unimpaired acquisition response (% over 300 sec) (b), 1 day later in a passive avoidance test. *p < 0.05, * p < 0.01.

Clinical chemistry parameters

Serum biochemistry data are shown in Table 1. No significant differences in any measured parameter were observed in mice exposed to 0, 200, or 500 ppm Zn. These results suggest that long-term administration of 200 or 500 ppm Zn does not induce hepatotoxicity, nephrotoxicity, or pancreatic toxicity.

Table 1. Serum biochemistry parameters in female aged mice after 33-week Zn exposure.
Serum biochemistry parameter Zn0 Zn200 Zn500
TP (g/dL) 5.1 ± 0.5 4.8 ± 0.3 4.8 ± 0.4
Alb (g/dL) 3.6 ± 0.6 3.3 ± 0.4 3.3 ± 0.4
A/G 2.6 ± 0.8 2.4 ± 0.6 2.3 ± 0.5
AST (U/L) 62 ± 12 73 ± 25 65 ± 18
ALT (U/L) 24 ± 5 29 ± 11 32 ± 18
ALP (U/L) 264 ± 98 193 ± 30 177 ± 64
Amy (U/L) 1176 ± 236 1115 ± 265 1022 ± 168
Cre (mg/dL) 0.12 ± 0.06 0.12 ± 0.04 0.15 ± 0.02
BUN (mg/dL) 24 ± 8 24 ± 7 24 ± 3

Data from 0, 200, or 500 ppm Zn-exposed mice (Zn0, Zn200, Zn500) are represented as mean ± SD. Alanine aminotransferase (ALT), albumin (Alb), albumin/globulin ratio (A/G), alkaline phosphatase (ALP), amylase (Amy), aspartate aminotransferase (AST), creatinine (Cre), total protein (TP), and urea nitrogen (BUN).

Histopathological changes in the hippocampus of long-term Zn-administered mice

There were no overt treatment-related histopathological findings in the brains of Zn-exposed mice. No apparent differences were observed in the expression of Aβ (Supplemental Fig. 1A and 1B) or the number of Iba1-positive microglia cells (Supplemental Fig. 1C and 1D) between Zn-exposed and control mice.

Gene expression in the hippocampus of long-term Zn-administered mice

A total of 171 genes were differentially expressed (87 upregulated and 84 downregulated) in the hippocampal tissues of Zn-exposed mice compared with control mice. Of these differentially expressed genes, 60 genes were categorised as related to nervous system development and function by Ingenuity Pathway Analysis, including TTR and olfactory receptors. Notably, 47 of these 60 genes were olfactory receptors (Table 2). Moreover, results of quantitative PCR indicated that TTR was increased in the hippocampus of mice administered 500 ppm Zn compared with controls, albeit without statistical significance, possibly as a result of the small number of animals used (Fig. 5).

Table 2. Nervous system development- and function-related gene expression changes in the hippocampus of 0 ppm and 500 ppm Zn-administered mice.
Symbol Gene Name Fold Change
TTR transthyretin 3.58
LOC102723532/OR4N4 olfactory receptor family 4 subfamily N member 4 3.47
Olfr1532-ps1/Olfr707 olfactory receptor 707 3.44
Olfr1446/Olfr1447 olfactory receptor 1446 3.03
Olfr1495 olfactory receptor 1495 3.00
Olfr1281 (includes others) olfactory receptor 1281 2.98
Olfr393 olfactory receptor 393 2.75
Olfr910/Olfr912 olfactory receptor 910 2.65
Olfr811 olfactory receptor 811 2.53
Olfr1413 olfactory receptor 1413 2.50
Olfr907 olfactory receptor 907 2.48
OR8A1 olfactory receptor family 8 subfamily A member 1 2.46
Olfr908 olfactory receptor 908 2.42
OR2Y1 olfactory receptor family 2 subfamily Y member 1 2.42
Olfr887 olfactory receptor 887 2.38
MAML1 mastermind like transcriptional coactivator 1 2.35
Olfr818 olfactory receptor 818 2.25
OR9G4 olfactory receptor family 9 subfamily G member 4 2.24
Olfr706 olfactory receptor 706 2.23
Olfr535 olfactory receptor 535 2.22
Olfr1354 (includes others) olfactory receptor 8 2.19
OR3A1 olfactory receptor family 3 subfamily A member 1 2.16
SALL1 Spalt-like transcription factor 1 2.16
NMNAT1 Nicotinamide nucleotide adenylyltransferase 1 2.14
ACTB actin beta 2.14
Olfr10 (includes others) olfactory receptor 10 2.11
Olfr1348 olfactory receptor 1348 2.11
Olfr117/Olfr130 olfactory receptor 117 2.10
OR1D2 olfactory receptor family 1 subfamily D member 2 2.10
Olfr893 olfactory receptor 893 2.09
Olfr1318 olfactory receptor 1318 2.08
Olfr1083-ps/Olfr1085 olfactory receptor 1085 2.07
NTN3 netrin 3 2.07
Olfr467/Olfr471 olfactory receptor 471 2.04
Olfr550 olfactory receptor 550 2.02
TNFSF14 tumour necrosis factor superfamily member 14 2.01
IFNG interferon gamma -2.04
Olfr725/Olfr726 olfactory receptor 726 -2.11
OR4S2 olfactory receptor family 4 subfamily S member 2 -2.12
Mup1 (includes others) major urinary protein 1 -2.16
OR6P1 olfactory receptor family 6 subfamily P member 1 -2.22
DLL4 delta like canonical Notch ligand 4 -2.25
Olfr311 (includes others) olfactory receptor 311 -2.31
OR1L6 olfactory receptor family 1 subfamily L member 6 -2.33
OR4A5 olfactory receptor family 4 subfamily A member 5 -2.34
Nat8f3 (includes others) N-acetyltransferase 8 (GCN5-related) family member 3 -2.39
Olfr401 olfactory receptor 401 -2.41
Olfr61 olfactory receptor 61 -2.54
Olfr1199 olfactory receptor 1199 -2.55
OR1Q1 olfactory receptor family 1 subfamily Q member 1 -2.59
Olfr484 olfactory receptor 484 -2.63
OR2H1 olfactory receptor family 2 subfamily H member 1 -2.65
Olfr1259 olfactory receptor 1259 -2.66
Olfr92 olfactory receptor 92 -2.75
Olfr775/Olfr776 olfactory receptor 776 -2.81
OR7G1 olfactory receptor family 7 subfamily G member 1 -2.98
Olfr1128 olfactory receptor 1128 -2.99
OR5K1 olfactory receptor family 5 subfamily K member 1 -3.02
Olfr153 olfactory receptor 153 -3.21
HDAC1 histone deacetylase 1 -5.09
Fig. 5

Messenger RNA expression of transthyretin (TTR) in the hippocampus of mice exposed to 500 ppm Zn for 33 weeks.

DISCUSSION

Recent studies have indicated that excess Zn is linked with several neurodegenerative diseases, such as AD and VD in humans (Kawahara et al., 2014). Although excitotoxicity, induction of oxidative stress, and impairment of cellular energy generation are three hypotheses proposed for the neurotoxic effects of excess Zn (Gower-Winter and Levenson, 2012), primary mechanisms are not well understood. Zn neurotoxicity has been reported in in vitro experiments using cells, but not in in vivo experiments using mice or rats, other than transgenic mice. Exposure to chronic high Zn in the drinking water resulted in only slight elevations of Zn in the brains of transgenic APP-C100 mice (Maynard et al., 2009). Akiyama et al. (2012) reported that oral intake of excess Zn in drinking water does not accelerate the deposition of Aβ or tau in transgenic mouse models for Aβ and tau accumulation. However, our data showed that Zn administration impaired long-term memory and object recognition memory in novel object recognition and step-through passive avoidance tests (Figs. 3 and 4). Impairments of long-term memory and object recognition memory were deemed to occur when aged mice were administered Zn for 30 weeks. An age-associated increase in oxidative damage to nucleic acids (both DNA and RNA) has been reported in CNS neurons, which may play a fundamental role in the development of age-associated neurodegeneration (Nunomura et al., 2012).

Our data did not show the apparent differences in the expression of Aβ between Zn-exposed and control mice (Supplemental Fig. 1A and 1B). It has been reported that the sequence of the rodent variety of Aβ(1–42) contains three amino acid substitutions compared to the human sequence, and rodent Aβ is less likely to form larger β-sheet structures, and consequently, large aggregates (Boyd-Kimball et al., 2004). As a consequence of the lack of deposition of the peptide in rodent brain, the expression of Aβ in Zn-exposed might have not been observed.

The BBB, which has important roles for brain physiology, is essential for normal neuronal function and information processing by limiting the entry of circulating blood, chemicals, pathogens, and cells into the brain. Recent studies have shown that breakdown of the BBB in the hippocampus is an early event in the ageing brain (Montagne et al., 2015). In this study, BBB breakdown might cause excessive inflow of Zn into the hippocampus. Many studies have reported that the hippocampus contributes to novel object recognition memory (Antunes and Biala, 2012; Broadbent et al., 2010). It is also widely accepted that the perirhinal cortex and entorhinal cortex play an important role in object recognition memory, at least in rodent brain (Antunes and Biala, 2012; Wilson et al., 2013). Zn is highly concentrated in the synaptic vesicles of a specific subset of glutamatergic neurons. The highest concentrations of these gluzinergic cells are in the perirhinal cortex, subjacent lateral amygdala, prosubiculum, and hippocampus (Frederickson et al., 2000). Thus, long-term administration of excess Zn in aged mice may have impaired both the hippocampus and perirhinal cortex.

To investigate the mechanism by which novel object recognition and long-term memory are impaired by long-term exposure to Zn in aged mice, we used microarray analysis to examine the effects of Zn administration on the expression of genes related to nervous system development and function in the hippocampus of aged mice. Expression levels of 64 related genes were changed in the hippocampus of mice exposed to 500 ppm Zn. Notably, large changes in gene expression were observed for many olfactory receptors in the hippocampus of Zn-treated mice (Table 2). Previous studies of olfactory function in patients with AD showed that AD often results in impaired olfactory perceptual acuity, including reduced abilities to detect, distinguish, and identify odours (Mesholam et al., 1998). Moreover, epidemiological studies of olfactory dysfunction in elderly individuals and patients and dementia indicate that olfactory function may be a useful clinical marker for AD severity and progression (Zou et al., 2016). Christen-Zaech et al. (2003) reported that both the olfactory system and areas of the brain with extensive connections to the olfactory system demonstrate pathological changes, such as Aβ plaques and neurofibrillary tangles. Moreover, AD pathological changes have been described in hippocampal and olfactory cortical regions, and pathological changes occurring in the entorhinal cortex and associated neural systems may be responsible for early memory deficits in AD (Van Hoesen et al., 1991). Wesson et al. (2010) also reported a correlation between olfactory deficits and Aβ deposition, that is, a correlation between the magnitude and occurrence olfactory deficits and the spatiotemporal pattern of Aβ deposition. Indeed, mice overexpressing a mutated form of the human Aβ precursor protein exhibited non-fibrillar Aβ deposition in the olfactory bulb earlier than in any other brain region at 3 months of age.

Notably, substantial changes in gene expression of TTR receptors were observed in the hippocampus of Zn-treated mice (Table 2, Fig. 5). TTR, a homotetrameric protein produced mainly in the liver and brain’s choroid plexus, circulates in plasma and cerebrospinal fluid. Schwarzman et al. (1994) showed that TTR sequesters Aβ and prevents amyloid formation. On the other hand, there are some reports that TTR causes amyloidosis. Its aggregation is associated with senile systemic amyloidosis (Westermark et al., 1990), familial amyloid cardiomyopathy (Rapezzi et al., 2010), and familial amyloid polyneuropathy (Hou et al., 2007). Indeed, the misassembly of soluble proteins like TTR into toxic aggregates, including amyloid fibrils, underlies a variety of human diseases. The results of our microarray analysis and novel object recognition testing suggest that excess Zn affects the olfactory system, resulting in impaired learning and memory.

In conclusion, this study demonstrated that long-term Zn administration to aged mice dose-dependently inhibited learning and memory in novel object recognition and step-through passive avoidance tests, suggesting that excess Zn administration impaired long-term memory and object recognition memory in aged mice (Figs. 3 and 4). Although Zn neurotoxicity has been reported in in vitro experiments using cells, it has not been examined in in vivo experiments using mice (Akiyama et al., 2012). This is the first study to show that exposure to chronic excess Zn in drinking water impaired learning and memory in in vivo experiments using mice. Zn dyshomeostasis and oxidative stress might act to promote ageing-related neurodegeneration in adult mice. We found that Zn exposure caused changes in gene expression of TTR and olfactory receptors in the hippocampus. Thus, it is possible that Zn affects the olfactory system, resulting in impaired learning and memory. Aggregation of TTR may be associated with impairment of learning and memory. However, the relationship between changes in gene expression and results of behavioral tests, as well as the detailed mechanism of zinc neurotoxicity, remains unknown. As such, further studies will be necessary to elucidate molecular mechanisms underlying learning and memory deficits elicited by long-term administration of excess Zn.

ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI Grant Number JP26350163 and JP16H06276.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2019 The Japanese Society of Toxicology
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