Deficits of learning and memory in Hemojuvelin knockout mice.

Iron is involved in various physiological processes of the human body to maintain normal functions. Abnormal iron accumulation in brain has been reported as a pathogenesis of several neurodegenerative disorders and cognitive impairments. Hemojuvelin (HVJ) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition known as juvenile hemochromatosis. Although iron accumulation in brain has been related to neurodegenerative diseases, it remains unknown the effect of mutation of HVJ gene on cognitive performance. In our studies, HJV(-/-) mice showed deficits in novel object recognition and Morris water maze tests. Furthermore, the expression ration of apoptotic marker Bax and anti-apoptotic marker Bcl-2 in the hippocampus and prefrontal cortex showed higher levels in HJV(-/-) mice. Our results suggested that deletion of HJV gene could increase apoptosis in brain which might contribute to learning and memory deficits in mutant mice. These results indicated that HJV(-/-) mice would be a useful model to study cognitive impairment induced by iron overload in brain.

Hemojuvelin (HJV) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition [17]. Juvenile hemochromatosis (JH) is an early-onset form of hereditary disease which is caused by mutations of HJV gene [17]. The progressive tissue iron overload is the most important character in patients. A previous study showed that HJV is highly expressed in the skeletal muscle, liver and heart and plays a role in iron absorption and release from cells [13]. HJV gene acts as co-receptor for bone morphogenetic protein (BMP) receptor and signals via the products of the Drosophila Mod and C. elegans Sma genes (SMAD) pathway to regulate hepcidin expression [1]. Hepcidin is a key regulator of dietary iron absorption to maintain systemic iron homeostasis [4]. Patients with HJV mutation fail to properly upregulate hepcidin in response to iron [14]. HJV(−/−) mice showed increased iron deposition in liver, pancreas and heart and reduced hepcidin expression level, but no abnormalities in fertility and no obvious cardiac or endocrine abnormalities [5,6,7].

Iron, one of the important trace elements, is widely involved in various physiological processes of the human body to maintain normal metabolism. Iron participates in many important biological processes related to the development of the nervous system, such as myelination, neurotransmitter synthesis and mitochondrial energy production [21]. Iron accumulation in brain areas has been reported as a pathogenesis of several neurodegenerative disorders, such as Parkinson’s [21] and Alzheimer’s diseases [19]. Clinical studies found that patients with iron deposition in brain could exacerbate neuronal functions significantly associated with cognitive impairments [19, 21]. Excess iron accumulation promotes formation of free radical and causes injury of cell or tissue structures. Although one of the common features in HJV(−/−) mice as an animal model of JH is iron overload [5,6,7], it has not been examined whether disruption of iron homeostasis in HJV(−/−) mice causes neurodegenerative phenotype. To address this question, we investigated behavioral phenotypes and cellular mechanism of iron-overload in brain of HJV(−/−) mice. Our present study suggested that iron accumulation in brain caused by HJV mutation could lead to learning or memory impairments.

MATERIALS AND METHODS

Animals: Male HJV(−/−) and HJV(+/+) mice were kindly provided by Dr. Fudi Wang from Zhejiang University, Hangzhou, China. The mice were kept at room temperature (23 ± 2°C) and 55 ± 5% humidity under a 12/12-hr light/dark cycle (light from 8:00 am to 8:00 pm). All experiments were approved by the Animal Experiments Committee of China Astronaut Research and Training Center.

Novel object recognition (NOR) test: The NOR test was performed according to the protocol of Ennaceur and Delacour [2], with slight modification. The apparatus consisted of a closed container (60 cm length, 40 cm width and 80 cm height) made of polyvinyl chloride with a black and white cardboard pattern nailed to one of the walls. Two row LED bulbs were fixed above two sides of the closed container. The protocol consisted of 3 trials: habituation, training and test. Each mouse was individually habituated to the apparatus for 10 min in the absence of objects (habituation trial). 24 hr after the habituation trial, the mouse was placed in the apparatus for the training trial, and 2 identical objects were placed in a symmetrical position 10 cm above the side wall. The order of objects used per subject per trial was determined randomly. The total time spent exploring the two objects was recorded for 10 min. Exploration of an object was defined as directing the nose to the object and/ or touching it with the nose. 24 hr after the training trial, the mouse was placed in the apparatus for the test trial, but with 2 dissimilar objects, a familiar one and a novel one. The object which didn’t use in the training trial was used as the novel object in the test trial. The mice were allowed to explore freely for 10 min, and the time spent exploring each object was recorded. If recognition memory was intact, the mice were expected to spend more time exploring the novel object. The proportion of the total exploration time that the mouse spent exploring the novel object was the “object preference index” expressed by the ratio TN/(TF+TN), TF=time spent exploring the familiar object and TN=time spent exploring the novel object.

Morris Water Maze (MWM) test: MWM test was slightly modified from the previous study [24]. MWM test was divided into 2 different phases: place navigation test (hidden platform) and spatial probe test (removal of the platform). In the place navigation test, mice daily performed 4 different starting points for 5 consecutive days. If a mouse fails to find the platform in 90 sec, it is usually picked up and placed on platform for 15 sec. Mice were placed in a randomized starting point with their head towards the board of the pool, and the time started recording after placing the mouse into the water and stopped recording when it found the submerged platform. All mice performed the spatial probe test on the sixth day. Time recording started after placing the mouse into the water, and the experiment was finished after 90 sec. Time to reach the platform and time spent in the quadrant which previously contained the platform were recorded. The tank was 100 cm in diameter, and all testing conditions and trials were identical for the two groups.

RNA Isolation and Real-time Reverse Transcription polymerase chain reaction (RT-PCR) analysis: Hippocampus and prefrontal cortex were frozen in liquid nitrogen, and the mRNA was extracted with TRIzol (Life Technologies, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol. Optimal polymerase chain reaction (PCR) conditions were determined for primer sets designed from the sequences of the Bax and Bcl-2 genes, which had the following sequences: Bax (forward, 5- GGGCCCACCAGCTCTGA-3, reverse, 5-TGGATGAAACCCTGTAGCAAAA-3; Accession No: NM 007527) and Bcl-2 (forward, 5-TGGGATGCCTTTGTGGAACT-3, reverse, 5-CAGCCAGGAGAAATCAAACAGA-3; Accession No: NM 009741). Each PCR mixture contained 21 µl of sterile water, 25 µl SYBR Green (Applied Biosystems, Foster City, CA, U.S.A.), 2 µl of cDNA (500 ng/µl), 1 µl of forward primer (10 pM/µl) and 1 µl of reverse primer (10 pM/µl). PCR was performed an initial denaturation at 95°C for 10 min, followed by 40 cycles each consisting of 95°C for 10 sec, 62°C for 15 sec and 72°C for 20 sec. Gene amplification was performed according to a melting program of 70°C for 15 sec, and fluorescence was monitored continuously during the change in the temperature of 0.3°C/sec from 60 to 95°C. The final level of mRNA expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a housekeeping gene. The sequences of primers for GAPDH gene were as follows:

GAPDH (forward, 5-TTGTGATGGGTGTGAACCACGAGA-3, reverse, 5- CATGAGCCCTTCCACAATGCCAAA-3; Accession No: NM 008084).

Protein extraction and western blot analysis: The extracts were prepared by lysing the hippocampus and the prefrontal cortex in 20 mM Tris-HCl, pH7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol and protease inhibitors on ice for 20 min. The cell debris was separated by centrifuging at 12,000 × g for 5 min at 4°C. Total protein concentration of the supernatant was determined using Bradford protein assay reagent (Bio-Rad Laboratories, Shanghai, China). Samples each equivalent to 30 µg of the total protein was loaded onto 12% polyacrylamide gels, electrophoresed in Tris/glycine buffer and then electro blotted onto the NC membranes. The blots were blocked in 5% non-fat dry milk dissolved in Tris-buffered saline (TBS, pH7.4) containing 0.1% Tween-20 (TBST) for 2 hr, then probed overnight at 4°C with primary antibodies against Bax (bs-0127R; Bioss Inc., Shanghai, China), Bcl-2 (bs-0032R; Bioss Inc.) and β-actin (sc1616; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A) and washed in TBST, followed by an incubation with HRP-conjugated secondary antibodies. The ECL-kit (Prod34080; Thermo Fisher Scientific, West Palm Beach, FL, U.S.A.) was used to detect the proteins bound to their respective antibody. Proteins were quantified using the densitometer, Image-Pro Plus 6.0 software.

Date analysis: The data for each experiment were presented as mean ± standard error of the mean (SEM) and were processed using SPSS 16.0 software. Data of escape latency in the MWM test were analyzed using repeated measures analysis of variance (ANOVA), and other data were analyzed using One-way ANOVA for comparison between groups. P<0.05 was considered as statistically significance.

RESULTS

Impaired novel object recognition memory: Clinical studies suggested that patients with iron deposition in brain might impair the neuronal functions and cause cognitive impairments [19, 21]. Although HJV(−/−) mice showed iron overload [5,6,7], it has not been examined whether disruption of iron homeostasis in HJV(−/−) mice causes cognitive phenotypes. To address this question, we first investigated the HJV(+/+) mice on novel object recognition test (NOR). After trained with two identical objects, mice were introduced to two distinct objects during the test session. HJV(+/+) mice (Fig. 1A) showed a significant preference for novel object comparing familiar object. In contrast, HJV(−/−) mice (Fig. 1B) showed no preference for novel object comparing familiar object. The data indicated the impairment of recognitive memory in HJV(−/−) mice.

Fig. 1.

Object recognition test. A and B) Data show object preference index of HJV(+/+) (A) and HJV(−/−) (B) mice. Data are represented as mean ± SEM. n=9 for each group. **P<0.01, compared with familiar object in HJV(+/+) and HJV(−/−) mice.

Impaired spatial learning and memory: Water maze is widely used to test the spatial learning and memory ability related to hippocampal function in mice. Then, we investigated the HJV (+/+) mice on water maze test. Deletion of HVJ gene caused increased time to reach the platform (Fig. 2A) in the place navigation test, decreased time spent in target quadrant (Fig. 2B) and decreased numbers of crossing the platform (Fig. 2C) in the spatial probe test. These data indicated the deficit of spatial learning and memory in HJV(−/−) mice.

Fig. 2.

Morris water maze test. A to C) Data show latency to reach the platform (A), percentage of time in target quadrant (B) and number of crossing the platform (C) in HJV(+/+) (WT) and HJV(−/−) (KO) mice. Data are represented as mean ± SEM. n=8 for each group. *P<0.05, **P<0.01 comparison between HJV (+/+) and HJV(−/−) mice.

Alternation of the expression patterns of apoptosis markers: Since HJV(−/−) mice exhibit iron-overload conditions in brain [5], rats fed with high iron-diet consumption result in brain iron accumulation and brain apoptosis [22]. We looked at the apoptosis status which might be one of the possible mechanisms responsible for the learning and memory impairments in HJV(−/−) mice. We confirmed the specificity of the primers we used in RT-PCR first (Supplementary Fig. 1: online only) and then checked mRNA expression patterns of anti-apoptotic marker Bcl-2 and apoptotic marker Bax. In hippocampus (Fig. 3A) and prefrontal cortex (Fig. 3B), we observed up-regulation of Bax mRNA and down-regulation of Bcl-2 mRNA in HJV(−/−) mice, suggesting increased signaling of apoptosis in the mutant mice. We then measured protein expression patterns of these markers using Western blot analysis. Although hippocampus (Fig. 4A, upper panel) and prefrontal cortex (Fig. 4B, upper panel) showed similar expression patterns of anti-apoptotic marker Bcl-2 between HJV(+/+) and HJV(−/−) mice, the expression of apoptotic marker Bax was increased in HJV(−/−) mice. In hippocampus (Fig. 4A, lower panel) and prefrontal cortex (Fig. 4B, lower panel), the ratio of Bax/Bcl-2 was significantly increased in HJV(−/−) mice compared with HJV(+/+) mice. These results indicated increased level of apoptosis in HJV(−/−) mice.

Fig. 3.

RT-PCR analysis. A and B) Data show relative mRNA levels of Bax and Bcl-2 in hippocampus (A) and prefrontal cortex (B) of HJV(+/+) (WT) and HJV(−/−) (KO) mice. Data are represented as mean ± SEM. n=4 for each group. *P<0.05, **P<0.01 comparison between HJV(+/+) and HJV(−/−) mice.

Fig. 4.

Western blot analysis. A and B) Data show protein expression patterns of Bax and Bcl-2 in hippocampus (A, upper panel) and prefrontal cortex (B, upper panel) of HJV(+/+) (WT) and HJV (−/−) (KO) mice. Data also show ratio of Bax/Bcl-2 in hippocampus (A, lower panel) and prefrontal cortex (B, lower panel) of HJV(+/+) (WT) and HJV(−/−) (KO) mice. Data are represented as mean ± SEM. n=4 for each group. **P<0.01 comparison between HJV(+/+) and HJV(−/−) mice.

DISCUSSION

Previous reports showed that male Wistar rats fed with high iron-diet consumption for 12 weeks resulted in brain iron accumulation, brain mitochondrial dysfunction, impaired brain synaptic plasticity and cognition, blood-brain-barrier breakdown and brain apoptosis [22]. Combined iron chelator and anti-oxidant therapy attenuated deleterious effects and restored brain functions [22]. In a previous study, iron overload resulting in iron accumulation in brain produces cognitive deficits in an animal model [19]. Recent studies described that neurodegenerative diseases had mutation in hemochromatosis, transferrin [10] and apolipoprotein E genes [15]. These findings provide evidences that disruption of iron homeostasis, resulting in iron accumulation in brain regions, is the pathogenesis of neurodegenerative disorders. However, the mechanism of neurodegenerative disorder induced by iron-overload remains unrevealed.

Juvenile hemochromatosis (JH) is an autosomal recessive disorder caused by mutation of HJV gene and characterized by progressive tissue iron overload [16]. It has been reported that HJV mRNA is widely expressed in brain, liver, heart, lung, stomach, spleen, kidney, duodenum, jejunum, ileum, colon, skeletal muscle, testis and blood [17]. The main symptoms of JH include absent or decreased function of the testes in males or ovaries in females (hypotrophic hypogonadism), heart disease, scarring of the liver (cirrhosis), joint disease, diabetes and dark discoloration of patches of skin (hyperpigmentation) [16]. However, the precise physiological role of HJV in brain has not been determined. Our present data showed that HJV(−/−) mice showed deficits of learning and memory and exhibited apoptosis in hippocampus and prefrontal cortex. In the NOR test, we observed that the amount of time spent exploring between novel and old objects was no significant differences between HJV(+/+) and HJV(−/−) mice, but HJV(+/+) mice exhibited robust preference for novel object. These result indicated that HJV (+/+) mice could remember and discriminate the old object, but HJV(−/−) mice could not. In the MWM test, HJV(−/−) mice performed longer escape latency in the place navigation test and showed decreased time spent and numbers of crossing the platform in spatial probe test. Therefore, loss function of HJV gene in brain could result in spatial learning and memory impairments. It is well known that apoptosis mediated neuronal death is responsible to cognitive and memory symptoms of neurodegenerative disorders in humans and animal models [9, 11]. Bcl-2 forms a heterodimer with Bax to inhibit from the apoptosis progress [26]. Thus, the Bax/Bcl-2 level indicates progress of apoptosis. Our RT-PCR analysis showed that Bax mRNA levels had a tendency of increase, and Bcl-2 mRNA levels were significantly decreased in hippocampus and prefrontal cortex in HJV(−/−) mice compared to HJV(+/+) mice. Western blot analysis exhibited that Bax of hippocampus and prefrontal cortex in HJV(−/−) mice showed significantly increased level and Bcl-2 showed similar levels between HJV(+/+) and HJV(−/−) mice. Although expression patterns were different between mRNA and protein levels, the ratio of Bax/Bcl-2 increased in hippocampus and prefrontal cortex of HJV(−/−) mice compared to HJV(+/+) mice. These results indicate that the signaling pathway of apoptosis is activated in hippocampus and prefrontal cortex of HJV(−/−) mice. Because previous studies have reported that the hippocampus and prefrontal cortex are involved in spatial memory and object recognition memory, respectively [3, 12, 18, 27], the results of relationship between behavioral test and expression analysis are reasonable. We noticed that there were different patterns between transcription and translation of BCL2 in HJV(−/−) mice. Several studies showed that protein level was not always proportional to mRNA and these could be caused by the processes of transcription, mRNA decay, translation and protein degradation and stability [8, 20, 23, 25]. Our data suggested that we might need a further study of the possibility that undefined mechanisms are responsible for post-transcriptional regulation of BCL2 expression.

In conclusion, the present study showed deficit of cognitive function and activation of apoptosis pathway in the hemojuvelin knockout mice. Our results indicate that hemojuvelin knockout mice would be a useful model to understand neurodegenerative diseases induced by iron overload brain.