Abstract
Manganese (Mn) superoxide dismutase (SOD) is mainly located in mitochondrial matrix and is responsible for scavenging about 80% free radicals from oxidative and phospharylative process in mitochondria. It was reported that the insufficiency of Mn SOD expression or activity was connected to the development of neurodegenerative diseases. In this article, we investigated the time course related to the changes of Mn SOD expression and its activity from mouse brain as well as the recognition dysfunction in chronic aluminum (Al) overloading mice. Aluminum gluconate solution (equal to Al 400 mg/kg) was given to mice once a day, 6 days per week for 12 weeks via intragastric gavage. The learning and memory function, malondialdehyde (MDA) level as well as expression and activity of Mn SOD in cortex were determined. It was found that function of passive learning and memory and spatial recognition decreased, MDA level and Mn SOD expression increased during the period of chronic Al loading, but the Mn SOD activity rose from the 4th week and then decreased from the 8th week in cortex in Al overloading mice compared with the control. The results indicated that the inconsistency between Mn SOD expression and its activity might contribute to the development of recognition dysfunction induced by chronic Al overload.
Introduction
With the coming of an aging society, more and more neurological diseases related to aging have been diagnosed, especially neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and so on. The main symptoms include a series of changes characterized by the presence of abnormal protein components that can cause neuronal damage or death in the certain regions of the central nervous system. However, the pathophysiological mechanisms of these neurodegenerative diseases have not been totally understood as yet. 1 –3
It has been confirmed that oxidative stress plays a pivotal role in the development of neurodegenerative diseases and the aging. 3 –5 Reactive oxygen species (ROS) are by-products from oxidative phosphorylation of mitochondrial respiratory chain, which were usually detoxified by some antioxidants, such as catalase, glutathione peroxidase, glutathione reductase, and manganese superoxide dismutase (Mn SOD). Dysfunction of the antioxidative stress system will result in insufficiency of ROS scavenging, which would lead to a high level of free radicals and subsequently lipid peroxidation and serious cell and tissue injury. 6 Federico et al. reviewed numerous evidences of oxidative stress that play a crucial role in neurodegeneration. 7 Malondialdehyde (MDA) as a product of lipid peroxidation caused by ROS also can be an indicator of lipid oxidation injury. 8 The dysfunction of spatial learning and memory is a notable phenomenon in neurodegeneration. The Morris water maze is a popular test for learning and memory deficits and has been widely used to study the recognition function in neurodegeneration rodent models. 9–10 In this study, MDA and the Morris water maze were used to evaluate the oxidative stress injury and spatial learning and memory function, respectively.
It is well known that mitochondria contain their own DNA that encodes polypeptide components of the respiratory chains as well as some ribosomal RNA and transfer RNA to support intramitochondrial protein synthesis. However, there exists an insufficiency of effectively repairing mechanisms and protecting factors in the mitochondria, which means that mitochondrial DNA are more susceptible to self-produced ROS damage. 3–4 Many researchers have confirmed that mitochondrial membrane dysfunction played an important role in the process of neurodegeneration. 3,4,11–12
Mn SOD is located predominantly in mitochondrial matrix and plays a key role in scanveging mitochondrial superoxide. 13–14 The overexpression of Mn SOD could improve antioxidant ability of many organs and reduce the permeability of mitochondrial membrane in mice or rats. 15–16 Curti et al. reported that upregulation of Mn SOD expression showed in vivo protective activities on human peripheral blood mononuclear cells. 17 On the other hand, inhibition of Mn SOD expression should lead to an accumulation of ROS, resulting in lipid peroxidation, DNA damage, and apoptosis. Paul et al. reported that Mn SOD-reduced expression would shorten the life span. 18 Lebovitz et al. and Kinoshita et al. reported that Mn-SOD knockout mice suffered from neurodegeneration, myocardial injury, perinatal death, and age-related hearing loss. 19,20
Aluminum (Al) is a nonessential metal element with obvious neurotoxicity. Chronic Al accumulation can induce oxidative stress and mitochondrial dysfunctions and consequently lead to the development of neurodegenerative diseases including AD, but the mechanism is still not understood. 21 –23
In this study, a chronic Al overloading model was established in mice through intragastric administration of aluminum gluconate solution in order to investigate the time course of Al-caused learning and memory dysfunction connected with Mn SOD expression and activity in the cortex. The results of this study might unveil a possible relation of Al-induced neurodegeneration and Mn SOD expression and/or activity.
Materials and methods
Animals and experimental design
A total of 210 male BALB/c mice (8 weeks old weighing 20–25 g) were provided by Animal Experimental Center of Chongqing Medical University, China. Mice were housed and habituated for 1 week before the experiments started and then divided into two groups. Group 1 was continuously treated with aluminum gluconate solution (equal to 400 mg/kg Al, once a day for 6 days per week) through intragastric gavage, while group 2 was given same volume vehicle instead. The aluminum gluconate solution was prepared by dissolving sodium gluconate (2.25 g) and aluminum chloride hexahydrate (4.07 g) in 100 ml water according to the method described by Ward et al. 24 The animal experiments were performed and data were obtained from 15 mice for each group at the 1st, 2nd, 3rd, 4th, 6th, 8th, and 12th week after continuous administration of aluminum gluconate solution, respectively. All procedures were performed in accordance with the Guidelines to the Care and Use of Experimental Animals (approval number: 20100055) and met the standards approved by the Animal Care and Use Committee of Chongqing Medical University.
Morris water maze test
The test was performed followed the methods described by Inman-Wood et al. 25 In the Morris water maze test, the mice were trained to seek a visible platform in water maze apparatus for 4 days; a total of four trials for each mouse were performed every day; on day 5, the platform was hidden below the surface of water and the time that mice took to cross the original platform first was used to evaluate the spatial recognition function.
Measurement of MDA level and SOD activity
After the mouse was anesthetized with 4% chloral hydrate through intraperitoneal injection, the brain was dissected and the cortex was isolated. The cortex was made 10% (wt/vol) homogenate with saline and then comparable MDA level and SOD activity were tested according to MDA and SOD kit manual provided by the manufacturer (Nanjing Jiancheng Bioengineering Company, Nanjing, China).
Measurement of mRNA expression by RT-PCR
After the mouse brain was dissected, the cortex was isolated to extract total RNA using the total RNA isolation kit (Invitrogen, Gaithersburg, Maryland, USA). Then, 4 µl of the total RNA extract was processed for complementary DNA (cDNA) synthesis using the first-strand cDNA synthesis kit (Invitrogen). Then, 4 µl of the cDNA synthesis was placed into the Bio-Rad cycler instrument (Bio-Rad, Hercules, California, USA). The polymerase chain reaction (PCR) conditions were as follows: for β-actin, 28 cycles at 94°C for 5 min, 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and 72°C for 5 min; for Mn SOD, 30 cycles at 94°C for 5 min, 94°C for 30 s, 60.5°C for 30 s, 72°C for 1 min, and 72°C for 5 min. Next, the PCR products were subjected to 2% agarose gel electrophoresis, scanned with Bio-Rad Gel Doc 100 (Bio-Rad) and analyzed using the Quantity One software.
The sequences of the primer used were as follows: 5′-GGCACCTTTCTCAGTAGCGG-3′ (forward) and 5′-CTAAGGGACCCAGACCCAAC-3′ (reverse) for Mn SOD; 5′-ATCATGTTTGAGACCTTCAACA-3′ (forward) and 5′-CATCTCTTGATCGAAGTCCA-3′ (reverse) for β-actin.
Western blotting
After the mouse brain was dissected on ice, the cortex was isolated and homogenized in buffer (50 mmol/L tris(hydroxymethyl)aminomethane)–hydrochloric acid (pH 8.0), 1% Triton X-100,150 mmol/L sodium chloride, and 100 µg/mL phenylmethanesulfonyl fluoride) for 30 min and then centrifuged at 12,000g for 5 min at 4°C (Sigma-3k30, St Louis, Missouri, USA). Protein level was measured using the Bradford protein assay reagent and protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were recognized using goat anti-mouse Mn SOD monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Californua, USA). The optical density of the Mn SOD bands was measured and analyzed using Gel DocTM XR (Bio-Rad).
Statistical analysis
All data were described with mean ± SEM. The difference between groups was compared by t-test, p < 0.05 was considered as significant difference.
Results
Morris water maze test
The results showed that the time which model mice took to search for the platform in Morris water maze test was prolonged significantly at the 4th week after experiment began, reached a plateau value at the 8th week, and kept it till the 12th week, compared with corresponding control mice (p < 0.01, Figure 1).
Effects of aluminum overloading on learning and memory function of mice in Morris water maze. Mean values, n = 15. □: control group and ▪: aluminum overloading group. *p < 0.05; ** p < 0.01: versus corresponding control.
MDA level and Mn SOD activity in cortex
Figure 2 showed that MDA level in cortices significantly increased at the 6th week and kept at high levels until the end of experiment at the 12th week in model mice compared with the control mice. Mn SOD activities in cortices also increased in a manner similar to that of MDA levels but marked increase in Mn SOD activities occurred at the 4th week, reached the maximum at the 8th week, and then clearly decreased in model mice in comparison with the corresponding control mice.
Effects of aluminum overloading on MDA level and Mn SOD activity in cortices of mice. Mean values, n = 10. □: control group and ▪: aluminum overloading group. *p < 0.05; **p < 0.01: versus corresponding control.
Expression of Mn SOD mRNA and protein in cortex
Figure 3 shows that messenger RNA (mRNA) and protein expression of Mn SOD significantly increased in a time-dependent manner at the 4th week after experiment started in model mice compared with the corresponding control mice (p < 0.05 and p < 0.01), with the high level of expression kept till the end of experiment.
Effects of aluminum overloading on Mn SOD mRNA and protein expression in cortices of mice. RT-PCR and Western blot were used to determine the mRNA and protein expressions of both Mn SOD from the cortices and internal control β-actin. Results were represented as a ratio of Mn SOD expressive amount to that of β-actin. Mean values, n = 5. □: control group and ▪: aluminum overloading group. *p < 0.05; **p < 0.01: versus corresponding control. Mn: manganese; SOD: superoxide dismutase; mRNA: messenger RNA; RT-PCR: reverse transcriptase polymerase chain reaction.
Discussion
Al is one of the most abundant elements in the Earth’s crust and also one of the metals frequently contacted in our daily life. It is well known that Al is a toxic element to neural system when it is chronically accumulated in the brain, but the mechanism is not quite clear. 21 –23 Our previous work evidenced that an Al overloading in mice through intracerebroventricular injection of aluminum chloride solution showed oxidative damage to brain, high expression of heme oxygenase-1, 26 and suggested that chronic Al overloading might trigger an unknown oxidative stress/antioxidative stress cascade that promoted a neurodegenerative process. But the key candidate factor involved in the cascade of neurodegenerative process still keeps to be illustrated. Kumar et al. reported that Al-induced oxidative stress resulted in decreasing activity of Mn SOD and damage to mitochondrial DNA in brain, which was associated with neurodegeneration. 27–28 But it was not clear why the antioxidative enzyme systems in the body failed to be against the attack of ROS when chronic Al accumulate in the brain. In this study, we established the chronic Al overloading model of mice by chronic administration of aluminum gluconate solution via intragastric gavage and found that chronic Al overloading induced learning and memory dysfunction (Figure 1), and there existed a sustained increase of not only MDA level but also Mn SOD expression in the brains, which indicated that chronic Al overloading induced elevation of oxidative reaction/antioxidative stress reaction in brain and antioxidant stress system (such as Mn SOD) failed to thoroughly scavenge free radicals (Figure 2).
SOD is one of main enzymatic antioxidants and a primary defense against oxidative stress injury in brain. There are three types of SOD, namely, copper/zinc SOD (Cu/Zn SOD), Mn SOD, and extracellular SOD (EC-SOD). The Cu/Zn SOD is a soluble cytosolic enzyme, Mn SOD is located in the matrix of cellular mitochondria, and EC-SOD is mainly found in extracellular fluids of mammals. The Cu/Zn SOD and Mn SOD are the main SOD types against antioxidative stress in human body. 13 Studies showed that the mitochondrial dysfunction played a critical role in the development of neurodegenerative disorders. The mitochondria are only organelles with unique genetic materials in the cells and responsible for making chemical energy and generating 90% of the endogenous ROS of the cell as a by-product. In normal condition, about 80% of these free radicals are cleared by Mn SOD. The mitochondria are easy to be attacked by free radicals if insufficiency of Mn SOD expression or activity occurs, which may be associated with the neurodegenerative diseases. 3–4 Therefore, Mn SOD plays a key role in the antioxidative stress damage. Our experimental results show that there is an elevation first, followed by a gradual decrease of the Mn SOD activity, and finally leading to an imbalance between MDA level and SOD activity in cortices (Figure 2). Constantly increasing expressions of Mn SOD mRNA and protein could not explain the decrease of its activity during the period of Al overloading. This phenomena mean that Al overloading may lead to some mistaken events in the process of transcription and/or translation of Mn SOD gene or modification of the Mn SOD protein, which in turn causes a decreasing activity of Mn SOD and consequently leading to an imbalance between oxidative stress and antioxidative stress systems in brain, triggering neurodegenerative cascade process. Our hypothesis was supported by Kumar et al. who reported that Al not only induced oxidative damage to Mn SOD and aconitase in mitochondria but also decreased expression of Lon protease that could remove the modified protein enzymes. 27 It has been evidenced that Mn is a trace element in the body and increases the formation of ROS. 29 This may be another reason for high MDA level to remain with a decreased Mn SOD activity at the 12th week of experiment if the enzyme was consumed and Mn was released from the enzyme. However, more experiments are needed to confirm the above hypothesis.
In conclusion, the results demonstrated that the mechanism of neurodegeneration induced by chronic Al overloading might be related to triggering oxidative stress system/antioxidative stress system cascade and causing inconsistency among Mn SOD expression, its activity, and MDA level in brain.
Footnotes
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.