Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized clinically by cognitive decline and memory loss. The pathological features are amyloid-β peptide (Aβ) plaques and intracellular neurofibrillary tangles. Many studies have suggested that oxidative damage induced by reactive oxygen species (ROS) is an important mechanism for AD progression. Our recent study demonstrated that oxidative stress could further impair mitochondrial function. In the present study, we adopted a transgenic mouse model of AD (mAPP, overexpressing AβPP/Aβ in neurons) and performed redox measurements using in vivo electron paramagnetic resonance (EPR) imaging with methoxycarbamyl-proxyl (MCP) as a redox-sensitive probe for studying oxidative stress in an early stage of pathology in a transgenic AD mouse model. Through assessing oxidative stress, mitochondrial function and cognitive behaviors of mAPP mice at the age of 8-9 months, we found that oxidative stress and mitochondrial dysfunction appeared in the early onset of AD. Increased ROS levels were associated with defects of mitochondrial and cognitive dysfunction. Notably, the in vivo EPR method offers a unique way of assessing tissue oxidative stress in living animals under noninvasive conditions, and thus holds a potential for early diagnosis and monitoring the progression of AD.
INTRODUCTION
Alzheimer’s disease (AD) is a predominant causeof dementia in the elderly population, affectingabout 44 million people worldwide in 2015 [1].AD is pathologically defined by aggregation of amyloid-β (Aβ) peptide that has been derived from pathologically processed amyloid-β protein precursor (AβPP) in extracellular senile plagues and neurofibrillary tangles which consist of hyperphosphorylated tau protein and lead to severe neuronal loss through oxidative stress and neurodegeneration [2, 3]. The mitochondrial dysfunction and increased oxidative stress have been observed in AD-affected brain and mouse model for AD [2, 5]. Oxidative stress leads to an alteration in redox state resulting from an imbalance between the generation and detoxification of reactive oxygen species (ROS), which can be detected in the blood, cerebrospinal fluid, and brain of AD patients [6–9]. ROS plays an important role in many chronic diseases including mitochondrial diseases [10], atherosclerosis [11], diabetes [12], cancer [13], as well as AD [14]. The brain’s high energy demand is supplied mostly by OXPHOS, the major producer of free radicals.When the free radical level overwhelms cellular antioxidant defense system, a deleterious conditionknown as oxidative stress occurs. Oxidative stressand mitochondrial dysfunction can emerge in thecourse of aging and AD [15, 16]. Considerabledata implicate a mechanistically relevant rolefor ROS in AD. In particular, evidence has suggested that elevated ROS level might increase Aβ production [17]. It is noteworthy that age and life-style related AD risk factors, such as hypertension, traumatic brain injury, diabetes mellitus, hypercholesterolemia, hyperhomocysteinemia, smoking, high calorie intake, and lack of exercise, are contributors to the increased oxidative stress. There are several reports indicating that oxidative damageprecedes the onset of AD pathology [3, 18]. In view of the important role ROS play in healthand disease, it is very useful to develop a reliable quantitative noninvasive method for the assessment of oxidative stress in live animals and humans. The present study was aimed at demonstrating electron paramagnetic resonance (EPR) imaging [19, 20] using methoxycarbamyl-proxyl (MCP) as a redox-sensitive probe for visualizing oxidative stress in living subjects and studying oxidative stress and mitochondrial function in the early stageof AD.
MATERIALS AND METHODS
Animals
All the animal studies were approved by the Animal Care and Use Committee of the University of Kansas and Ohio State University in accordance with the National Institutes of Health guidelines for animal care. Transgenic mice (Tg mAPP), both male and female, that overexpress a human mutant form of AβPP bearing both the Swedish (K670N/M671L) and the Indiana (V717F) mutations (APPSwInd, J-20 line, obtained from Jackson Lab) were used in the experiments. The investigators were blinded to the mouse genotype in performing experiments.
Electron paramagnetic resonance measurements
In vitro EPR measurements
Intracellular ROS levels were measured using EPR spectroscopy as described in our previous study [21]. Hippocampal slices were incubated with CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine; 100μM) for 30 min, and then washed with cold PBS. The cerebral cortex from Tg mAPP mice and control WT mice were collected and homogenized with 100μl of PBS for EPR measurement. Samples were loaded into 50μL BLAUBRAND micropipettes (Sigma), which were sealed with Critoseal (Fisher) and placed in 4 mm EPR tubes (Wilmad Labglass, Vineland, NJ, USA). The EPR spectra were collected, stored, and analyzed with a Bruker EMXplus EPR spectrometer (Billerica, MA, USA) and Bruker software Xenon. The EPR spectrometer was operated at 9.63 GHz and 100 kHz field modulation at room temperature. The spectra were recorded with the following parameters: number of scans, 6; magnetic field center, 344 mT; scan range, 10 mT; microwave power, 2 mW; modulation amplitude, and 0.1 mT; time constant, 0.08 s.
In vivo EPR imaging measurements on live animals
EPR imaging measurements were performed using EPR instrumentation consisting of an L-band EPR spectrometer, three sets of water-cooled gradient coils, and a personal computer-based data acquisition system [19, 20]. EPR spectra were recorded using a custom-built surface resonator. The resonator was capable of sampling a cylindrical volume measuring a diameter of 10 mm and a depth of 5 mm. The open structure of this resonator was ideal for localized measurements on large objects and thus was not limited by the size of the object.
Tg mAPP and littermate control (nonTg) mice at age 8-9 months were anesthetized by administration of ketamine and xylazine. The carotid artery was cannulated with a heparin-filled 30-gauge catheter for infusion of the redox (nitroxide) probe. The hair of the skin on the observation spot (head) was shaved and the animal was placed on a bedplate with a circular slot (20 mm diameter) in such a way that the head was centered at the slot. The animal was secured to the plate with adhesive tape and placed on top of the resonator. An IR lamp was used to maintain normal body temperature, which was measured using a rectal thermistor probe. MCP probe (Alexis) was administered at a dose of 1μl/g bw at 200 mM concentration via carotid artery. EPR measurements were started immediately up on completion of probe infusion. The common carotid artery was occluded during EPR measurement to minimize the effects of blood flow. Projection data were acquired using angular sampling method. The projections were acquired as single scans (1024 points/projection) using constant sweep time. The measured projections were corrected for removal of hyperfine-based artifacts and deconvoluted with the corresponding zero-gradient projection [22]. The deconvoluted projections were then convoluted with a Shepp-Logan filter and subsampled to 128 points for back projection. A single-stage, filtered back projection reconstruction algorithm was used to recover the image.
ROS refer to a number of reactive oxidants including superoxide, hydroxyl, and peroxyl radicals as well as non-radical species including hydrogen peroxide. In most of the cases, such as the study here, superoxide free radicals are the first species generated by the cell/mitochondria while all other species are derived as by-products of superoxide. The superoxide free radical is known to reach with CMH probe, which is EPR silent, and converts it to a nitroxide radical (paramagnetic), which is EPR active and exhibits a characteristic triplet EPR signal. The specificity of CMH for superoxide free radicals has been well established. Thus the appearance of a triplet EPR signal is indicative of superoxide (ROS) and the peak height is a measure of the amount of superoxide accumulation in the tissue.
Aβ measurement
Brain cortical homogenates were incubated in 5-M guanidine HCl and 50-mM Tris HCl (pH 8.0) overnight and then subjected to Aβ concentration detection using human Aβ1 - 40 and Aβ1 - 42 ELISA kits (Invitrogen) following the manufacturer’s instructions [18].
Cytochrome c oxidase (CcO) activity assay
Cytochrome c oxidase (COX IV) activity was spectrophotometrically determined using Cytochrome c Oxidase Assay Kit (Sigma). In brief, cortex of transgenic mAPP mice and non Tg mice were homogenized in the lysis buffer, incubated on ice for 15 min, and centrifuged at 14,000 g for 15 min. Suitable volume of supernatants and enzyme solutions were added into 950-μl assay buffer. The reaction was triggered by the addition of 50μl ferrocytochrome c substrate solution (0.22 mM) into the cuvette. The changes in absorbance of cytochrome c at 550 nm were recorded immediately using a kinetic program with 5-s delay, 10-s interval, and total 6 readings on an Ultrospect 3100 Pro spectrophotometer.
Measurement of ATP level
ATP levels in cortex were determined using an ATP Bioluminescence Assay Kit (Roche) following the manufacturer’s instruction. Mice brain tissues were homogenized in lysis buffer provided in the kit, incubated on ice for 15 min, and centrifuged at 14,000 g for 15 min. Subsequent supernatants were measured for the ATP levels using a Luminescence plate reader (Molecular Devices) with an integration time of 10 s.
Behavioral test
The Morris water maze (MWM) test was performed according to the published method [23]. The platform was hidden 0.5–1 cm below the water surface and the white paint was used to better cover the platform. In spatial acquisition session, mice were trained for 6 consecutive days with 4 trials each mouse per day. A trial started with releasing one mouse facing the pool wall and the mouse was allowed to swim freely and search for the escape platform. If the mouse could not reach the platform within 60 s, it was guided to the platform and allowed to stay on for 15 s before the next trial. After all trials, each mouse was dried with paper towels and returned to its own cage. The escape latency was analyzed by the behavior software system (HVS water 2020). On day 7, a probe trial was performed to assess the spatial memory of mice. The platform was removed from the pool and the mice were allowed to swim freely for 60 s. Traces of mice were recorded and data were analyzed by HVS water 2020.
Statistical analysis
One-way ANOVA was used for repeated measure analysis, followed by Fisher’s protected least significant difference for post hoc comparisons. p < 0.05 was considered significant. StatView statistics software was used. All data were expressed as the mean±SEM.
RESULTS
Increased ROS levels in the brain of transgenic mAPP mice
To determine whether oxidative status correlates with AD pathology and mitochondrial dysfunction, transgenic mice overexpressing human Aβ and mutant AβPP (Tg mAPP) were used for our study. Tg mAPP mice have been well-characterized with respect to mitochondrial dysfunction and its associated neuropathological, behavioral, and electrophysiological alterations [5, 24–29]. We used EPR spectroscopy and imaging to measure and map the ROS-mediated tissue oxidative stress in the brain, both in vitro and in vivo, using a redox-sensitive nitroxyl spin probe MCP. MCP has been used as a blood-brain barrier-permeable nitroxide probe to evaluate redox status [30]. In vivo bioreduction of MCP in some tissues, such as tumor tissue, is mostly caused by its reaction with intracellular reductants such GSH and ascorbic acid [20]. However, MCP is also known to react with reactive oxygen radicals, such as superoxide and peroxyl radicals that occur in tissues under oxidative stress; therefore, MCP reduction can be used as a measure of the oxidative stress. The bioreduction of MCP can be measured conveniently in real-time by in vivo EPR spectroscopy [30]. The change of EPR signal of MCP injected into animals has been observed as “enhanced signal decay” in various diseases [31–33]. The initial reduction rate of the probe in the brain was determined from a plot of signal intensity versus time, 2–7 min after injection. EPR image of the probe distribution in the brain, in vivo, was obtained at approximately 30 min after infusion of the probe. The rate of bioreduction in the Tg mAPP mice was significantly higher than that in the nonTg littermates (Fig. 1), suggesting that the brain tissue of Tg mAPP mice had higher oxidative stress as compared with nonTg mice. To validate the in vivo EPR results, we measured ROS in brain tissue homogenates by in vitro EPR spectroscopy. The EPR results showed that Tg mAPP mice had significantly enhanced oxidative stress in Aβ-enriched hippocampus and cortex but not in Aβ-spared cerebellum compared to the nonTg littermates at 8-9 months of age in cerebral cortex (Fig. 2). Furthermore, compared to nonTg brain at age of 3 months prior to amyloid deposit in the brain, no significant changes in EPR signal were found in mAPP cortex including hippocampus (date not shown), suggesting that ROS levels correlate to the Aβ accumulation in the brain. The enhanced oxidative stress level measured in vitro was similar to the results obtained from the in vivo measurements of the redox decay and distribution by EPR spectroscopy and imaging (Fig. 1B). Aβ levels were increased in Tg mAPP cerebral cortex compared with nonTg littermate controls(Fig. 3).
Decline in cytochrome c oxidase activity and ATP levels in mAPP mice
Given that mitochondria are the main sources for ROS and that mitochondrial dysfunction is involved in abnormal accumulation and generation of ROS, we evaluated mitochondrial respiratory function by determining CcO, a key enzyme for complex IV of the mitochondrial respiratory chain, in Tg mAPP mice and nonTg littermate controls. Compared to nonTg mice, mAPP mice showed a significant reduction in CcO activity in the cortex (Fig. 4A). ATP levels, a sign for mitochondrial bioenergy, were significantly decreased in mAPP brain compared with nonTg brain (Fig. 4B). These data suggest the impairment in the mitochondrial respiration and energy metabolism in Aβ-rich brain of AD mice.
Spatial learning and memory impairments in transgenic mAPP mice
In view of the contribution of mitochondrial dysfunction and oxidative stress to the synaptic and cognitive decline, we next examined whether mAPP mice also had impairments in spatial learning and memory. We assessed the spatial learning memory and target searching strategy using MWM. MWM test showed an average latency to locate the hidden platform during each day of training sessions. The transgenic mAPP mice revealed longer latency to locate the hidden platform during the training (Fig. 5A) and reduced the number of times crossing the target (Fig. 5B) and time in the target quadrant (Fig. 5C-D) in the recording period compared to nonTg mice. The mAPP and nonTg mice had similar swimming speed by the visual swimming speed test (Fig. 5E). Thus, the observed difference in spatial learning and memory is a result of defects in cognition but not motility or altered motivation. These data indicate that mAPP mice had significantly impaired spatial learning and memory.
DISCUSSION
AD is an age-related progressive neurodegenerative disease characterized clinically by cognitive decline and memory loss. So far there are no effective treatments to prevent, slow down, or reverse the relentless progression of AD, though significant progresses have been made in understanding its pathogenesis over the past decades. AD can be divided into early versus late onset forms as well as sporadic and autosomal-dominant familial variants. It is necessary to identify persons who are at high risk for developing progressive disease, which are most likely to benefit from early therapeutic intervention. Plenty of data suggest that primary pathological changes including oxidative stress and mitochondria impairment occur prior to Aβ accumulation and tau pathology [34–36]. To verify these assumptions, the key point is whether these changes indeed occur at early AD stages. Using a well-established AD mouse model (mAPP mice) [24, 37] and advanced EPR techniques, we demonstrated that cerebral ROS levels were elevated in mAPP brain at early stage, which negatively correlates tomitochondrial function and learning memory. Oxidative stress is defined by harmful over-production of reactive oxygen/nitrogen species and ROS is mainly derived from the electron transport chain at the mitochondrial inner membrane. Mounting evidences suggest that oxidative stress is an important pathogenic factor in AD and the study for different disease stages showed that 4-hydroxynonenal (a marker of lipid peroxidation) increased significantly in the early stages of AD [4, 38]. Similarly, another marker, F2-isoprostane levels, increased significantly in frontal poles and cerebrospinal fluid from AD patients [39]. In fact, there is evidence showing that oxidative damage is the earliest event in AD progression among all AD hallmarks. Moreover, Aβ can induce oxidative stress and increase production of ROS, both of which impair mitochondrial function. In turn, oxidative stress also promotes Aβ deposition [2, 40], forming a vicious cycle of promoting neurodegeneration. Given the key role of ROS in early onset and progression of AD, developing a highly sensitive and specific probe/method to detect oxidative stress would be critical for studying the initiation of AD and monitoring AD progression. Currently, the most commonly used methods are based on the determination of specific end-products of the damage resulting from oxidative stress including proteins, membrane lipids, and DNA [41, 42]. All these methods are invasive and return an indirect ROS determination. In the present study, we showed in vitro and in vivo measurements based on EPR spectroscopy and imaging for detecting ROS in the brain of living animals.
The EPR method has become a unique and indispensable tool for the specific detection of reactive oxygen free radicals in biological systems. It has the potential to be used for assessing oxidative stress in humans because of its non-invasiveness. As shown in Figs. 1 and 2, EPR can measure the intracellular ROS in the brain homogenates and in live animals. Given that mitochondria are the major resource of ROS generation and that mitochondrial dysfunction occurs in Aβ-rich mAPP brain, damaged mitochondria from mAPP brain could be a major resource of ROS relevant to amyloid pathology. Indeed, mitochondria-derived superoxide was significantly elevated in mAPP hippocampal and cortical neurons, indicating increased ROS accumulation in Aβ-loaded mitochondria [2, 5]. We have successfully detected increased levels of ROS in Aβ-enriched mAPP brain compared with those from nonTg brain at the age of 8-9 months. Importantly, the ROS levels were not elevated in Aβ- and AD-unaffected cerebellum of the same age of mice, suggesting that oxidative stress occurs in the early onset of AD before severe amyloid accumulation. Mitochondrial dysfunction is one of the early features of the AD-affected brain [3, 43–51]. Impairment of mitochondrial energy metabolism, generation of ROS, and altered activity of the key respiratory enzymes such as CcO are among the earliest detectable defects in AD [3, 52–54]. Studies have highlighted the role of mitochondrial Aβ accumulation in the AD pathogenesis. Accumulation of Aβ in mitochondria precedes the extracellular Aβ deposition in the AD brain and increases with age, which is associated with an early onset of loss of synapses, synaptic damage and mitochondrial oxidative damage [18, 55–65]. CcO is a key enzyme involved in the complex IV of the mitochondrial respiratory electron-transport chain. Impairment of CcO disrupts electron flow and may cause mitochondrial respiratory dysfunction [66, 67]. CcO reduction is well-documented at various stages of AD, including early mild cognitive impairment stage [4, 47]. Consistent with our previous results [2], there was a significant reduction in CcO activity in transgenic mAPP mice at the age of 8-9 month, compared to nonTg mice. Similarly, ATP level was reduced significantly in transgenic mAPP mice, suggesting impairment of mitochondrial energy metabolism. Accordingly, oxidative stress and mitochondrial dysfunction intervene in learning and memory as demonstrated by mAPP mice with the impairment of spatial learning and memory. With this highly sensitive and specific EPR imaging protocol using MCP as a redox-sensitive probe for assessing oxidative stress level in the brain of a living animal of AD-type mouse model, the present study demonstrated that elevated levels of ROS in Aβ-rich brain negatively correlate to the extent of mitochondrial and behavioral dysfunction. Except for the minimallyinvasively surgical intervention to infuse the redox probe into the brain, this in vivo EPR method offers a unique way of assessing tissue oxidative stress in living animals under noninvasive conditions. Thus, this in vivo approach–the EPR imaging approach–has a great advantage on early diagnostic and monitoring for the progression and treatment of AD by assessing oxidative stress associated with AD pathology.
