Mitochondrial Complex I Deficit in the Olfactory Systems of Age-related Neurodegenerative Monkey Models: A PET Study using 18F-BCPP-EF

Objective: Dysfunction of olfactory bulb area (OBA) is reported in several types of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Although pathophysiological mechanisms responsible for changes in olfactory function remain unclear, the quantitative parameters for olfactory function are expected for diagnose of neurodegenerative diseases. We developed a novel probe for positron emission tomography (PET), 18F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one (18F-BCPP-EF), for quantitative analysis of mitochondrial complex I (MC-I) activity in the living brain. In the present study, the applicability of 18F-BCPP-EF to predict age-related neurodegenerative damage in the monkey brain as an MC-I deficit in the OBA was investigated. Methods: PET measurements with 11C-PiB for amyloid--β (Aβ), 11C-DPA-713 for translocator protein (TSPO) and 18F-BCPP-EF were performed in aged monkeys. The binding specificity of 18F-BCPP-EF to MC-I in the OBA was evaluated with rotenone, a specific MC-1 inhibitor, in young animals. 11C-PiB binding to Aβ and 11C-DPA-713 binding to TSPO were calculated as standard uptake value ratios (SUVRs). The total distribution volume (VT) of 18F-BCPP-EF was calculated using a Logan graphical analysis using metabolite-corrected plasma input function, and correlations between the olfactory VT of 18F-BCPP-EF and SUVRs of 11C-PiB or 11C-DPA-713 in several brain regions were analyzed. Results: Pre-dosing of rotenone resulted in the significant reduction of VT values in all brain regions including the OBA. MC-I activity in the OBA exhibited age-related reduction, which positively correlated with MC-I activity in the olfactory-related and cortical regions. OBA MC-I and TSPO as measured using 11C-DPA-713 were inversely correlated in the olfactory-related and cortical regions, but association between OBA MC-I and Aβ deposition as measured using 11C-PiB were observed only in olfactory-related regions. Conclusion: The present study demonstrated that OBA MC-I activity could be a potential predictive parameter of neurodegenerative damages related Aβ deposition and TSPO/neuroinflammation in the living brain. Citation: Hashimoto F, Ohba H, Kanazawa M, Nishiyama S, Kakiuchi T, et al. (2018) Mitochondrial Complex I Deficit in the Olfactory Systems of Agerelated Neurodegenerative Monkey Models: A PET Study using 18F-BCPP-EF. J Alzheimers Dis Parkinsonism 8: 433. doi: 10.4172/21610460.1000433


Introduction
Olfactory ability to correctly identify a smell was previously reported to be defective not only in the course of physiologically normal aging but also in the patients with Alzheimer's disease, Parkinson's disease, and Huntington's disease [1][2][3][4]. University of Pennsylvania smell identification test (UPSIT) has been applied to assess olfactory function and clinical reports using behavioral testing showed a correlation between cognitive status and baseline olfactory test scores in the elderly, those with mild cognitive impairment (MCI), and patients with Alzheimer's disease [5,6]. In contrast, most cases of preclinical Alzheimer's disease have no subjective symptoms of olfactory dysfunction, suggesting a poor understanding of the prevalence of olfactory dysfunction in the normally aging population and those with related diseases [7].
Previous studies indicated that neurodegenerative diseases target specific networks in the brain, and connectivity within brain networks may partly explain the spread of neurodegeneration. The olfactory system and connected areas provide a neural network that may be involved in selective network-driven neuronal vulnerability in relation to a neurodegenerative Alzheimer's disease-related pathology, suggesting that the olfactory system has potential as a diagnostic index of Alzheimer's disease. The pathophysiological mechanisms responsible for changes in olfactory function currently remain unclear; however, previous studies were conducted on Alzheimer's disease patients in order to elucidate these mechanisms using positron emission tomography (PET) [8][9][10]. PET applied target-oriented PET probes for the quantitative imaging probe, 18 F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoroethoxy)-ethoxy]pyridin-3-ylmethoxy}-2H-pyridazin-3-one ( 18 F-BCPP-EF), to image mitochondrial complex I (MC-I) function [23]. MC-I is the first enzyme of the respiratory electron transport chain in living cells. In vitro assessment using isolated mitochondria confirmed the specificity of BCPP-EF and its related compounds to MC-I, and the specific binding of 18 F-BCPP-EF to MC-I was also confirmed by pre-administration of rotenone, a specific MC-I inhibitor, in the living brain [17,24,25]. 18 F-BCPP-EF-PET detected ischemic neuronal damage 7 days after an ischemic insult by which time higher 18 F-FDG uptake was observed in the damaged area because of microglial activation [16][17][18]. Our findings demonstrated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced diffuse reductions in 18 F-BCPP-EF binding to MC-I throughout the brain, not only in dopaminergic neurons in the nigrostriatal regions but also in presynaptic serotonergic neurons in the cortex [26,27]. Significant age-related reductions in MC-I activity were detected in the living brains of monkeys, and MC-I activity in the cortex of aged monkeys inversely correlated with the deposition of Aβ, measured using 11 C-PiB, and neuroinflammation, measured using 11 C-DPA-713 [21].
In the present study, changes in MC-I activity in the olfactory bulb area (OBA) of the living brains of monkeys (Macaca mulatta) were measured using 18 F-BCPP-EF in order to assess its potential as a parameter for age-related neurodegenerative damage. The specificity of 18 F-BCPP-EF binding to MC-1 was evaluated by the pre-administration of rotenone, a specific MC-1 inhibitor, in young animals. Age-related effects on MC-I activity in the OBA were evaluated in the living brains of young and aged monkeys. Furthermore, the connectivity of MC-I activity between the OBA and olfactory-related and cortical regions and the relationship between an MC-I deficit in the OBA and Aβ deposition or neuroinflammation in the olfactory-related and cortical regions were examined.

Anatomical definition
The olfactory bulb area (OBA) means the olfactory bulb from the adjacent gyrus rectus of the frontal base. The olfactory related area includes hippocampus, amygdala and hypothalamus.

Animals and drugs
Ten young male (3-5 years old) and 10 aged male (20-24 years old) rhesus monkeys (Macaca mulatta) were used for the PET measurements. Magnetic resonance images (MRI) of monkeys were obtained with a 3.0 T MR imager (Allegra; Siemens, Erlangen, Germany) under pentobarbital anesthesia.
Rotenone and the precursor of 11 C-PiB were obtained from Sigma-Aldrich Japan (Tokyo, Japan) and PharmaSynth (Tartu, Estonia), respectively. The standard compound of PiB was from ABX Advanced Biochemical Compounds (Radeberg, Germany). The precursors of 11 C-DPA-713, 18 F-BCPP-EF and their corresponding standard compounds were purchased from NARD Institute Ltd. (Amagasaki, Japan).

Drug treatments
In order to assess the binding specificity of 18 F-BCPP-EF to MC-I, rotenone, an MC-1 inhibitor, in 0.5 mL/kg of N, N-dimethylformamide/ polyethylene glycol 400/saline (1:1:2) was infused into monkeys through a posterior tibial vein cannula for 1h, and 18 F-BCPP-EF was then injected as a bolus into monkeys for PET measurement [17,25].

PET measurements in monkey brain
Almost all PET scans were conducted for measurements under conscious states, as reported previously [21,25,27]. After overnight fasting, a venous cannula for the PET ligand injection and an arterial cannula for blood sampling were inserted in both inferior limbs. The animal's head was rigidly fixed to the upper frame of a monkey chair using an acrylic head-restraining device. The animal sitting in the restraining chair was placed at a fixed position in the PET gantry with stereotactic coordinates aligned parallel to the orbito-meatal plane.
For PET measurements under anesthetic state in 4 young control and 4 young rotenone-treated animals, PET scans were conducted as reported previously [18]. Since our previous study confirmed no significant anesthetic effects of isoflurane on 18 F-BCPP-EF binding to MC-I in the living monkey brain both conscious and anesthetized data were analyzed together [21]. After overnight fast, monkeys were sedated with 5 mg/kg i.m. of ketamine, intubated, immobilized with 0.05 mg/ kg/h i.v. of pancuronium bromide, and artificially ventilated (Cato, Drager, Germany). Anesthesia was continued with 0.8 vol. % isoflurane in a N 2 O/O 2 (2:1) gas mixture during the entire experiment. A venous cannula for PET ligand injection and an arterial cannula for blood sampling were inserted in both inferior limbs. The mean arterial blood pressure, heart rate, rectal temperature, arterial PO 2 and PCO 2 and pH were continuously or regularly monitored. During the experiments, the animal's body temperature was maintained within normal limits with heating blankets. The animal was set in a supine position on a bed with head holder (SFCT-RB-PR-2, Hamamatsu Photonics, Hamamatsu, Japan) was placed at a fixed position in the PET gantry with stereotactic coordinates aligned parallel to OM plane.
After a transmission scan for 30 min using a 68 Ge-68 Ga rotation rod source, a dynamic emission scan for 91 min with each PET probe was conducted using an animal PET scanner (SHR-7700, Hamamatsu Photonics, Hamamatsu, Japan). The PET data obtained were reconstructed by the FBP method with a Hanning filter of 4.5 mm FWHM and attenuation correction using transmission scan data. Individual PET and MRI images were co-registered. Volumes of interest (VOIs) in brain regions were drawn manually on MRI with reference to regional information from BrainMaps.org and the VOIs of MRI were superimposed on co-registered PET images in order to measure the time activity curves of each PET probe for kinetic analyses [28]. In order to avoid excessive stress on animals, arterial blood sampling was only performed in scans with 18 F-BCPP-EF.

PET data analysis
In the quantitative analysis of 18 F-BCPP-EF, arterial blood samples were obtained every 8 sec up to 64 sec, followed by 90 and 150 sec, and then 4, 6, 10, 20, 30, 45, 60, 75 and 90 min after the tracer injection, and blood samples were centrifuged to separate plasma, weighed, and their radioactivity was measured. In the metabolite analysis, methanol was added to some plasma samples (sample/methanol=1/1) obtained 16, 40, and 64 s and 6, 10, 30, 45, 60, 75 and 90 min after the injection, followed by centrifugation. The supernatants obtained were developed with thin layer chromatography plates (AL SIL G/UV, Whatman, Kent, UK) with a mobile phase of ethyl acetate. The ratio of the unmetabolized fraction was assessed using a phosphoimaging plate (FLA-7000, Fuji Film, Tokyo, Japan). The input function of unmetabolized 18 F-BCPP-EF was calculated using data obtained by corrections of the ratio of the unmetabolized fraction to total radioactivity, which was used as the arterial input function. Kinetic analyses of 18 F-BCPP-EF were performed by a Logan graphical analysis using PMOD software (PMOD Technologies Ltd., Zurich, Switzerland) [29].
In analyses of 11 C-PIB for Aβ deposition and 11 C-DPA-713 for translocator protein (TSPO) activity, PET images were normalized by the injected dose and the subject weight to produce standardized uptake value (SUV) images. Then, SUV images averaged from 60 to 90 min and from 40 to 60 min, respectively, after the injection were created, and VOIs were set on each SUV image with the aid of MRI of individual monkeys. SUV ratios (SUVR) of each region (SUV (region)) against SUV in the cerebellum (SUV (cerebellum)) were calculated [30].

Statistical analysis
Results are expressed as means + SD. Comparisons between conditions were carried out using the paired, two-tailed Student's t-test. A probability level of less than 5% (p<0.05) was considered to indicate statistical significance. In the quantitative analysis of MC-1 activity using 18 F-BCPP-EF, a Logan graphical analysis was applied for total distribution volume (V T ) image generation and regional V T value calculations, and the effects of rotenone and aging on MC-I activity were assessed in the living monkey brain. Logan-V T values were the highest in the occipital cortex, higher in the parietal cortex, intermediate in the hippocampus, amygdala, hypothalamus, and frontal and temporal cortices, and the lowest in the OBA of young normal monkeys ( Figure 2). As shown in Figures 1A, 1B  and 2, the pre-administration of rotenone, a specific MC-1 inhibitor, at a dose of 0.1 mg/kg/h markedly reduced V T values in olfactory-related (hippocampus, amygdala, and hypothalamus; average=60.3% of the vehicle condition) and cortical regions (parietal, temporal, occipital, and frontal cortices; 58.1%) including the OBA (59.9%). Aged animals had significantly lower V T values in olfactory-related (average=79.8% of young) and cortical regions (75.5%) than those in young animals ( Figure 2). It is important to note that aging effects on MC-I activity were the most prominent in the OBA, being 44.7% in young animals ( Figure 2).

Results
In order to examine regional interactions in MC-I activity between the OBA and several brain regions, correlation analyses were performed using V T values in the olfactory-related and cortical regions of young and aged animals. The plotting results obtained demonstrated that the regional V T values of 18 F-BCPP-EF in olfactoryrelated (hippocampus r 2 =0.561; amygdala r 2 =0.536; hypothalamus r 2 =0.676, P<0.01 in all areas) and cortical regions (parietal r 2 =0.672; temporal r 2 =0.545; occipital r 2 =0.614; frontal r 2 =0.662; P<0.01 in all areas) correlated well with the V T values of the OBA in living monkey brains ( Figures 3A and 3B). When the regional SUVR of 11 C-PiB was plotted against the V T values of 18 F-BCPP-EF in the OBA of aged animals, the results obtained indicated an inverse relationship, with correlations being observed between OBA MC-I and Aβ deposition in the hippocampus (r 2 =0.800, P<0.01), amygdala (r 2 =0.563, P<0.01) and hypothalamus (r 2 =0.572, P<0.01) (Figure 4).
The plot of the regional SUVR of 11

Discussion
This is the first study to demonstrate that OBA MC-I activity measured using 18 F-BCPP-EF-PET could be a potential predictive parameter of neurodegenerative damages related Aβ deposition and TSPO/neuroinflammation in the living brains of aged nonhuman primate (Macaca mulatta) using animal PET.
The present study showed the suppression of 18 F-BCPP-EF uptake by the pre-administration of rotenone, a specific MC-I inhibitor, in all brain regions analyzed, including the OBA, with uptake being inhibited to a similar extent in all regions. Although a dose-escalation study for full displacement was not performed because of the lethal effects of rotenone on cardiac function, significant reductions in V T were observed even at a relatively low dose of 0.1 mg/kg/h, suggesting that the uptake of 18 F-BCPP-EF reflected specific binding to MC-I, even in the OBA.
The present study confirmed our previous findings that 18 F-BCPP-EF detected the age-related reduction of MC-1 activity in the all cortical regions of living monkey brain using PET [25]. Mitochondrial respiratory chain failures have been implicated as factors in the aging process, and this is called the "mitochondrial free radical theory of aging" [31]. Increases in the production of mitochondrial reactive oxygen species (ROS) with aging are regarded as toxic byproducts of aerobic metabolism that induce oxidative damage to various cellular macromolecules. Mitochondria are the main intracellular source of ROS and also the main target of oxyradical-mediated damage and cumulative free radical damage, leading to significant changes in brain mitochondrial function. Since MC-I is an important factor in the regulation of mitochondrial respiration as well as a source of ROS in mitochondria, we previously demonstrated that assessments of MC-I activity using 18 F-BCPP-EF may be useful for the detection of age-related changes in cortical function, which may be involved in the age-related memory impairment in monkeys [21,25,32].  Furthermore, the remarkable finding of the present study was that age-related reductions in MC-I activity were observed also in the OBA in a group comparison between young and aged animals. The extent of reductions in MC-I activity in the OBA was significantly greater than those in any other brain regions. This result is consistent with previous findings showing that patients progressing to Alzheimer's disease showed the greater loss of gray matter than MCI patients remaining stable, and this loss mainly occurred in the olfactory network [33]. Olfactory ability has been reported to become defective in the course of physiologically normal aging and olfactory dysfunction occurs even before the clinical presentation of Alzheimer's disease [1,6]. In patients with Alzheimer's disease, the olfactory system and areas with extensive connections to this system exhibit several pathological changes. Collectively, these results suggest that specific and sensitive parameters of OBA function, such as MC-I activity, are useful as predictors of preclinical Alzheimer's disease.
In assessments of the OBA in aged animals, Aβ deposition, a pathological hallmark of Alzheimer's disease, measured using 11 C-PiB did not reflect the MC-I deficit assessed with 18 F-BCPP-EF-PET, which is consistent with previous findings of a weak relationship between UPSIT performance and Aβ deposition in the OBA [8,10]. Inconsistent findings have been reported in postmortem studies; some studies did not detect Aβ deposition in the OBA, while others showed the presence of Aβ in the OBA, mostly in severe Alzheimer's disease patients [34][35][36]. In contrast, the deposition of hyperphosphorylated tau, another pathological hallmark of Alzheimer's disease, in the OBA was more consistent than the presence of Aβ deposition [35,36]. Mitochondrial transport along microtubules within axons is crucial for axonal maintenance, and its dysregulation by hyperphosphorylated tau may contribute to neurodegenerative diseases [37]. Therefore, it may be more useful to assess the deposition of hyperphosphorylated tau in the OBA of the living brain, as previously elucidated using 18 F-Flortaucipir and PET [9].
Several in vivo imaging studies reported a weak relationship between UPSIT performance and cortical Aβ deposition; however, an in vivo imaging study using functional MRI revealed that blood oxygen level-dependent signal activation induced by a lavender oil odorant in the primary olfactory cortex and hippocampus correlated with UPSIT in MCI and AD patients [8,10,38]. In order to ensure the capability of OBA MC-I as a predictive parameter of neurodegenerative damage in cortical regions, correlation analyses were performed between MC-I activity in the OBA and those in olfactory-related and cortical regions. The combined data of young and aged animals revealed positive correlations between MC-I activity in the OBA and those in olfactory-related and cortical regions. These findings suggested strong connectivity between the OBA and olfactory-related and cortical regions.
When the relationship between OBA MC-I activity, measured using 18 F-BCPP-EF, and regional Aβ deposition, measured using 11 C-PiB, was investigated in aged monkeys, inverse correlations were observed in olfactory-related regions (hippocampus, amygdala, and hypothalamus), but not in cortical regions. Previous studies reported that no species other than humans show marked neuron loss or cognitive decline approaching clinical grade AD in humans, and aged monkeys did not exhibit as high Aβ deposition and 11 C-PiB uptake in cortical regions as those reported in AD patients. The present results support the histopathological finding that cortical Aβ deposition was not directly linked with olfactory ability; however, 11 C-PiB does not directly measure the more toxic soluble forms of Aβ, which disrupt neural network function [21,[39][40][41]. If direct measurements of soluble Aβ were performed, a clearer relationship between OBA MC-I activity and cortical Aβ deposition may have been found.
In the correlation analysis in the OBA, TSPO activity, as a neuroinflammation marker, measured using 11 C-DPA-713, showed an inverse relationship with the MC-I deficit assessed using 18 F-BCPP-EF. Furthermore, when the relationship between OBA MC-I activity and regional neuroinflammation was evaluated, inverse correlations were noted in all olfactory-related regions (hippocampus, amygdala, and hypothalamus) as well as all cortical regions (parietal, temporal, occipital, and frontal cortices). TSPO activity measured using 11 C-DPA-713 was sufficiently high for correlation analyses, even in the limited sample size of aged monkeys used in the present study. In mammalian species, the olfactory system has strong connections with several brain regions, such as the hippocampus and neocortical areas that are related to memory function and display AD pathology. The inverse correlation observed between OBA MC-I and TSPO in olfactory-related and cortical regions and the positive correlations between OBA MC-I and MC-I olfactory-related and cortical regions are mirror-image relationships; both support our working hypothesis that MC-I assessments in the OBA may be useful for investigating connectivity and activity in the spread of pathology in AD.
The present study had a few limitations. Due to the limited sample size of aged animals, a replicative study with a larger sample may be required for more definite conclusions, particularly regarding the relationship between OBA MC-I activity and cortical Aβ deposition measured as low-level 11 C-PiB uptake. In addition, since this study included only resting-state MC-I activity, the task-based MC-I imaging of olfactory responses may provide important insights into early changes in olfaction.

Conclusion
The present study demonstrated that OBA MC-I activity measured using 18 F-BCPP-EF-PET has potential as a predictive parameter of neurodegenerative damage related to Aβ deposition, as measured using 11 C-PiB, and TSPO/neuroinflammation, as measured using 11 C-DPA-713, in the living brain. Further studies on the relationship between OBA MC-I activity and olfactory ability assessed by UPSIT need to be performed in order to confirm the capability of 18 F-BCPP-EF-PET for the preclinical detection of AD.