Mitochondrial complex-I Inhibitor Research Articles

Partial Inhibition of Mitochondrial Complex I Reduces Tau Pathology and Improves Energy Homeostasis and Synaptic Function in 3xTg-AD Mice.

Background:Accumulation of hyperphosphorylated tau (pTau) protein is associated with synaptic dysfunction in Alzheimer’s disease (AD). We previously demonstrated that neuroprotection in familial mouse models of AD could be achieved by targeting mitochondria complex I (MCI) and activating the adaptive stress response. Efficacy of this strategy on pTau-related pathology remained unknown.Objective:To investigate the effect of specific MCI inhibitor tricyclic pyrone compound CP2 on levels of human pTau, memory function, long term potentiation (LTP), and energy homeostasis in 18-month-old 3xTg-AD mice and explore the potential mechanisms.Methods:CP2 was administered to male and female 3xTg-AD mice from 3.5–18 months of age. Cognitive function was assessed using the Morris water maze. Glucose metabolism was measured in periphery using a glucose tolerance test and in the brain using fluorodeoxyglucose F18 positron-emission tomography (FDG-PET). LTP was evaluated using electrophysiology in the hippocampus. The expression of key proteins associated with neuroprotective mechanisms were assessed by western blotting.Results:Chronic CP2 treatment restored synaptic activity in female 3xTg-AD mice; cognitive function, levels of synaptic proteins, glucose metabolism, and energy homeostasis were improved in male and female 3xTg-AD mice. Significant reduction of human pTau in the brain was associated with increased activity of protein phosphatase of type 2A (PP2A), and reduced activity of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β).Conclusion:CP2 treatment protected against synaptic dysfunction and memory impairment in symptomatic 3xTg-AD mice, and reduced levels of human pTau, indicating that targeting mitochondria with small molecule specific MCI inhibitors represents a promising strategy for treating AD.

Piceatannol promotes hepatic and renal AMPK/SIRT1/PGC-1α mitochondrial pathway in rats exposed to reserpine or gamma-radiation.

Human exposure to radio-therapeutic doses of gamma rays can produce late effects, which negatively affect cancer patients’ quality of life, work prospects, and general health. This study was performed to explore the role of Piceatannol (PIC) in the process of “mitochondrial biogenesis” signaling pathway as possible management of disturbances induced in stressed animal model(s) either by gamma-irradiation (IR) or administration of reserpine (RES); as a mitochondrial complex-I inhibitor. PIC (10 mg/kg BW/day; orally) were given to rats for 7 days, after exposure to an acute dose of γ-radiation (6 Gy), or after a single reserpine injection (1 g/kg BW; sc). Compared to reserpine or γ-radiation, PIC has attenuated hepatic and renal mitochondrial oxidative stress denoted by the significant reduction in the content of lipid peroxides and NO with significant induction of SOD, CAT, GSH-PX, and GR activities. PIC has also significantly alleviated the increase of the inflammatory markers, TNF-α and IL-6 and apoptotic markers, cytochrome c, and caspase-3. The decrease of oxidative stress, inflammation, and apoptotic responses were linked to a significant amelioration in mitochondrial biogenesis demonstrated by the increased expression and proteins’ tissue contents of SIRT1/p38-AMPK, PGC-1α signaling pathway. The results are substantiated by the significant amelioration in mitochondrial function verified by the higher levels of ATP content, and complex I activity, besides the improvement of hepatic and renal functions. Additionally, histopathological examinations of hepatic and renal tissues showed that PIC has modulated tissue architecture after reserpine or gamma-radiation-induced tissue damage. Piceatannol improves mitochondrial functions by regulating the oxidant/antioxidant disequilibrium, the inflammatory and apoptotic responses, suggesting its possible use as adjuvant therapy in radio-therapeutic protocols to attenuate hepatic and renal injuries.

Andrographolide suppresses NLRP3 inflammasome activation in microglia through induction of parkin-mediated mitophagy in in-vitro and in-vivo models of Parkinson disease

Cellular communication linking microglia activation and dopaminergic neuronal loss play an imperative role in the progression of Parkinson’s disease (PD); however, underlying molecular mechanisms are not precise and require further elucidation. NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation is extensively studied in context to microglial activation and progressive dopaminergic neuronal loss in PD. Several pathophysiological factors such as oxidative stress, mitochondrial dysfunction impaired mitophagy plays a crucial role in activating NLRP3 inflammasome complex. Hence, regulation of microglial activation through mitophagy could be a valuable strategy in controlling microglia mediated neurodegeneration. In this study we have developed a model of inflammasome activation by combining LPS with a mitochondrial complex-I inhibitor MPP+. The idea of using MPP+ after priming mouse microglia with LPS was to disrupt mitochondria and release reactive oxygen species, which act as Signal 2 in augmenting NLRP3 assembly, thereby releasing potent inflammatory mediators such as active interleukin-1 beta (IL-1β) and IL-18. LPS-MPP+ combination was seen to impaired the mitophagy by inhibiting the initial step of autophagosome formation as evidenced by protein expression and confocal imaging data. Treatment with Andrographolide promoted the parkin-dependent autophagic flux formation in microglia; resulting in the removal of defective mitochondria which in turn inhibit NLRP3 inflammasome activation. Additionally, the neuroprotective role of Andrographolide in inhibiting NLRP3 activation together with salvage ATP level via promoting parkin-dependent mitophagy was seen in the substantial nigra par compacta (SNpc) region of mice brain. Furthermore, Andrographolide rescued the dopaminergic neuron loss and improved the behavioural parameters in animal model. Collectively, our results reveal the role of mitophagy in the regulation of NLRP3 inflammasome by removing defective mitochondria. In addition, andrographolide was seen to abate NLRP3 inflammasome activation in microglia and rescue dopaminergic neuron loss.

Metabolism of hyperpolarized 13 C-acetoacetate to β-hydroxybutyrate detects real-time mitochondrial redox state and dysfunction in heart tissue.

Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter-conversion of β-hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3-13 C]acetoacetate to [1,3-13 C]β-hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) 13 C-acetoacetate (AcAc) to 13 C-β-hydroxybutyrate (β-HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP-AcAc to β-HB was investigated using 13 C magnetic resonance spectroscopy in Langendorff-perfused rat hearts in four groups: control, global ischemic reperfusion, low-flow ischemic, and rotenone (mitochondrial complex-I inhibitor)-treated hearts. We observed that more β-HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in β-HB, as detected by 13 C MRS, was validated by a direct measure of tissue β-HB by 1 H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD+ /NADH, measured by enzyme assays of homogenized tissue, also paralleled production of β-HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP-AcAc to HP-β-HB detected by 13 C-MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.

Abstract B37: Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma

Abstract Despite increasing prevalence and lethality for hepatocellular carcinoma (HCC), the need for effective treatment remains unmet. Metabolic reprogramming during hepatocarcinogenesis promotes cancer cell growth and survival, but also suggests the possibility of identifying as yet unexplored drug targets. In this study, we developed a synthetic lethal drug combination to treat HCC through the inhibition of the energy generation triangle. Most cancers, including HCC, increase glycolysis (the Warburg effect) to ensure sufficient supplies of energy (ATP), reducing equivalents (NADPH), and biochemical building blocks for cell growth and proliferation. Unlike most tumors, which express both hexokinase 1 (HK1) and HK2, many HCC tumors express only HK2, whereas normal hepatocytes only express HK4. We investigated the role of HK2 in HCC using a doxycycline (DOX)-inducible shRNA knockdown system. The HK4-to-HK2 isoform switch during hepatocarcinogenesis results in high sensitivity to HK2 silencing-induced glycolysis inhibition and cytostasis in HCC cells, in contrast to cancer cells expressing both HK1 and HK2. We performed a high throughput screen (HTS) of 3,205 drug-like small molecules and 119 FDA-approved oncology drugs to identify synthetic lethal partners of HK2 knockdown, using the CellTiter-Glo assay for primary HTS and confirmed with the alamarBlue assay. Diphenyleneiodonium (DPI), a mitochondrial complex-I inhibitor, is the best compound in our HTS that shows synthetic lethality in combination with HK2 silencing/inhibition in HCC cells that express only HK2. Cell proliferation was measured by the MTT assay and cell counting. Cytotoxicity was measured using Trypan blue to distinguish live and dead cells. HK2 activity in xenograft tumors was monitored non-invasively using 18F-fludeoxyglucose PET/CT scans. Perhexiline (PER), a fatty acid oxidation inhibitor, further sensitizes HCC cells to the complex-I/HK2-targeted combination. Cellular ATP levels were examined by a luciferase activity assay, oxygen consumption was examined by a Seahorse respirometry assay, and cell metabolism was examined by a metabolomics analysis. The inclusion PER significantly enhances the reduction of HCC cellular energy production caused by the combination of HK2 inhibition and DPI, and the efficacy of the complex-I/HK2-targeted combination in treating HCC tumors in vivo. Our findings indicate that selective inhibition of the energy generation triangle (glycolysis, oxidative phosphorylation, and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising for HCC therapy. Citation Format: Shili Xu, Arthur Catapang, Daniel Braas, Linsey Stiles, Jason T. Lee, Thomas G. Graeber, Robert Damoiseaux, Orian Shirihai, Harvey R. Herschman. Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B37.

Effects of acetaminophen on mitochondrial complex I activity in the rat liver and kidney: a PET study with 18F-BCPP-BF.

BackgroundIn the present study, 2-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF), a PET probe for mitochondrial complex I (MC-I), was used to validate whether MC-I is a useful biomarker for detecting acetaminophen-induced dysfunctions in the liver and kidney.The kinetic and distribution of 18F-BCPP-BF were assessed in rats using high-resolution animal PET in vivo. The binding specificity of 18F-BCPP-BF to MC-I in the liver and kidney was confirmed by the pre-administration of rotenone, a specific MC-I inhibitor. The effects of acetaminophen on MC-I activity were assessed 2 and 24 h after the administration of vehicle or acetaminophen at a dose of 100 or 300 mg/kg. Biochemical parameters in plasma and urine were assessed 2, 6, and 24 h after the administration of vehicle or acetaminophen.ResultsThe uptake of 18F-BCPP-BF by the liver and kidney was significantly inhibited by the pre-administration of rotenone. Two and more hours after the administration of acetaminophen, the uptake of 18F-BCPP-BF was dose-dependently reduced in the liver, even at 100 mg/kg, and in the kidney at 300 mg/kg, whereas biological parameters started to be affected 6 h or later at doses of 300 mg/kg.ConclusionsThe present study demonstrated that 18F-BCPP-BF has potential as a PET probe for the quantitative imaging of hepatic and renal dysfunction as impaired MC-I activity in the early phase of the treatment for an overdose of acetaminophen in the living body with PET.

The AMPK activator R419 improves exercise capacity and skeletal muscle insulin sensitivity in obese mice

ObjectiveSkeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects.MethodsGlucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK β1β2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation.ResultsThere was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice.ConclusionsTreatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity.

AMPA receptor activation causes preferential mitochondrial Ca2+ load and oxidative stress in motor neurons

It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca2+ loading. Earlier we have reported that in motor neurons a generic rise in [Ca2+]i does not always lead to mitochondrial Ca2+ loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca2+ regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca2+]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca2+]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca2+]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca2+]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na+/Ca2+ exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca2+ sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na+/Ca2+ exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.

Novel PET Probes18F-BCPP-EF and18F-BCPP-BF for Mitochondrial Complex I: A PET Study in Comparison with18F-BMS-747158-02 in Rat Brain

We developed novel PET probes, 2-tert-butyl-4-chloro-5-{6-[2-(2-(18)F-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ((18)F-BCPP-EF) and 2-tert-butyl-4-chloro-5-[6-(4-(18)F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one ((18)F-BCPP-BF), for quantitative imaging of mitochondrial complex I (MC-I) activity in the brain and preliminarily evaluated their properties in comparison with (18)F-BMS-747158-02 ((18)F-BMS). The affinity of (18)F-BCPP-EF, (18)F-BCPP-BF, and (18)F-BMS to MC-I was analyzed using in vitro binding assays with (3)H-dihydrorotenone and bovine cardiomyocyte submitochondrial particles. (18)F-BCPP-EF, (18)F-BCPP-BF, or (18)F-BMS was intravenously injected into rats, and the uptake (standardized uptake value) in each organ was determined by dissection method. The effects of rotenone, a specific MC-I inhibitor, on the uptake of each probe were assessed by whole-body PET imaging in rats. Ischemic brain model rats were imaged using (18)F-BCPP-EF. The rank order of affinity to MC-I was (18)F-BCPP-BF > (18)F-BMS > (18)F-BCPP-EF. The uptake of (18)F-BCPP-EF and (18)F-BMS was high in the heart, intermediate in brain, and low in muscle and bone 60 min after the injection. (18)F-BCPP-BF provided increasing bone uptake with time after the injection. The uptake of (18)F-BCPP-EF and (18)F-BMS into the brain and heart was significantly decreased by preadministration of rotenone; however, the reduction degree of (18)F-BCPP-EF was more pronounced than that of (18)F-BMS. Rotenone did not affect (18)F-BCPP-BF uptake in either the brain or the heart. (18)F-BCPP-EF imaged the cortical ischemic neuronal damage without any disturbance by microglial activation even on day 7 when (18)F-FDG showed high uptake in the damaged area. The present study demonstrated that (18)F-BCPP-EF could be a potential PET probe for quantitative imaging of MC-I activity and its ischemic damage in the living brain with PET.

Polyphenols Protect the Epithelial Barrier Function of Caco-2 Cells Exposed to Indomethacin through the Modulation of Occludin and Zonula Occludens-1 Expression

The aim of this study was to determine the protective effect of quercetin, epigallocatechingallate, resveratrol, and rutin against the disruption of epithelial integrity induced by indomethacin in Caco-2 cell monolayers. Indomethacin decreased the transepithelial electrical resistance and increased the permeability of the monolayers to fluorescein-dextran. These alterations were abolished by all the tested polyphenols but rutin, with quercetin being the most efficient. The protective effect of quercetin was associated with its capacity to inhibit the redistribution of ZO-1 protein induced in the tight junction by indomethacin or rotenone, a mitochondrial complex-I inhibitor, and to prevent the decrease of ZO-1 and occludin expression induced by indomethacin. The fact that the antioxidant polyphenols assayed in this study differ in their protective capacity against the epithelial damage induced by indomethacin suggests that this damage is due to the ability of this agent to induce not only oxidative stress but also mitochondrial dysfunction.

Nitric oxide synthase inhibitors protect against rotenone-induced, oxidative stress mediated parkinsonism in rats

Rotenone is known to cause progressive dopaminergic neuronal loss in rodents, but it remains unclear how this mitochondrial complex-I inhibitor mediates neurodegeneration specific to substantia nigra pars compacta (SNpc). One of the proposed mechanisms is increased free radical generation owing to mitochondrial electron transport chain dysfunction following complex-I inhibition. The present study examined the role of nitric oxide (NO) and hydroxyl radicals (OH) in mediating rotenone-induced dopaminergic neurotoxicity. Indications of NO involvement are evidenced by inducible nitric oxide synthase (NOS) over-expression, and increased NADPH-diaphorase staining in SNpc neurons 96h following rotenone administration. Treatment of these animals with specific neuronal NOS inhibitor, 7-nitroindazole (7-NI) and non-specific NOS inhibitor, N-ω-nitro-l-argenine methyl ester (l-NAME) caused reversal of rotenone-induced striatal dopamine depletion, and attenuation of the neurotoxin-induced decrease in the number of tyrosine hydroxylase immunoreactive neurons in SNpc, as well as in apomorphine and amphetamine-induced unilateral rotations. Interestingly, the study also demonstrated the contribution of OH in mediating rotenone nigral toxicity since there appeared a significant generation of the reactive oxygen species in vivo 24h following rotenone administration, a copious loss of reduced and oxidized glutathione, and increased superoxide dismutase and catalase activities in the cytosolic fractions of the ipsilateral SNpc area on the 5th day. An OH scavenging capacity of 7-NI and l-NAME in a Fenton-like reaction, as well as complete reversal of the rotenone-induced increases in the antioxidant enzyme activities, and the loss in reduced and oxidized glutathione contents in the SNpc supported OH involvement in rotenone-induced dopaminergic neurotoxicity. While these results strongly suggest the contribution of both OH and NO, resulting in acute oxidative stress culminating in dopaminergic neurodegeneration caused by rotenone, the course of events indicated generation of OH as the primary event in the neurotoxic processes.

Preparation and biodistribution of [ 18F]FP2OP as myocardial perfusion imaging agent for positron emission tomography

Myocardial extractions of pyridaben, a mitochondrial complex I (MC-I) inhibitor, is well correlated with blood flow. Based on the synthesis and characterization of pyridaben analogue 2- tert-butyl-5-[2-(2-[ 18F]fluroethoxy)ethoxy]benzyloxy]-4-chloro-2 H-pyridazin-3-one ([ 18F]FP2OP), this study assessed its potential to be developed as myocardial perfusion imaging (MPI) agent. Methods: The tosylate labeling precursor 2-(2-(4-( tert-butyl-5-chloro-6-oxo-1,6-dihydro-pyridazin-4-yloxymethyl)benzyloxy)ethoxy)ethyl ester (OTs-P2OP) and the nonradioactive 2- tert-butyl-5-[2-(2-[ 19F]fluroethoxy)ethoxy]benzyloxy]-4-chloro-2 H-pyridazin-3-one ([ 19F]FP2OP) were synthesized and characterized by IR, 1H NMR, 13C NMR and MS analysis. By substituting tosyl of precursor OTs-P2OP with 18F, the radiolabeled complex [ 18F]FP2OP was prepared and further evaluated for its in vitro physicochemical properties, in vivo biodistribution, the metabolic stability in mice, ex vivo autoradiography and cardiac PET/CT imaging. Results: Starting with [ 18F]F − Kryptofix 2.2.2./K 2CO 3 solution, the total reaction time for [ 18F]FP2OP was about 100 min, with final high-performance liquid chromatography purification included. Typical decay-corrected radiochemical yield stayed at 41 ± 5.3%, the radiochemical purity, 98% or more. Biodistribution in mice showed that the heart uptake of [ 18F]FP2OP was 41.90 ± 4.52%ID/g at 2 min post-injection time, when the ratio of heart/liver, heart/lung and heart/blood reached 6.83, 9.49 and 35.74, respectively. Lipophilic molecule was further produced by metabolized [ 18F]FP2OP in blood and urine at 30 min. Ex vivo autoradiography demonstrates that [ 18F]FP2OP may have high affinity with MC-I and that can be blocked by [ 19F]FP2OP or rotenone (a known MC-I inhibitor). Cardiac PET images were obtained in a Chinese mini-swine at 5, 15, 30 and 60 min post-injection time with high quality. Conclusion: [ 18F]FP2OP was synthesized with high radiochemical yield. The promising biological properties of [ 18F]FP2OP suggest high potential as MPI agent for positron emission tomography in the future.

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

A clinically-related animal model of Parkinson's disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research.

NADPH oxidase-derived reactive oxygen species involved in angiotensin II-induced monocyte chemoattractant protein-1 expression in mesangial cells

To investigate the origin of oxidative stress induced by angiotensin II (AngII) in human mesangial cells and the role of reactive oxygen species (ROS) in AngII-induced monocyte chemoattractant protein-1 (MCP-1) expression. MCP-1 expression was determined by real time RT-PCR. ROS production was measured by DCFDA fluorescence. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence. p47phox and p67phox translocation was assayed by Western blot. Twenty-four male mice were randomly divided into three groups: the control, the AngIIinfusion [AngII 400 ng/(kg.min)], and the apocynin treatment. AngII was infused by subcutaneously osmotic minipump for 14 days. Urinary albumin and 8-isoprostane excretion were measured by ELISA. In cultured human mesangial cells, AngII induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control. AngII increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent. Incubation with different dosages of AngII (1 nmol/L, 10 nmol/L, and 100 nmol/L AngII) for 60 min, ROS production increased at 1.82, 2.92, and 4.08 folds respectively. AngII-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI, 10 micromol/L) and apocynin (500 micromol/L), two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex Iinhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect. AngII-induced ROS generation was inhibited by the AT1 antagonist losartan (10 micromol/L) but not the AT2 antagonist PD123319 (10 micromol/L). AngII treatment induced translocation of cytosolic of p47phox and p67phox to the membrane. The antioxidants almost abolished AngII-induced MCP-1 expression. AngII infusion increased urinary and p67 translocation by 2.69-, 2.97-, and 2.67-fold, respectively. NADPH oxidase-derived ROS is involved in AngII-induced MCP-1 expression. Inhibition of NADPH oxidase alleviates AngII-induced renal injury.

Phosphorylated-ERK 1/2 and neuronal degeneration induced by rotenone in the hippocampus neurons

Rotenone, a mitochondrial complex-I inhibitor, has been verified to cause dopaminergic neurons degeneration in vivo and in vitro, and the substantia nigra pars compacta (SNc) and the striatum are the main target organs of rotenone in the rat brain. However, whether rotenone could cause damage to other regions in the brain has been unclear till now. To address this question, the rotenone-induced neurotoxicity in the hippocampal neurons was investigated in the present study. Rotenone (4 mg/kg) was given to the male Sprague–Dawley rats per day for up to 4 weeks by using the osmotic minipumps. Results showed that neurodegeneration was formed and phosphorylated ERK1/2 (p-ERK1/2) was induced in the hippocampus of rats following rotenone treatment. In additionally, Ras, PKA and PKC were also activated and free [Ca 2+] i was increased in the cytoplasm of the hippocampus neurons. To determine how ERK cascade was activated, studies in the primary cultured hippocampus neurons were carried out in a further. Cell viability was reduced, and also apoptosis was induced in vitro following rotenone administration. Expressions of p-ERK1/2 were also enhanced evidently in the cultured neurons treated by rotenone. Free [Ca 2+] i was also increased in the cultured neurons induced by rotenone. However, this influx might not take main effect in ERK1/2 phosphorylation. In conclusion, Ras-Raf-1-MEK-ERK1/2 classic signal pathway, not by PKA/PKC alternative pathway may be the mainly contributor to the ERK1/2 phosphorylation. And also, Ras protein is the dominant activator in the ERK phosphorylation induced by rotenone.

Assessment of 18F-labeled mitochondrial complex I inhibitors as PET myocardial perfusion imaging agents in rats, rabbits, and primates

Myocardial extractions of mitochondria complex I (MC-I) inhibitors were high and well correlated with flow. This study assessed the potential of MC-I inhibitors to be developed as myocardial perfusion imaging (MPI) agents. RP1003, RP1004, and RP1005 representing three classes of MC-I inhibitor were synthesized and radio-labeled with (18)F. These agents were evaluated for IC(50) values, tissue biodistribution, and cardiac PET imaging. (18)F-RP1004 was further examined for first-pass extraction and by imaging in non-human primates (NHP) and rats following coronary ligation. RP1003, RP1004, and RP1005 exhibited high MC-I inhibitory activity with IC(50) of 3.7, 16.7, and 14.4 nM. Heart uptakes in rats (percent injected dose per gram tissue) at 15 and 60 min after injection were 3.52 +/- 0.36 and 2.68 +/- 0.20 for (18)F-RP1003, 2.40 +/- 0.21 and 2.67 +/- 0.27 for (18)F-RP1004, and 2.28 +/- 0.12 and 1.81 +/- 0.17 for (18)F-RP1005. The heart to lung and liver uptake ratios were favorable for cardiac imaging with these agents. In isolated perfused rabbit hearts, the uptake of (18)F-RP1004 increased from 0.74 +/- 0.19 to 1.68 +/- 0.39 mL/min/g at flow rates of 1.66 to 5.06 mL/min/g. These values were higher than or similar to that of (99m)Tc-sestamibi. Cardiac imaging with these agents in rats and rabbits allowed visualization of the heart with minimal lung interference and rapid liver activity clearance. Imaging with (18)F-RP1004 also showed clear myocardium and marked liver activity washout in the NHP and clear detection of the perfusion-deficit area associated with left coronary artery ligation in the rat. MC-I inhibitors have the potential to be a new class of MPI agent.

Interleukin-1β Mediates Sleep Alteration in Rats With Rotenone-Induced Parkinsonism

Recently, the pathogenesis of Parkinson disease (PD) has been focused on microglial activation, especially the subsequent increase of cytokines. A body of clinical evidence suggests that sleep is altered in patients with PD; however, there is a lack of understanding of the basic cellular mechanism. This study was designed to elucidate the influence of brain interleukin (IL)-1beta on sleep changes, in addition to the dopaminergic and gamma-aminobutyric acid (GABA)-ergic systems, in an animal PD model. We employed a long-term subcutaneous infusion of rotenone, a mitochondrial complex-I inhibitor, to induce a parkinsonism-like model in rats. Behavioral tests and tyrosine hydroxylase immunocytochemistry were used for confirmation of PD in this animal model. Pharmacologic agonist and antagonists were administered centrally to test the involvement of dopamine, GABA, and IL-1 in rotenone-induced sleep alteration. Protein expression of cytokines, ie, IL-1beta and tumor necrosis factor alpha (TNF-alpha), in 5 distinct brain regions was also determined by Western blot and enzyme-linked immunosorbent assay (ELISA). Sleep-recording equipment in the National Taiwan University and China Medical University. PARTICIPANT AND INTERVENTIONS: Male Sprague-Dawley rats were implanted with electroencephalogram electrodes, a thermistor, and an intracerebroventricular guide cannula. Chronic infusion of rotenone was given by an Alzet minipump implanted subcutaneously on the back of each rat. We found that locomotion activity was reduced, slow-wave sleep (SWS) was increased during the dark (active) phase and decreased during the light (rest) period, and rapid eye movement sleep (REM) was enhanced in the dark period after rotenone treatment. This rotenone PD animal model successfully causes loss of tyrosine hydroxylase-immunopositive neurons in the substantia nigra; induces the events of sleep disturbance, such as excessive daytime sleepiness and insomnia during the nighttime, that are seen in patients with PD; and suppresses locomotion. Our results that intracerebroventricular administration of dopamine and blockade of GABA in the brain have less significant effect on rotenone-induced sleep alteration suggest that the sleep disturbance is not primarily mediated by the disruption of dopaminergic and GABAergic systems in the current PD rat model. The expression of TNF-alpha was not altered by rotenone. However, the results of enhanced expression of IL-1beta in the hypothalamus after rotenone and that of the blockade of sleep alteration, but not the locomotion activity, by intracerebroventricular administration of an IL-1 receptor antagonist implies that increased IL-1beta in the hypothalamus mediates sleep alteration, but not the locomotion, in rats with rotenone-induced parkinsonism. These observations suggest that rotenone-induced sleep-wake alteration is dominated by central increase of somnogenic IL-1.

L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats

The present study investigated oxidative damage and neuroprotective effect of the antiparkinsonian drug, l-deprenyl in neuronal death produced by intranigral infusion of a potent mitochondrial complex-I inhibitor, rotenone in rats. Unilateral stereotaxic intranigral infusion of rotenone caused significant decrease of striatal dopamine levels as measured employing HPLC-electrochemistry, and loss of tyrosine hydroxylase immunoreactivity in the perikarya of ipsilateral substantia nigra (SN) neurons and their terminals in the striatum. Rotenone-induced increases in the salicylate hydroxylation products, 2,3- and 2,5-dihydroxybenzoic acid indicators of hydroxyl radials in mitochondrial P 2 fraction were dose-dependently attenuated by l-deprenyl. l-deprenyl (0.1–10 mg/kg; i.p.) treatment dose-dependently attenuated rotenone-induced reductions in complex-I activity and glutathione (GSH) levels in the SN, tyrosine hydroxylase immunoreactivity in the striatum or SN as well as striatal dopamine. Amphetamine-induced stereotypic rotations in these rats were also significantly inhibited by deprenyl administration. The rotenone-induced elevated activities of cytosolic antioxidant enzymes superoxide dismutase and catalase showed further significant increase following l-deprenyl. Our findings suggest that unilateral intranigral infusion of rotenone reproduces neurochemical, neuropathological and behavioral features of PD in rats and l-deprenyl can rescue the dopaminergic neurons from rotenone-mediated neurodegeneration in them. These results not only establish oxidative stress as one of the major causative factors underlying dopaminergic neurodegeneration as observed in Parkinson's disease, but also support the view that deprenyl is a potent free radical scavenger and an antioxidant.

Behavioral differences in a rotenone-induced hemiparkinsonian rat model developed following intranigral or median forebrain bundle infusion

A mitochondrial complex-I inhibitor, rotenone was unilaterally infused into the substantia nigra pars compacta (SNpc) or median forebrain bundle (MFB) to create hemiparkinsonian animal models and investigated spontaneous and drug-induced stereotypic rotations, as well as certain postural behaviors in Sprague–Dawley rats. Animals infused intranigrally, but not intra-MFB, with rotenone exhibited spontaneous contralateral rotations immediately after recovery from anesthesia. Head position bias and elevated body swing test showed insignificant contralateral bias in animals with nigral damage but a significant ipsilateral bias in MFB-lesioned rats. General motor activity of the animals was reduced in both the groups as indicated by reduced performance on a Plus-Maze. Intranigrally, rotenone-infused animals exhibited progressive ipsilateral rotations when challenged with d-amphetamine on the 7th, 14th, 21st, and 28th days or with apomorphine on 9th, 16th, 23rd, and 30th days. However, animals that received rotenone in MFB exhibited ipsilateral or contralateral rotations when challenged respectively with d-amphetamine or apomorphine only in the 5th week (28th and 30th days). Stereotaxic administration of rotenone into SNpc or MFB caused a significant loss of dopamine in the ipsilateral striatum (>80% in SNpc; >95% in MFB), when assayed employing an HPLC equipped with electrochemical detector on the 32nd day. Neuronal loss in SNpc was confirmed in coronal sections stained with cresyl violet and revealed extension of lesion towards SN pars reticulata, in SNpc-infused animals. Our results demonstrate that rotenone-induced neurodegeneration is a slow, yet progressive process similar to that in idiopathic Parkinson's disease and unlike that observed in other classical neurotoxin-mediated lesions which are abrupt and developed in few hours to days. Thus, intranigral or intra-MFB infusion of rotenone could be used for producing hemiparkinsonian animal models in rats. These findings further suggest that, while both d-amphetamine and apomorphine-induced stereotypic rotations could be used as a valuable behavioral assay procedure to test novel drugs against Parkinson's disease, yet apomorpine-induced contralateral bias in turning is a reliable indicator of specific destruction in nigrostriatal pathway and development of postsynaptic dopamine receptor supersensitivity.