Investigating the role of rotenone on human blood platelets: Molecular insights into abnormal platelet functions in Parkinson's disease.

Parkinson's disease (PD) is a predominant neuromotor disorder characterized by the selective death of dopaminergic neurons in the midbrain. The majority of PD cases are sporadic or idiopathic, with environmental toxins and pollutants potentially contributing to its development or exacerbation. However, clinical PD patients are often associated with a reduced stroke frequency, where circulating blood platelets are indispensable. Although platelet structural impairment is evident in PD, the platelet functional alterations and their underlying molecular mechanisms are still obscure. Therefore, we investigated rotenone (ROT), an environmental neurotoxin that selectively destroys dopaminergic neurons mimicking PD, on human blood platelets to explore its impact on platelet functions, thus replicating PD conditions in vitro. Our study deciphered that ROT decreased thrombin-induced platelet functions, including adhesion, activation, secretion, and aggregation in human blood platelets. As ROT is primarily responsible for generating intracellular reactive oxygen species (ROS), and ROS is a key player regulating the platelet functional parameters, we went on to check the effect of ROT on platelet ROS production. In our investigation, it became evident that ROT treatment resulted in the stimulation of ROS production in human blood platelets. Additionally, we discovered that ROT induced ROS production by augmenting Ca2+ mobilization from inositol 1,4,5-trisphosphate receptor. Apart from this, the treatment of ROT triggers protein kinase C associated NADPH oxidase-mediated ROS production in platelets. In summary, this research, for the first time, highlights ROT-induced abnormal platelet functions and may provide a mechanistic insight into the altered platelet activities observed in PD patients.

No Evidence of Sensory Neuropathy in a Traditional Mouse Model of Idiopathic Parkinson's Disease.

Parkinson's disease (PD) is the second-most common neurodegenerative disorder worldwide and is diagnosed based on motor impairments. Non-motor symptoms are also well-recognised in this disorder, and peripheral neuropathy is a frequent but poorly appreciated non-motor sign. Studying how central and peripheral sensory systems are affected can contribute to the development of targeted therapies and deepen our understanding of the pathophysiology of PD. Although the cause of sporadic PD is unknown, chronic exposure to the pesticide rotenone in humans increases the risk of developing the disease. Here, we aimed to investigate whether peripheral neuropathy is present in a traditional model of PD. Mice receiving intrastriatal rotenone showed greatly reduced dopamine terminals in the striatum and a reduction in tyrosine hydroxylase-positive neurons in the Substantia nigra pars compacta and developed progressive motor impairments in hindlimb stepping and rotarod but no change in spontaneous activity. Interestingly, repeated testing using gold-standard protocols showed no change in gut motility, a well-known non-motor symptom of PD. Importantly, we did not observe any change in heat, cold, or touch sensitivity, again based upon repeated testing with well-validated protocols that were statistically well powered. Therefore, this traditional model fails to replicate PD, and our data again reiterate the importance of the periphery to the disorder.

Malvidin attenuates behavioral and inhibits the TNF-α/Caspase-3/Nrf-2 expression in rotenone-induced Parkinson's disease in rats: insights from molecular docking.

OBJECTIVE: Malvidin is a natural, biologically active polyphenol found in several fruits. It exhibits several therapeutic benefits; however, limited studies are available on its effects on neurodegenerative clinical conditions, including Parkinson's disease. The study aimed to investigate the therapeutic properties of malvidin on rotenone-triggered Parkinson's disease in an animal model. MATERIALS AND METHODS: To determine the effects of malvidin, rotenone (1.5 mg/kg) was injected subcutaneously into Wistar rats for 21 days, followed by a dose of malvidin (200 and 100 mg/kg). Behavioral tests were performed on the experimental animals before sacrifice. On the 22nd day of the experiment, biochemical tests were performed, including superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and catalase (CAT). The activity of neurotransmitters and their metabolites, including acetylcholine (ACh), acetylcholinesterase (AChE), dopamine (DA), norepinephrine (NE), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) along with neuroinflammatory markers including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor- α (TNF-α), and nuclear factor erythroid 2-related factor 2 (Nrf-2) were estimated. Moreover, the level of the apoptotic marker, caspase-3, was also estimated. In addition, molecular docking was performed. RESULTS: The administration of rotenone resulted in oxidative stress, cholinergic imbalances, dopaminergic alternations, and increased expression of inflammatory compounds. The docking analysis revealed that malvidin displayed a favorable binding affinity for AChE, showcasing a binding energy of -9.329 Kcal/mol. CONCLUSIONS: The investigation concludes that malvidin exhibits neuroprotective effects due to its curative effects against inflammation and oxidative stress. These findings suggest that malvidin possesses therapeutic potential against rotenone-triggered behavioral, oxidative, and inflammatory abnormalities in rodents.

LRRK2 kinase inhibition protects against Parkinson's disease-associated environmental toxicants.

Idiopathic Parkinson's disease (PD) is epidemiologically linked with exposure to toxicants such as pesticides and solvents, which comprise a wide array of chemicals that pollute our environment. While most are structurally distinct, a common cellular target for their toxicity is mitochondrial dysfunction, a key pathological trigger involved in the selective vulnerability of dopaminergic neurons. We and others have shown that environmental mitochondrial toxicants such as the pesticides rotenone and paraquat, and the organic solvent trichloroethylene (TCE) appear to be influenced by the protein LRRK2, a genetic risk factor for PD. As LRRK2 mediates vesicular trafficking and influences endolysosomal function, we postulated that LRRK2 kinase activity may inhibit the autophagic removal of toxicant damaged mitochondria, resulting in elevated oxidative stress. Conversely, we suspected that inhibition of LRRK2, which has been shown to be protective against dopaminergic neurodegeneration caused by mitochondrial toxicants, would reduce the intracellular production of reactive oxygen species (ROS) and prevent mitochondrial toxicity from inducing cell death. To do this, we tested in vitro if genetic or pharmacologic inhibition of LRRK2 (MLi2) protected against ROS caused by four toxicants associated with PD risk - rotenone, paraquat, TCE, and tetrachloroethylene (PERC). In parallel, we assessed if LRRK2 inhibition with MLi2 could protect against TCE-induced toxicity in vivo, in a follow up study from our observation that TCE elevated LRRK2 kinase activity in the nigrostriatal tract of rats prior to dopaminergic neurodegeneration. We found that LRRK2 inhibition blocked toxicant-induced ROS and promoted mitophagy in vitro, and protected against dopaminergic neurodegeneration, neuroinflammation, and mitochondrial damage caused by TCE in vivo. We also found that cells with the LRRK2 G2019S mutation displayed exacerbated levels of toxicant induced ROS, but this was ameliorated by LRRK2 inhibition with MLi2. Collectively, these data support a role for LRRK2 in toxicant-induced mitochondrial dysfunction linked to PD risk through oxidative stress and the autophagic removal of damaged mitochondria.

Rotenone activates the LKB1-AMPK-ULK1 signaling pathway to induce autophagy and apoptosis in rat thoracic aortic endothelial cells.

BACKGROUND: The specific mechanism by which rotenone impacts thoracic aortic autophagy and apoptosis is unknown. We aimed to investigate the regulatory effects of rotenone on autophagy and apoptosis in rat thoracic aortic endothelial cells (RTAEC) via activation of the LKB1-AMPK-ULK1 signaling pathway and to elucidate the molecular mechanisms of rotenone on autophagy and apoptosis in vascular endothelial cells. METHODS: In vivo, 60 male SD rats were randomly selected and divided into 5 groups: control (Con), DMSO, 1, 2, and 4 mg/kg groups, respectively. After 28 days of treatment, histopathological and ultrastructural changes in each group were observed using HE and transmission electron microscopy; Autophagy, apoptosis, and LKB1-AMPK-ULK1 pathway-related proteins were detected by Western blot; Apoptosis levels in the thoracic aorta were detected by TUNEL. In vitro, RTAEC were cultured and divided into control (Con), DMSO, 20, 100, 500, and 1000 nM groups. After 24 h of intervention, autophagy, apoptosis, and LKB1-AMPK-ULK1 pathway-related factors were detected by Western blot and qRT-PCR; Flow cytometry to detect apoptosis levels; Autophagy was inhibited with 3-MA and CQ to detect apoptosis levels, and changes in autophagy, apoptosis, and downstream factors were detected by the AMPK inhibitor CC intervention. RESULTS: Gavage in SD rats for 28 days, some degree of damage was observed in the thoracic aorta and heart of the rotenone group, as well as the appearance of autophagic vesicles was observed in the thoracic aorta. TUNEL analysis revealed higher apoptosis in the rotenone group's thoracic aorta; RTAEC cultured in vitro, after 24 h of rotenone intervention, showed increased ROS production and significantly decreased ATP production. The flow cytometry data suggested an increase in the number of apoptotic RTAEC. The thoracic aorta and RTAEC in the rotenone group displayed elevated levels of autophagy and apoptosis, and the LKB1-AMPK-ULK1 pathway proteins were activated and expressed at higher levels. Apoptosis and autophagy were both suppressed by the autophagy inhibitors 3-MA and CQ. The AMPK inhibitor CC reduced autophagy and apoptosis in RTAEC and suppressed the production of the AMPK downstream factors ULK1 and P-ULK1. CONCLUSIONS: Rotenone may promote autophagy in the thoracic aorta and RTAEC by activating the LKB1-AMPK-ULK1 signaling pathway, thereby inducing apoptosis.

Microglial gp91phox-mediated neuroinflammation and ferroptosis contributes to learning and memory deficits in rotenone-treated mice.

Apart from dopaminergic neurotoxicity, exposure to rotenone, a commonly used insecticide in agriculture, also adversely affects hippocampal and cortical neurons, resulting in cognitive impairments in mice. We recently established a role of microglia-mediated neuroinflammation in rotenone-elicited deficits of cognition, yet the mechanisms remain elusive. Here, we investigated the involvement of NADPH oxidase 2 (NOX2) catalytic subunit gp91phox in rotenone-induced cognitive deficits and the associated mechanisms. Our study demonstrated that rotenone exposure elevated expression of gp91phox and phosphorylation of the NOX2 cytosolic subunit p47phox, along with NADPH depletion in the hippocampus and cortex of mice, indicating NOX2 activation. Specific knockdown of gp91phox in microglia via adeno-associated virus delivery resulted in reduced microglial activation, proinflammatory gene expression and improved learning and memory capacity in rotenone-intoxicated mice. Genetic deletion of gp91phox also reversed rotenone-elicited cognitive dysfunction in mice. Furthermore, microglial gp91phox knockdown attenuated neuronal damage and synaptic loss in mice. This intervention also suppressed iron accumulation, disruption of iron-metabolism proteins and iron-dependent lipid peroxidation and restored the balance of ferroptosis-related parameters, including GPX4, SLC711, PTGS2, and ACSL4 in rotenone-lesioned mice. Intriguingly, pharmacological inhibition of ferroptosis with liproxstatin-1 conferred protection against rotenone-induced neurodegeneration and cognitive dysfunction in mice. In summary, our findings underscored the contribution of microglial gp91phox-dependent neuroinflammation and ferroptosis to learning and memory dysfunction in rotenone-lesioned mice. These results provided valuable insights into the pathogenesis of cognitive deficits associated with pesticide-induced Parkinsonism, suggesting potential therapeutic avenues for intervention.

Neuroprotective potential of Mentha piperita extract prevents motor dysfunctions in mouse model of Parkinson's disease through anti-oxidant capacities.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Neurodegeneration of the substantia nigra (SN) and diminished release of dopamine are prominent causes of this progressive disease. The current study aims to evaluate the protective potential of ethanolic extract of Mentha piperita (EthMP) against rotenone-mediated PD features, dopaminergic neuronal degeneration, oxidative stress and neuronal survival in a mouse model. Swiss albino male mice were assigned to five groups: control (2.5% DMSO vehicle), PD (rotenone 2.5 mg/kg), EthMP and rotenone (200mg/kg and 2.5mg/kg, respectively), EthMP (200 mg/kg), and Sinemet, reference treatment containing levodopa and carbidopa (20 mg/kg and rotenone 2.5mg/kg). Behavioral tests for motor functional deficit analysis were performed. Anti-oxidant capacity was estimated using standard antioxidant markers. Histopathology of the mid-brain for neurodegeneration estimation was performed. HPLC based dopamine level analysis and modulation of gene expression using quantitative real-time polymerase chain reaction was performed for the selected genes. EthMP administration significantly prevented the rotenone-mediated motor dysfunctions compared to PD group as assessed through open field, beam walk, pole climb down, stepping, tail suspension, and stride length tests. EthMP administration modulated the lipid peroxidation (LPO), reduced glutathione (GSH), and superoxide dismutase (SOD) levels, as well as glutathione-s-transferase (GST) and catalase (CAT) activities in mouse brain. EthMP extract prevented neurodegeneration in the SN of mice and partially maintained dopamine levels. The expression of genes related to dopamine, anti-oxidant potential and synapses were modulated in M. piperita (MP) extract treated mice brains. Current data suggest therapeutic capacities of MP extract and neuroprotective capacities, possibly through antioxidant capacities. Therefore, it may have potential clinical applications for PD management.

Neuroprotective potential of traditionally used medicinal plants of Manipur against rotenone-induced neurotoxicity in SH-SY5Y neuroblastoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera sessilis (L.) R. Br. ex DC., Eryngium foetidum L., and Stephania japonica (Thunb.) Miers plants are traditionally used to treat various central nervous system disorders like paralysis, epilepsy, seizure, convulsion, chronic pain, headache, sleep disturbances, sprain, and mental disorders. However, their possible neuroprotective effects have not been evaluated experimentally so far. AIM OF THE STUDY: The study aims to examine the neuroprotective potential of the three plants against cytotoxicity induced by rotenone in SH-SY5Y neuroblastoma cells and assess its plausible mechanisms of neuroprotection. MATERIALS AND METHODS: The antioxidant properties of the plant extracts were determined chemically by DPPH and ABTS assay methods. The cytotoxicity of rotenone and the cytoprotective activities of the extracts were evaluated using MTT assays. Microtubule-associated protein 2 (MAP2) expression studies in cells were performed to assess neuronal survival after rotenone and extract treatments. Mitochondrial membrane potential and intracellular levels of reactive oxygen species were evaluated using Rhodamine 123 and DCF-DA dye, respectively. Catalase, glutathione peroxidase, and superoxide dismutase activities were also measured. Apoptotic nuclei were examined using DAPI staining. Liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS) analysis of the plant extracts was also performed. RESULTS: The methanol extracts of A. sessilis, S. japonica, and E. foetidum showed excellent free radical scavenging activities. MAP2 expression studies show that A. sessilis and S. japonica have higher neuroprotective effects against rotenone-induced neurotoxicity in SH-SY5Y cells than E. foetidum. Pre-treating cells with the plant extracts reverses the rotenone-induced increase in intracellular ROS. The plant extracts could also restore the reduced mitochondrial membrane potential induced by rotenone treatment and reinstate rotenone-induced increases in catalase, glutathione peroxidase, and superoxide dismutase activities. All the extracts inhibited rotenone-induced changes in nuclear morphology and DNA condensation, an early event of cellular apoptosis. LC-QTOF-MS analysis of the plant extracts shows the presence of neuroprotective compounds. CONCLUSIONS: The plant extracts showed neuroprotective activities against rotenone-treated SH-SY5Y cells through antioxidant and anti-apoptotic mechanisms. These findings support the ethnopharmacological uses of these plants in treating neurological disorders. They probably are a good source of neuroprotective compounds that could be further explored to develop treatment strategies for neurodegenerative diseases like Parkinson's disease.

Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

Dopamine- and Grape-Seed-Extract-Loaded Solid Lipid Nanoparticles: Interaction Studies between Particles and Differentiated SH-SY5Y Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, primarily associated with dopaminergic neuron depletion in the Substantia Nigra. Current treatment focuses on compensating for dopamine (DA) deficiency, but the blood-brain barrier (BBB) poses challenges for effective drug delivery. Using differentiated SH-SY5Y cells, we investigated the co-administration of DA and the antioxidant Grape Seed Extract (GSE) to study the cytobiocompability, the cytoprotection against the neurotoxin Rotenone, and their antioxidant effects. For this purpose, two solid lipid nanoparticle (SLN) formulations, DA-co-GSE-SLNs and GSE-ads-DA-SLNs, were synthesized. Such SLNs showed mean particle sizes in the range of 187-297 nm, zeta potential values in the range of -4.1--9.7 mV, and DA association efficiencies ranging from 35 to 82%, according to the formulation examined. The results showed that DA/GSE-SLNs did not alter cell viability and had a cytoprotective effect against Rotenone-induced toxicity and oxidative stress. In addition, this study also focused on the evaluation of Alpha-synuclein (aS) levels; SLNs showed the potential to modulate the Rotenone-mediated increase in aS levels. In conclusion, our study investigated the potential of SLNs as a delivery system for addressing PD, also representing a promising approach for enhanced delivery of pharmaceutical and antioxidant molecules across the BBB.

A High-Throughput Screening of a Natural Products Library for Mitochondria Modulators.

Mitochondria, the energy hubs of the cell, are progressively becoming attractive targets in the search for potent therapeutics against neurodegenerative diseases. The pivotal role of mitochondrial dysfunction in the pathogenesis of various diseases, including Parkinson's disease (PD), underscores the urgency of discovering novel therapeutic strategies. Given the limitations associated with available treatments for mitochondrial dysfunction-associated diseases, the search for new potent alternatives has become imperative. In this report, we embarked on an extensive screening of 4224 fractions from 384 Australian marine organisms and plant samples to identify natural products with protective effects on mitochondria. Our initial screening using PD patient-sourced olfactory neurosphere-derived (hONS) cells with rotenone as a mitochondria stressor resulted in 108 promising fractions from 11 different biota. To further assess the potency and efficacy of these hits, the 11 biotas were subjected to a subsequent round of screening on human neuroblastoma (SH-SY5Y) cells, using 6-hydroxydopamine to induce mitochondrial stress, complemented by a mitochondrial membrane potential assay. This rigorous process yielded 35 active fractions from eight biotas. Advanced analysis using an orbit trap mass spectrophotometer facilitated the identification of the molecular constituents of the most active fraction from each of the eight biotas. This meticulous approach led to the discovery of 57 unique compounds, among which 12 were previously recognized for their mitoprotective effects. Our findings highlight the vast potential of natural products derived from Australian marine organisms and plants in the quest for innovative treatments targeting mitochondrial dysfunction in neurodegenerative diseases.

The effect of common paralytic agents used for fluorescence imaging on redox tone and ATP levels in Caenorhabditis elegans.

One aspect of Caenorhabditis elegans that makes it a highly valuable model organism is the ease of use of in vivo genetic reporters, facilitated by its transparent cuticle and highly tractable genetics. Despite the rapid advancement of these technologies, worms must be paralyzed for most imaging applications, and few investigations have characterized the impacts of common chemical anesthetic methods on the parameters measured, in particular biochemical measurements such as cellular energetics and redox tone. Using two dynamic reporters, QUEEN-2m for relative ATP levels and reduction-oxidation sensitive GFP (roGFP) for redox tone, we assess the impact of commonly used chemical paralytics. We report that no chemical anesthetic is entirely effective at doses required for full paralysis without altering redox tone or ATP levels, and that anesthetic use alters the detected outcome of rotenone exposure on relative ATP levels and redox tone. We also assess the use of cold shock, commonly used in combination with physical restraint methods, and find that cold shock does not alter either ATP levels or redox tone. In addition to informing which paralytics are most appropriate for research in these topics, we highlight the need for tailoring the use of anesthetics to different endpoints and experimental questions. Further, we reinforce the need for developing less disruptive paralytic methods for optimal imaging of dynamic in vivo reporters.

A polarity-responsive lysosomes-nucleus translocation probe for the dual-emissive visualization of cell apoptosis.

Visualization of cell apoptosis is a critical task playing central roles in the fundamental studies in biology, pathology, and biomedicine. Dual-emissive fluorescent probes are desired molecular tools for study on apoptosis, which however were rarely reported. Herein, utilizing the polarity differences between lysosomes and nucleus, a translocation type of fluorescent probe (NA-S) was developed for the dual-color visualization of cell apoptosis. NA-S was designed to be polarity sensitive, bearing alkalescence group, and with DNA affinity. In living cells, NA-S targeted the lysosomes to give blue fluorescence, which translocated into the nucleus during cell apoptosis to give green emission. Thereby, the cell apoptosis could be visualized with NA-S in dual-emissive manner. With the unique probe, the cell apoptosis induced by oxidative stress, UV irradiation, rotenone, colchicine, and paclitaxel have been successfully visualized.

Syagrus coronata fixed oil prevents rotenone-induced movement disorders and oxidative stress in Drosophila melanogaster.

One prominent aspect of Parkinson's disease (PD) is the presence of elevated levels of free radicals, including reactive oxygen species (ROS). Syagrus coronata (S. coronata), a palm tree, exhibits antioxidant activity attributed to its phytochemical composition, containing fatty acids, polyphenols, and flavonoids. The aim of this investigation was to examine the potential neuroprotective effects of S. coronata fixed oil against rotenone-induced toxicity using Drosophila melanogaster. Young Drosophila specimens (3-4 d old) were exposed to a diet supplemented with rotenone (50 µM) for 7 d with and without the inclusion of S. coronata fixed oil (0.2 mg/g diet). Data demonstrated that rotenone exposure resulted in significant locomotor impairment and increased mortality rates in flies. Further, rotenone administration reduced total thiol levels but elevated lipid peroxidation, iron (Fe) levels, and nitric oxide (NO) levels while decreasing the reduced capacity of mitochondria. Concomitant administration of S. coronata exhibited a protective effect against rotenone, as evidenced by a return to control levels of Fe, NO, and total thiols, lowered lipid peroxidation levels, reversed locomotor impairment, and enhanced % cell viability. Molecular docking of the oil lipidic components with antioxidant enzymes showed strong binding affinity to superoxide dismutase (SOD) and glutathione peroxidase (GPX1) enzymes. Overall, treatment with S. coronata fixed oil was found to prevent rotenone-induced movement disorders and oxidative stress in Drosophila melanogaster.

Lipid Carrier Nanostructured Astilbin Ameliorates Rotenone-Induced Neurodegeneration in Mice Brain via Modulation of GSK3ß-Nrf2 Signaling Pathways.

Astilbin is a flavanonol, found in St John's wort (Hypericum perforatum) and many other plants. It has been demonstrated that astilbin contains anti-inflammatory, antioxidant, and immune-suppressive properties. However, the bioavailability of astilbin remains a question for which drug delivery-based nanoparticles can be utilized. We formulated a nanostructured lipid carrier loaded with astilbin (NLC-AS) and tested its effects on the rotenone exposed PC12 cells and in a neurodegenerative mice model of Parkinson's disease (PD) induced by rotenone. Results show that rotenone caused dose-dependent inhibition of PC12 cell growth with about 50% cell death at 2 µM rotenone. Rotenone caused apoptosis in PC12 cells which was reduced to a notable level by NLC-AS through suppression of oxidative stress, especially via elevation of GSH and total antioxidant capacity, and inhibition of monoamine oxidase. Rotenone significantly augmented neurodegeneration in mouse brains by triggering apoptosis and oxidative damage, while NLC-AS treatment halted these processes. Rotenone-exposed mice showed neuronal deficits and impaired neurocognitive functions like loss of memory and learning restrictions which were restored to a remarkable level by NLC-AS administration. The protective effect of NLC-AS was mediated through the inhibition of GSK3ß and induction of Nrf2 genes in the brain tissues. These findings suggest that NLC-AS administration may efficiently regulate the signs of PD in mice and prevent neurodegeneration and neurocognitive dysfunctions.

ASSESSMENT OF RAT BRAIN MORPHOFUNCTIONAL STATE IN A PARKINSON'S MODEL: INFLUENCE OF THERAPEUTIC AGENTS OF ANIMAL AND SYNTHETIC ORIGINS.

In neurodegenerative diseases, particularly in Parkinson's disease (PD), antinociceptive centers are often implicated in neurodegeneration, leading to persistent pain unresponsive to narcotic substances. This study investigated the periaqueductal gray matter (PAG) and the nucleus raphe magnus (NRM), components of the brain's antinociceptive system. In conditions of rotenone intoxication (an experimental PD model), morphological changes in intracellular structures were observed in PAG and NRM neurons, indicating metabolic disorders characteristic of PD (alterations in the shape and size of neuronal bodies and processes, disruption of acid phosphatase activity in neuron cytoplasm). Under the influence of bacterial melanin and in combination with synoestrol, positive changes in structural properties were observed in PAG and NRM neurons compared to the rotenone model of PD. This included the preservation of the morphological characteristics typical of these brain regions, with cells exhibiting shapes and sizes close to normal. Furthermore, under the influence of these therapeutic agents, an increase in phosphatase activity in cell cytoplasm was detected, indicating an acceleration of metabolic processes (metabolic activation) disrupted by rotenone intoxication. The data obtained suggests that bacterial melanin and synoestrol may act as potential neuroprotective agents against PAG and NRM neurons in the rat brain in the rotenone model of PD. Further research is needed to elucidate the mechanisms of action of therapeutic doses and propose their use in the treatment of PD, either in isolation or combination therapy.

H2S Protects from Rotenone-Induced Ferroptosis by Stabilizing Fe-S Clusters in Rat Cardiac Cells.

Hydrogen sulfide (H2S) has been recently recognized as an important gasotransmitter with cardioprotections, and iron is vital for various cellular activities. This study explored the regulatory role of H2S on iron metabolism and mitochondrial functions in cultured rat cardiac cells. Rotenone, a mitochondrial complex I inhibitor, was used for establishing an in vitro model of ischemic cell damage. It was first found that rotenone induced oxidative stress and lipid peroxidation and decreased mitochondrial membrane potential and ATP generation, eventually causing cell death. The supplement of H2S at a physiologically relevant concentration protected from rotenone-induced ferroptotic cell death by reducing oxidative stress and mitochondrial damage, maintaining GPx4 expression and intracellular iron level. Deferiprone, an iron chelator, would also protect from rotenone-induced ferroptosis. Further studies demonstrated that H2S inhibited ABCB8-mediated iron efflux from mitochondria to cytosol and promoted NFS1-mediated Fe-S cluster biogenesis. It is also found that rotenone stimulated iron-dependent H2S generation. These results indicate that H2S would protect cardiac cells from ischemic damage through preserving mitochondrial functions and intracellular Fe-S cluster homeostasis.

Three chemosensory proteins enriched in antennae and tarsi of Rhaphuma horsfieldi differentially contribute to the binding of insecticides.

Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 µM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.