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In addition to the canonical Gs adenylyl cyclase pathway, the serotonin type 6 receptor (5-HT6R) recruits additional signaling pathways that control cognitive function, brain development, and synaptic plasticity in an agonist-dependent and independent manner. Considering that aberrant constitutive and agonist-induced active states are involved in various pathological mechanisms, the development of biased ligands with different functional profiles at specific 5-HT6R-elicited signaling pathways may provide a novel therapeutic perspective in the field of neurodegenerative and psychiatric diseases. Based on the structure of SB-258585, an inverse agonist at 5-HT6R-operated Gs and Cdk5 signaling, we designed a series of 1-(arylsulfonyl-isoindol-2-yl)piperazine derivatives and synthesized them using a sustainable mechanochemical method. We identified the safe and metabolically stable biased ligand 3g, which behaves as a neutral antagonist at the 5-HT6R-operated Gs signaling and displays inverse agonist activity at the Cdk5 pathway. Inversion of the sulfonamide bond combined with its incorporation into the isoindoline scaffold switched the functional profile of 3g at Gs signaling with no impact at the Cdk5 pathway. Compound 3g reduced the cytotoxicity of 6-OHDA and produced a glioprotective effect against rotenone-induced toxicity in C8-D1A astrocyte cell cultures. In view of these findings, compound 3g can be considered a promising biased ligand to investigate the role of the 5-HT6R-elicited Gs and Cdk5 signaling pathways in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Ethnopharmacological Relevance: Traditional harvest of Achyrocline satureioides (AS) occurs at dawn on Good Friday in some South American countries. Inflorescences are traditionally used as infusions for several disorders, including neuropsychiatric disorders. Pillows and cushions are popularly filled with AS to attenuate the symptoms of depression, anxiety, and sleep disturbances. However, evidence for the potential beneficial effects of AS on human neural cells remains unclear., Aim of the Study: An in vitro model of SH-SY5Y human neural cells was applied to evaluate the effect of AS infusion, prepared as commonly used, on cells exposed to rotenone and to investigate its potential for neuropsychiatric disorders., Materials and Methods: A hot aqueous extract was obtained from a traditionally prepared AS inflorescence infusion and chemically characterized by high-resolution mass spectrometry and spectrophotometric quantification of total polyphenols, tannins, and flavonoids. The SH-SY5Y cell cultures were treated with AS extract at concentrations of 1, 3, 5, 10, 50, 100, and 300 μL/mL to determine the potential cyto- and genotoxic effects of AS on neural cells using MTT, Neutral Red, and GEMO assays. Apoptosis modulation was assessed using flow cytometry and apoptosis-modulating genes were evaluated by qRT-PCR. The protective effect of AS on the neurotoxicity triggered by rotenone exposure (30 nM) was determined by analyzing cellular viability and oxidative markers such as lipid peroxidation and protein carbonylation, and DNA damage was assessed by micronucleus assay., Results: The AS extract, as traditionally prepared, had estimated concentrations of 409.973 ± 31.107 μg/mL, 0.1041 ± 0.0246 mg GAE/mL, and 63.309 ± 3.178 mg QE/mL of total tannins, total polyphenols, and flavonoids, respectively. At concentrations of 30 and 100 μl/mL, AS decreased apoptotic events, whereas the highest concentration (300 μl/mL) increased apoptosis compared to that in the control (p < 0.05). In cells exposed to rotenone, AS treatment induced cell proliferation, reduced DNA damage (as evaluated by micronuclei), and reduced lipid and protein oxidation., Conclusions: The data indicate the non-cytotoxic and beneficial effects of AS extract on human neural cells by reducing cellular mortality and oxidative stress in neural cells triggered by rotenone exposure., Competing Interests: Declaration of competing interest The authors declare no conflict of interest in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is an idiopathic disease caused by the loss or degeneration of the dopaminergic (dopamine-producing) neurons in the brain and characterized by various inflammatory and apoptotic responses in the neuronal cells. Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) axis is responsible for neuronal survival by providing a number of anti-inflammatory and anti-apoptotic milieu that prevent the progression of PD. Alpha-lipoic acid (ALA) is a natural cofactor that has antioxidant capacity and contributes to various metabolic processes. ALA can penetrate the blood-brain barrier and contribute to numerous neuroprotective effects. It can activate PI3K/AKT pathway with consequent reduction of different inflammatory and oxidative biomarkers. Our work aims to unfold the neuroprotective effects of ALA via targeting PI3k/AKT pathway. Forty male mice were divided into four groups: control, ALA (100 mg/kg/day; i.p.), rotenone (ROT) (1.5 mg/kg/2 days, i.p.) and rotenone + ALA for 21 days. ALA showed obvious neuroprotective effects via significant activation of PI3K/AKT pathway with subsequent decreasing level of Caspase-3. ALA resulted in prominent anti-inflammatory actions by decreasing interlukin-1β (IL-1β), tumor necrosis factor (TNF)-α and nuclear factor kabba (NFk)-B. ALA remarkably induced antioxidant activities via increasing reduced glutathione (GSH) and superoxide dismutase (SOD) levels as well as decreasing malondialdehyde (MDA) level. The substantial behavioral improvement reflected in these results was noticed in the ALA-treated mice as a reflection of the neuroprotective activities of ALA. In conclusion, ALA showed promising neuroprotective effects in rotenone-induced PD via activating the PI3K/AKT pathway and consequent inhibition of apoptotic and inflammatory biomarkers., Competing Interests: Declaration of competing interest none., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rotenone (ROT), a widely used natural pesticide, has an uncertain effect on reproductive toxicity. In this study, we used 20 mice distributed randomly into four groups, with each group receiving ROT doses of 0, 2, 4, and 8 mg/kg/day for 28 days. The results demonstrated that ROT induced significant testicular damage, including impaired spermatogenesis, inhibition of testosterone synthesis, and apoptosis of Leydig cells. Additionally, ROT disrupted the normal ultrastructure of the endoplasmic reticulum (ER) in testicular tissue, leading to ER stress in Leydig cells. To further explore whether ROT-induced apoptosis in Leydig cells is related to ER stress, the mouse Leydig cell line (TM3 cells) was treated with ROT at 0, 250, 500, and 1000 nM. ROT inhibited TM3 cell viability, induced cytotoxicity, and reduced testosterone content in the culture supernatants. Furthermore, ROT treatment triggered apoptosis in TM3 cells by activating ER stress and the PERK-eIF2α-CHOP signalling pathway. Pre-treatment of TM3 cells exposed to ROT with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated these effects, decreasing apoptosis and preserving testosterone levels. Further intervention with the PERK inhibitor GSK2606414 reduced ROT-induced apoptosis and testosterone reduction by inhibiting PERK activity. In summary, ROT-induced male reproductive toxicity is specifically driven by apoptosis, with the PERK-eIF2α-CHOP signalling pathway activated by ER stress playing a crucial role in the apoptosis of Leydig cells triggered by ROT., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guanghua Li reports financial support was provided by Ningxia Hui Autonomous Region Natural Science Foundation (2024A1105）If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

In this study, the antiparkinson effect of khellin (KL) on rotenone-induced Parkinson's disease (PD) was examined in zebrafish. Initially, In silico evaluations, such as drug likeness and ADME/T analysis, confirmed the pharmacological viability of KL. Molecular docking and molecular dynamics (MD) analysis revealed stable binding interactions between KL and monamine oxidase B (MAO-B). Molecular docking results for KL and pioglitazone (CCl) revealed binding energies of -6.5 and -10.4 kcal/mol, respectively. Later, molecular dynamics (MD) studies were performed to assess the stability of these complexes, which yielded binding energies of -36.04 ± 55.21 and -56.2 ± 80.63 kJ/mol for KL and CCl, respectively. These results suggest that KL exhibits considerable binding affinity for MAO-B. In In vitro studies, according to the DPPH free radical scavenging assay, KL exhibited significant antioxidant effects, indicating that it can promote redox balance with an IC 50 value of 22.68 ± 0.5 μg/ml. In vivo studies and evaluation of locomotor activity, social interaction, histopathology and biochemical parameters were conducted in KL-treated zebrafish to measure SOD and GSH antioxidant activity, the oxidative stress marker malondialdehyde (MDA), the inflammatory marker myeloperoxidase (MPO) and MAO-B. However, while the locomotor and social interaction abilities of the rotenone-treated zebrafish were significantly reduced, KL treatment significantly improved locomotor activity (p < 0.001) and social interaction (p < 0.001). KL alleviated PD symptoms, as indicated by significant increases in SOD (p < 0.01), GSH (p < 0.001), MDA (p < 0.001), MAO-B (p < 0.001) and MPO (p < 0.001) in rotenone-induced PD fish (p<0.001) significantly reduced activities. Histopathological studies revealed that rotenone-induced brain hyperintensity and abnormal cellularity of the periventricular gray matter in the optic tectum were significantly reduced by KL treatment. This study provides a strong basis for developing KL as a new candidate for the treatment of Parkinson's disease, with the prospect of improved safety profiles and efficacy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sheikh Fayaz Ahmad reports financial support was provided by King Saud University. Sheikh Fayaz Ahmad reports a relationship with King Saud University that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heat shock protein 70 (HSP70) is activated under stress response. Its involvement in cell protection, including energy metabolism and quality control makes it a promising pharmacological target. A strategy to increase HSP70 levels inside the cells is the application of recombinant HSP70. However, cell permeability and functionality of these exogenously applied proteins inside the cells is still disputable. Here, using fluorescence- labeled HSP70, we have studied permeability and distribution of HSP70 inside primary neurons and astrocytes, and how exogenous HSP70 changes mitochondrial metabolism and mitophagy. We have found that exogenous recombinant HSP70 can penetrate the neurons and astrocytes and distributes in mitochondria, lysosomes and in lesser degree in the endoplasmic reticulum. HSP70 increases mitochondrial membrane potential in control neurons and astrocytes, and in fibroblasts of patients with familial Parkinson´s disease (PD) with PINK1 and LRRK2 mutations. Increased mitochondrial membrane potential was associated with higher mitochondrial ROS production and activation of mitophagy. Importantly, preincubation of the cells with HSP70 protected neurons and astrocytes against cell death in a toxic model of PD induced by rotenone, and in the PINK1 and LRRK2 PD human fibroblasts. Thus, exogenous recombinant HSP70 is cell permeable, and acts as endogenous HSP70 protecting cells in the case of toxic model and familial forms of Parkinson's Disease., (© 2024. The Author(s).)

Naringenin, a potent antioxidant with anti-apoptotic effects, holds potential in counteracting rotenone-induced neurotoxicity, a model for Parkinson's disease, by reducing oxidative stress and supporting mitochondrial function. Rotenone disrupts ATP production in SH-SY5Y cells through mitochondrial complex-I inhibition, leading to increased reactive oxygen species (ROS) and cellular damage. However, the therapeutic use of naringenin is limited by its poor solubility, low bioavailability, and stability concerns. Nano crystallization of naringenin (NCs), significantly improved its solubility, dissolution rates, and stability for targeted drug delivery. The developed NAR-NC and HSA-NAR-NC formulations exhibit particle sizes of 95.23 nm and 147.89 nm, with zeta potentials of -20.6 mV and -28.5 mV, respectively. These nanocrystals also maintain high drug content and show stability over time, confirming their pharmaceutical viability. In studies using the SH-SY5Y cell line, these modified nanocrystals effectively preserved mitochondrial membrane potential, sustained ATP production, and regulated ROS levels, counteracting the neurotoxic effects of rotenone. Naringenin nanocrystals offer a promising solution for improving the stability and bioavailability of naringenin, with potential therapeutic applications in neurodegenerative diseases., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

In the present study, we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely, in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons, while TH-positive catecholamine neurons were significantly underrepresented. Furthermore, high NMDAR1 expression was also detected in the KS spheroid, similarly to other patients-derived neuronal cultures, denoting high NMDAR1 expression as a general, KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat, rotenone, bardoxolone, and doxorubicin), and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions, drug development, and screening towards individual therapeutic strategies., (© 2024. The Author(s).)

Parkinson's disease (PD) is a common motor neurodegenerative disease that still lacks effective therapeutic options. Previous studies have reported that lactoferrin exhibited neuroprotective effects in cellular and animal models of PD, typically induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP + ) synthetic toxin. However, the neuroprotective capacity of lactoferrin in the rotenone-induced cellular model of PD remains relatively less established. Unlike MPTP/MPP + , rotenone is a naturally occurring environmental toxin known to induce chronic toxicity and increase the risk of PD in humans. In this study, we constructed a cellular model of PD by differentiating SH-SY5Y neuroblastoma cells with retinoic acid into mature dopaminergic neurons with increased β-tubulin III and tyrosine hydroxylase expression, followed by 24 h of rotenone exposure. Using this cellular model of PD, we showed that lactoferrin (1-10 µg/ml) pre-treatment for 48 h decreased loss of cell viability, mitochondrial membrane potential impairment, reactive oxygen species generation and pro-apoptotic activities (pan-caspase activation and nuclear condensation) in cells exposed to rotenone (1 and 5 µM) using biochemical assays, Hoechst 33342 staining and immunocytochemical techniques. We further demonstrated that 48 h of lactoferrin (10 µg/ml) pre-treatment decreased Bax:Bcl2 ratio and p42/44 mitogen-activated protein kinase expression but increased pAkt expression in 5 µM rotenone-exposed cells. Our study demonstrates that lactoferrin neuroprotective capacity is present in the rotenone-induced cellular model of PD, further supporting lactoferrin as a potential PD therapeutic that warrants further studies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Parkinson's disease (PD) is a complex disorder, primarily of idiopathic origin, with environmental stressors like rotenone and manganese linked to its development. This study explores their potential interaction and resulting neurotoxicity, aiming to understand how environmental factors contribute to PD. In an eight-day experiment, male Wistar rats weighing 280-300 g were subjected to rotenone, manganese, or a combination of both. Various parameters were assessed, including body weight, behavior, serum markers, tissue damage, protein levels (tyrosine hydroxylase, Dopamine- and cAMP-regulated neuronal phosphoprotein -DARPP-32-, and α-synuclein), and mitochondrial function. Manganese heightened rotenone's impact on reducing food intake without causing kidney or liver dysfunction. However, the combined exposure intensified neurotoxicity, which was evident in augmented broken nuclei and decreased tyrosine hydroxylase and DARPP-32 levels in the striatum. While overall mitochondrial function was preserved, co-administration reduced complex IV activity in the midbrain and liver. In conclusion, our findings revealed a parallel toxic effect induced by rotenone and manganese. Notably, while these substances do not target the same dopaminergic regions, a notable escalation in toxicity is evident in the striatum, the brain region where their toxic effects converge. This study highlights the need for further exploration regarding the interaction of environmental factors and their possible impact on the etiology of PD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: Bitter taste receptors (TAS2Rs) and their downstream signaling pathways are expressed not only in the oral tissues but also in extraoral tissues. Emerging data has demonstrated the beneficial effect of ghrelin in neurodegenerative diseases. Gaining more insight into the interaction between TAS2Rs and gut hormones may expand their therapeutic applications. Herein, we aimed to assess the possible effect of TAS2R activation by denatonium benzoate (DB) in modulating functional and neurobiochemical alterations in a model of Parkinson's disease (PD)., Main Methods: PD model was induced by daily injection of rotenone (2 mg/kg). Rats received DB (5 mg/kg), atenolol (10 mg/kg), or both concomitantly with rotenone, daily for 28 days. Evaluation of the motor abnormalities and histological examination of brain tissues were conducted. In addition, striatal dopamine contents, immunohistochemical expression of tyrosine hydroxylase, plasma ghrelin level, and biochemical analysis of markers of inflammation and oxidative stress were assessed., Key Findings: Treatment with DB increased serum levels of ghrelin and striatal dopamine contents with consequent amelioration of oxidative stress and attenuation of inflammatory cytokines. Moreover, DB treatment significantly ameliorated motor disturbance and histological abnormalities compared to untreated rats. Atenolol inhibited ghrelin release and abolished the positive effect of DB suggesting the involvement of ghrelin on such effects., Significance: The current study suggests that TAS2Rs agonists are promising candidates for ameliorating rotenone-induced PD pathology in rats, an action that could be linked to the enhancement of ghrelin release with consequent antioxidant and anti-inflammatory activities., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Under regulations such as REACH, testing of novel and established compounds for their (neuro)toxic potential is a legal requirement in many countries. These are largely based on animal-, cost-, and time-intensive in vivo models, not in line with the 3 Rs' principle of animal experimentation. Thus, the development of alternative test methods has also received increasing attention in neurotoxicology. Such methods focus either on physiological alterations in brain development and neuronal pathways or on behavioral changes. An example of a behavioral developmental neurotoxicity (DNT) assay is the zebrafish (Danio rerio) embryo coiling assay, which quantifies effects of compounds on the development of spontaneous movement of zebrafish embryos. While the importance of embryo-to-embryo contact prior to hatching in response to environmental contaminants or natural threats has been documented for many other clutch-laying fish species, little is known about the relevance of intra-clutch contacts for zebrafish. Here, the model neurotoxin rotenone was used to assess the effect of grouped versus separate rearing of the embryos on the expression of the coiling behavior. Some group-reared embryos reacted with hyperactivity to the exposure, to an extent that could not be recorded effectively with the utilized software. Separately reared embryos showed reduced activity, compared with group-reared individuals when assessing. However, even the control group embryos of the separately reared cohort showed reduced activity, compared with group-reared controls. Rotenone could thus be confirmed to induce neurotoxic effects in zebrafish embryos, yet modifying one parameter in an otherwise well-established neurotoxicity assay such as the coiling assay may lead to changes in behavior influenced by the proximity between individual embryos. This indicates a complex dependence of the outcome of behavior assays on a multitude of environmental parameters., (© 2024. The Author(s).)

Background: Parkinson's patients have significant autonomic dysfunction, early detect the disorder is a major challenge. To assess the autonomic function in the rat model of rotenone induced Parkinson's disease (PD), Blood pressure and ECG signal acquisition are very important., New Method: We used telemetry to record the electrocardiogram and blood pressure signals from awake rats, with linear and nonlinear analysis techniques calculate the heart rate variability (HRV) and blood pressure variability (BPV). we applied nonlinear analysis methods like sample entropy and detrended fluctuation analysis to analyze blood pressure signals. Particularly, this is the first attempt to apply nonlinear analysis to the blood pressure evaluate in rotenone induced PD model rat., Results: HRV in the time and frequency domains indicated sympathetic-parasympathetic imbalance in PD model rats. Linear BPV analysis didn't reflect changes in vascular function and blood pressure regulation in PD model rats. Nonlinear analysis revealed differences in BPV, with lower sample entropy results and increased detrended fluctuation analysis results in the PD group rats., Comparison With Existing Methods and Conclusions: our experiments demonstrate the ability to evaluate autonomic dysfunction in models of Parkinson's disease by combining the analysis of BPV with HRV, consistent with autonomic impairment in PD patients. Nonlinear analysis by blood pressure signal may help in early detection of the PD. It indicates that the fluctuation of blood pressure in the rats in the rotenone model group tends to be regular and predictable, contributes to understand the PD pathophysiological mechanisms and to find strategies for early diagnosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rotenone is a mitochondrial complex I inhibitor that causes retinal degeneration. A study of a rat model of rotenone-induced retinal degeneration suggested that this model is caused by indirect postsynaptic N-methyl-D-aspartate (NMDA) stimulation triggered by oxidative stress-mediated presynaptic intracellular calcium signaling. To elucidate the mechanisms by which rotenone causes axonal degeneration, we investigated morphological changes in optic nerves and the change in retinal ganglion cell (RGC) number in rats. Optic nerves and retinas were collected 3 and 7 days after the intravitreal injection of rotenone. The cross-sections of the optic nerves were subjected to a morphological analysis with axon quantification. The axons and somas of RGCs were analyzed immunohistochemically in retinal flatmounts. In the optic nerve, rotenone induced axonal swelling and degeneration with the incidence of reactive gliosis. Rotenone also significantly reduced axon numbers in the optic nerve. Furthermore, rotenone caused axonal thinning, fragmentation, and beading in RGCs on flatmounts and decreased the number of RGC soma. In conclusion, the intravitreal injection of rotenone in rats induced morphological abnormities with a reduced number of optic nerve axons and RGC axons when the RGC somas were degenerated. These findings help elucidate the pathogenesis of optic neuropathy induced by mitochondrial dysfunction.

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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Parkinsonism in rats induced by the pesticide rotenone is one of the most adequate models of Parkinson's disease (PD). Isatin (indole-2,3-dione) is an endogenous regulator found in mammals and humans and exhibiting a wide range of biological activities mediated by numerous isatin-binding proteins, including those associated with neurodegenerative pathology. A course of rotenone administration to rats caused behavioral impairments and changes in the profile and relative content of isatin-binding proteins in the brain. In this study, we have investigated the delayed neuroprotective effect of isatin (5 days after completion of the course of rotenone administration) on behavioral reactions and the relative content of isatin-binding proteins in the brain of rats with rotenone-induced experimental parkinsonism. Although during this period the rats retained locomotor dysfunction, the proteomic analysis data (profile of isatin-binding proteins in the brain and changes in their relative content) differed from the results obtained immediately after completion of the course of rotenone administration. Moreover, all isatin-binding proteins with altered relative content changed during this period are associated to varying degrees with neurodegeneration (many with Parkinson's and Alzheimer's diseases).

Parkinson's disease is a progressive neurodegenerative disorder marked by the death of dopaminergic neurons in the substantia nigra region of the brain. Aggregation of alpha-synuclein (α-synuclein) is a contributing factor to Parkinson's disease pathogenesis. The objective of this study is to investigate the neuroprotective effects of gut microbes on α-synuclein aggregation using both in silico and in vivo approaches. We focussed on the interaction between α-synuclein and metabolites released by gut bacteria that protect from PD. We employed three probiotic microbe strains against α-synuclein protein: Lactobacillus casei, Escherichia coli, and Bacillus subtilis, with their chosen PDB IDs being Dihydrofolate reductase (3DFR), methionine synthetase (6BM5), and tryptophanyl-tRNA synthetase (3PRH), respectively. Using HEX Dock 6.0 software, we examined the interactions between these proteins. Among the various metabolites, methionine synthetase produced by E. coli showed potential interactions with α-synuclein. To further evaluate the neuroprotective benefits of E. coli, an in vivo investigation was performed using a rotenone-induced Parkinsonian mouse model. The motor function of the animals was assessed through behavioural tests, and oxidative stress and neurotransmitter levels were also examined. The results demonstrated that, compared to the rotenone-induced PD mouse model, the rate of neurodegeneration was considerably reduced in mice treated with E. coli. Additionally, histopathological studies provided evidence of the neuroprotective effects of E. coli. In conclusion, this study lays the groundwork for future research, suggesting that gut bacteria may serve as potential therapeutic agents in the development of medications to treat Parkinson's disease. fig. 1., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The abnormal accumulation of fibrillar α-synuclein in the substantia nigra contributes to Parkinson's disease (PD). Chemical chaperones like 4-phenyl butyric acid (4PBA) show neuroprotective potential, but high doses are required. A derivative, 5-phenyl valeric acid (5PVA), has reported therapeutic potential for PD by reducing Pael-R expression. This study assessed 5PVA's efficacy in PD animals and its molecular mechanism. In vitro studies revealed 5PVA's anti-aggregation ability against alpha-synuclein and neuroprotective effects on SHSY5Y neuroblastoma cells exposed to rotenone. PD-like symptoms were induced in SD rats with rotenone, followed by 5PVA treatment at 100 mg/kg and 130 mg/kg. Behavioral analysis showed significant improvement in memory and motor activity with 5PVA administration. Histopathological studies demonstrated normal neuronal histoarchitecture in mid-brain tissue sections of 5PVA-treated animals compared to the PD group. mRNA studies revealed significant suppression in the expression of various protein folding and heat-shock protein markers in the 5PVA-treated group. In conclusion, 5PVA, with its anti-aggregation ability against alpha-synuclein, acts as a chemical chaperone, showing potential as a therapeutic candidate for PD treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Given that global prevalence of Parkinson's disease (PD) is expected to rise over the next few decades, understanding the mechanisms and causes of PD is critical. With emphasis on gut-brain axis, we sought to assess the impact of gentisic acid (GA), a diphenolic compound generated from benzoic acid, in rotenone (Rot) induced PD model in zebrafish. For thirty days, adult zebrafish were exposed to GA and rotenone. Tox-Track program was used to analyze locomotor behaviors in the control, GA, Rot, and Rot + GA groups. LC-MS/MS was performed in brain and intestinal tissues. Proteome Discoverer 2.4 was used to analyze raw files, peptide lists were searched against Danio rerio proteins. Protein interactions or annotations were obtained from STRING database. Tyrosine hydroxylase (Th) staining was performed immunohistochemically in the brain. PD-related gene expressions were determined by RT-PCR. Lipid peroxidation, nitric oxide, superoxide dismutase, glutathione S-transferase, and acetylcholinesterase were measured spectrophotometrically. Improved locomotor behaviors were observed by GA treatment in Rot group as evidenced by increased average speed, exploration rate, and total distance. 5214 proteins were identified in intestinal tissues, 4114 proteins were identified in brain by LC-MS/MS. Rotenone exposure altered protein expressions related to oxidative phosphorylation in brain and intestines. Protein expressions involved in ferroptis and actin cytoskeleton changed in brain and intestines. Altered protein expressions were improved by GA. GA ameliorated Th-immunoreactivity in brain, improved park2, park7, pink1, and lrrk2 expressions. Our results show that GA may be a candidate agent to be evaluated for its potential protective effect for PD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 gp91 phox in rotenone-induced cognitive deficits and the associated mechanisms. Our study demonstrated that rotenone exposure elevated expression of gp91 phox and phosphorylation of the NOX2 cytosolic subunit p47 phox , along with NADPH depletion in the hippocampus and cortex of mice, indicating NOX2 activation. Specific knockdown of gp91 phox 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 gp91 phox also reversed rotenone-elicited cognitive dysfunction in mice. Furthermore, microglial gp91 phox 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 gp91 phox -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., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Spermidine is a natural biologically active substance that has widespread influences on the body., Objective: This study aims to enhance our understanding of the potential effect of spermidine on long non-coding RNA MALAT1 and explore the underlying mechanism in the rotenone-induced rat model of Parkinson's disease., Methods: Rats were sacrificed after locomotor behavioral testing. Striatal tissues were used to assess the expression of MALAT1, oxidative stress markers, and autophagy markers., Results: Our study found that treatment with spermidine for 2 weeks during the induction of the model significantly improved behavioral assessment, dopamine levels, and attenuated the histopathological changes that occurred in PD in comparison to the non-treated group., Conclusion: Our preliminary study supports the protective effect of spermidine on the activation of autophagy and its antioxidant properties. Part of the antioxidant activity is due to the inhibition of MALAT1. However, MALAT1 does not correlate with the spermidine-induced autophagy pathway., (© 2024 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.)

This study examined the effect of dihydroquercetin (DHQ), also knofigurewn as taxifolin, on rotenone‐induced Parkinsonism in rats. Male Wistar rats were administered 1.5 mg/kg rotenone for 10 days and subsequently treated with 0.25–1.0 mg/kg DHQ for 3 days followed by the assessment of parkinsonian symptoms. Brain striatal redox stress and neurochemical dysfunction markers were assessed spectrophotometrically. Histopathological evaluation of the striatum was done by hematoxylin and eosin staining technique. The expression of genes involved in the activation of NF‐κB signaling pathway (IL‐1β, TNF‐α, NF‐κB and IκKB), and the p53 gene in the striatum were determined by RT‐qPCR. DHQ attenuated parkinsonian symptoms as well as striatal redox stress, neurochemical dysfunction, and histological alterations occasioned by rotenone toxicity. Importantly, DHQ significantly suppressed the rotenone‐induced upregulation of IL‐1β, NF‐κB, and IκKB expression (p < 0.05) in the striatum of parkinsonian rats. DHQ demonstrated notable neurotherapeutic potential against rotenone‐induced Parkinsonism in rats by improving parkinsonian symptoms (bradykinesia, catalepsy, postural instability, impaired locomotor behavior, and tremor) and neurochemical dysfunctions via modulation of genes involved in the activation of the canonical pathway of NF‐κB‐mediated inflammation. [ABSTRACT FROM AUTHOR]

Ethnopharmacological Relevance: 'Karkataka Taila (KT), an ancient Ayurvedic Rasayana comprising the edible freshwater crab Scylla serrata Forskal flesh, is still used by local traditional practitioners in Kerala state to treat tremors and palsy. In the scientific community, it becomes less exposed due to the lack of adequate scientific validations and brief reports. There has been no published research on the effectiveness of KT in treating Parkinson's disease (PD)., Purpose: The purpose of the current research work was to investigate the anti-Parkison's potential of KT against rotenone-induced neurotoxicity in SH-SY5Y cell lines and rat model of PD and investigate underlying molecular mechanisms., Materials and Methods: The components of KT have been identified by gas chromatography-mass spectroscopy (GC-MS). The neuroprotective activity of KT was assessed using SH-SY5Y cell lines and rats against rotenone-induced PD. The parameters used for asses the neuroprotection are antioxidant markers (ROS and SOD), anti-inflammatory markers (IL-6, IL-1β, TNF-α, and nitrite), and dopamine levels. Behavioral evaluation and rat brain histopathology were carried out to further support the neuroprotection., Result: Analysis using GC-MS revealed 36 constituents in KT. In vitro, the KT displayed considerable neuroprotective effects in terms of decreasing oxidative stress (ROS and SOD), neuroinflammation (IL-6, IL-1β, TNF-α, and nitrite), and elevating dopamine concentration. In vivo data showing improvements in histopathological and biochemical parameters confirmed the in vitro study findings, and in terms of behavioral assays, KT displayed significant activity., Conclusion: GC-MS profiling was used to identify the bioactive compounds of KT with antioxidant, anti-inflammatory, and neuroprotective properties. As a result, they may be responsible for the therapeutic effects of KT on PD., Competing Interests: Declaration of competing interest The authors declare no financial or personal interests that may have influenced the work presented in this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rotenone is a pesticide that causes complex I inhibition and is widely known to induce motor disability and experimental Parkinson's disease (PD) in rodents. Evidence suggests a crucial role for sirtuin/nuclear factor-kappaB/nod-like receptor family, pyrin domain-containing 3 (SIRT1/NFκB/NLRP3) signaling and inflammation in PD and rotenone neurotoxicity. Hesperetin (C16H14O6) is a citrus flavonoid with documented anti-inflammatory activity. We investigated the value of hesperetin in delaying rotenone-induced PD in mice and the possible modulation of inflammatory burden. PD was induced in mice via rotenone injections. Groups were assigned as a vehicle, PD, or PD + hesperetin (50 or 100 mg/kg) and compared for the motor function, protein level (by ELISA), and gene expression (by real-time PCR) of the target proteins, histopathology, and immunohistochemistry for tyrosine hydroxylase enzyme. Hesperetin (50 or 100 mg/kg) alleviated the motor disability and the striatal dopamine level and decreased the expression of NLRP3 and NF-κB but increased SIRT1 expression ( p  < 0.05). Further, it enhanced the neural viability and significantly decreased neural degeneration in the substantia nigra, hippocampus, and cerebral cortex ( p  < 0.05). Taken together, we propose that hesperetin mediates its neuroprotective function via alleviating modulation of the SIRT1/NFκB/NLRP3 pathway. Therefore, hesperetin might delay the PD progression.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis., (© 2024 International Society for Neurochemistry.)

Mitochondrial dysfunction is critically involved in the degeneration of dopamine (DA) neurons in the substantia nigra, a common pathological feature of Parkinson's disease (PD). Previous studies have demonstrated that the NAD + -dependent acetylase Sirtuin 3 (SIRT3) participates in maintaining mitochondrial function and is downregulated in aging-related neurodegenerative disorders. The exact mechanism of action of SIRT3 on mitochondrial bioenergetics in PD pathogenesis, however, has not been fully described. In this study, we investigated the regulatory role of SIRT3-mediated deacetylation of mitochondrial complex II (succinate dehydrogenase) subunit A (SDHA) and its effect on neuronal cell survival in rotenone (ROT)-induced rat and differentiated MN9D cell models. The results revealed that SIRT3 activity was suppressed in both in vivo and in vitro PD models. Accompanying this downregulation of SIRT3 was the hyperacetylation of SDHA, impaired activity of mitochondrial complex II, and decreased ATP production. It was found that the inhibition of SIRT3 activity was attributed to a reduction in the NAD + /NADH ratio caused by ROT-induced inhibition of mitochondrial complex I. Activation of SIRT3 by icariin and honokiol inhibited SDHA hyperacetylation and increased complex II activity, leading to increased ATP production and protection against ROT-induced neuronal damage. Furthermore, overexpression of SDHA also exerted potent protective benefits in cells treated with ROT. In addition, treatment of MN9D cells with the NAD + precursor nicotinamide mononucleotide increased SIRT3 activity and complex II activity and promoted the survival of cells exposed to ROT. These findings unravel a regulatory SIRT3-SDHA axis, which may be closely related to PD pathology. Bioenergetic rescue through SIRT3 activation-dependent improvement of mitochondrial complex II activity may provide an effective strategy for protection from neurodegeneration., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Parkinson's disease (PD) is the most common neurodegenerative movement disorder worldwide. Current treatments for PD largely center around dopamine replacement therapies and fail to prevent the progression of pathology, underscoring the need for neuroprotective interventions. Approaches that target neuroinflammation, which occurs prior to dopaminergic neuron (DAn) loss in the substantia nigra (SN), represent a promising therapeutic strategy. The glucocorticoid receptor (GR) has been implicated in the neuropathology of PD and modulates numerous neuroinflammatory signaling pathways in the brain. Therefore, we investigated the neuroprotective effects of the novel GR modulator, PT150, in the rotenone mouse model of PD, postulating that inhibition of glial inflammation would protect DAn and reduce accumulation of neurotoxic misfolded ⍺-synuclein protein. C57Bl/6 mice were exposed to 2.5 mg/kg/day rotenone by intraperitoneal injection for 14 days. Upon completion of rotenone dosing, mice were orally treated at day 15 with 30 mg/kg/day or 100 mg/kg/day PT150 in the 14-day post-lesioning incubation period, during which the majority of DAn loss and α-synuclein (α-syn) accumulation occurs. Our results indicate that treatment with PT150 reduced both loss of DAn and microgliosis in the nigrostriatal pathway. Although morphologic features of astrogliosis were not attenuated, PT150 treatment promoted potentially neuroprotective activity in these cells, including increased phagocytosis of hyperphosphorylated α-syn. Ultimately, PT150 treatment reduced the loss of DAn cell bodies in the SN, but not the striatum, and prohibited intra-neuronal accumulation of α-syn. Together, these data indicate that PT150 effectively reduced SN pathology in the rotenone mouse model of PD., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ronald Tjalkens reports financial support was provided by Colorado State University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In addition to the canonical Gs adenylyl cyclase pathway, the serotonin type 6 receptor (5-HT6R) recruits additional signaling pathways that control cognitive function, brain development, and synaptic plasticity in an agonist-dependent and independent manner. Considering that aberrant constitutive and agonist-induced active states are involved in various pathological mechanisms, the development of biased ligands with different functional profiles at specific 5-HT6R-elicited signaling pathways may provide a novel therapeutic perspective in the field of neurodegenerative and psychiatric diseases. Based on the structure of SB-258585, an inverse agonist at 5-HT6R-operated Gs and Cdk5 signaling, we designed a series of 1-(arylsulfonyl-isoindol-2-yl)piperazine derivatives and synthesized them using a sustainable mechanochemical method. We identified the safe and metabolically stable biased ligand 3g, which behaves as a neutral antagonist at the 5-HT6R-operated Gs signaling and displays inverse agonist activity at the Cdk5 pathway. Inversion of the sulfonamide bond combined with its incorporation into the isoindoline scaffold switched the functional profile of 3g at Gs signaling with no impact at the Cdk5 pathway. Compound 3g reduced the cytotoxicity of 6-OHDA and produced a glioprotective effect against rotenone-induced toxicity in C8-D1A astrocyte cell cultures. In view of these findings, compound 3g can be considered a promising biased ligand to investigate the role of the 5-HT6R-elicited Gs and Cdk5 signaling pathways in neurodegenerative diseases.

Rotenone, as a common pesticide and insecticide frequently found in environmental samples, may be present in aquatic habitats worldwide. Exposure to low concentrations of this compound may cause alterations in the nervous system, thus contributing to Parkinsonian motor symptoms in both vertebrates and invertebrates. However, the effects of chronic exposure to low doses of rotenone on the activity of neurotransmitters that govern motor functions and on the specific molecular mechanisms leading to movement morbidity remain largely unknown for many aquatic invertebrates. In this study, we analyzed the effects that rotenone poisoning exerts on the activity of dopamine (DA) and acetylcholine (ACh) synthesis enzymes in the central nervous system (CNS) of Asian shore crab, Hemigrapsus sanguineus (de Haan, 1835), and elucidated the association of its locomotor behavior with Parkinson's-like symptoms. An immunocytochemistry analysis showed a reduction in tyrosine hydroxylase (TH) in the median brain and the ventral nerve cord (VNC), which correlated with the subsequent decrease in the locomotor activity of shore crabs. We also observed a variation in cholinergic neurons' activity, mostly in the ventral regions of the VNC. Moreover, the rotenone-treated crabs showed signs of damage to ChAT-lir neurons in the VNC. These data suggest that chronic treatment with low doses of rotenone decreases the DA level in the VNC and the ACh level in the brain and leads to progressive and irreversible reductions in the crab's locomotor activity, life span, and changes in behavior.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder currently affecting the ageing population. Although the aetiology of PD has yet to be fully elucidated, environmental factors such as exposure to the naturally occurring neurotoxin rotenone has been associated with an increased risk of developing PD. Rotenone inhibits mitochondrial respiratory chain (MRC) complex I activity as well as induces dopaminergic neuronal death. The aim of the present study was to investigate the underlying mechanisms of rotenone-induced mitochondrial dysfunction and oxidative stress in an in vitro SH-SY5Y neuronal cell model of PD and to assess the ability of pre-treatment with Coenzyme Q 10 (CoQ 10 ) to ameliorate oxidative stress in this model. Spectrophotometric determination of the mitochondrial enzyme activities and fluorescence probe studies of reactive oxygen species (ROS) production was assessed. Significant inhibition of MRC complex I and II-III activities was observed, together with a significant loss of neuronal viability, CoQ 10 status, and ATP synthesis. Additionally, significant increases were observed in intracellular and mitochondrial ROS production. Remarkably, CoQ 10 supplementation was found to reduce ROS formation. These results have indicated mitochondrial dysfunction and increased oxidative stress in a rotenone-induced neuronal cell model of PD that was ameliorated by CoQ 10 supplementation.

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Parkinson's disease with dementia (PDD) is a neurological disorder that clinically and neuropathologically overlaps with Parkinson's disease (PD) and Alzheimer's disease (AD). Although it is assumed that alpha-synuclein ( α -Syn), amyloid beta (A β ), and the protein Tau might synergistically induce cholinergic neuronal degeneration, presently the pathological mechanism of PDD remains unclear. Therefore, it is essential to delve into the cellular and molecular aspects of this neurological entity to identify potential targets for prevention and treatment strategies. Cholinergic-like neurons (ChLNs) were exposed to rotenone (ROT, 10 μ M) for 24 h. ROT provokes loss of Δ Ψ m , generation of reactive oxygen species (ROS), phosphorylation of leucine-rich repeated kinase 2 (LRRK2 at Ser 935 ) concomitantly with phosphorylation of α -synuclein ( α -Syn, Ser 129 ), induces accumulation of intracellular A β (iA β ), oxidized DJ-1 (Cys 106 ), as well as phosphorylation of TAU (Ser 202 /Thr 205 ), increases the phosphorylation of c-JUN (Ser 63 /Ser 73 ), and increases expression of proapoptotic proteins TP53, PUMA, and cleaved caspase 3 (CC3) in ChLNs. These neuropathological features resemble those reproduced in presenilin 1 (PSEN1) E280A ChLNs. Interestingly, anti-oxidant and anti-amyloid cannabidiol (CBD), JNK inhibitor SP600125 (SP), TP53 inhibitor pifithrin- α (PFT), and LRRK2 kinase inhibitor PF-06447475 (PF475) significantly diminish ROT-induced oxidative stress (OS), proteinaceous, and cell death markers in ChLNs compared to naïve ChLNs. In conclusion, ROT induces p- α -Syn, iA β , p-Tau, and cell death in ChLNs, recapitulating the neuropathology findings in PDD. Our report provides an excellent in vitro model to test for potential therapeutic strategies against PDD. Our data suggest that ROT induces a neuropathologic phenotype in ChLNs similar to that caused by the mutation PSEN1 E280A., (© 2024. The Author(s).)

NAD(P)H-quinone oxidoreductase 1 (NQO1) is an essential redox enzyme responsible for redox balance and energy metabolism. Despite of its importance, the brain contains high capacity of polyunsaturated fatty acids and maintains low levels of NQO1 expression. In this study, we examined how levels of NQO1 expression affects cell survival in response to toxic insults causing mitochondrial dysfunction and ferroptosis, and whether NQO1 has a potential as a biomarker in different stressed conditions. Following treatment with rotenone, overexpressed NQO1 in SH-SY5Y cells improved cell survival by reducing mitochondrial reductive stress via increased NAD + supply without mitochondrial biogenesis. However, NQO1 overexpression boosted lipid peroxidation following treatment with RSL3 and erastin. A lipid droplet staining assay showed increased lipid droplets in cells overexpressing NQO1. In contrast, NQO1 knockdown protected cells against ferroptosis by increasing GPX4, xCT, and the GSH/GSSG system. Also, NQO1 knockdown showed lower iron contents and lipid droplets than non-transfectants and cells overexpressing NQO1, even though it could not attenuate cell death when exposed to rotenone. In summary, our study suggests that different NQO1 levels may have advantages and disadvantages depending on the surrounding environments. Thus, regulating NQO1 expression could be a potential supplementary tool when treating neuronal diseases.

Introduction: Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e.g., epilepsy); however, their neuroprotective potential has not been widely investigated. Materials and Methods: Using primary cultures of cerebellar granule cells, we evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, trace levels of Δ9-tetrahydrocannabinol, and the acid form of CBD. We determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). Results: The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se . EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions between individual substances present in EPI occurred. In contrast, CBD did show a different profile to EPI and XAL because a neurotoxic effect per se was observed at higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Conclusion: Our data support a neuroprotective effect of EPI that may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based products that could be critical to avoid neurotoxicity at very high doses.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, characterized by motor and psychological dysfunction. Palliative treatment and dopamine replenishment therapy are the only available therapeutic options. Calcium channel blockers (CCBs) have been reported to protect against several neurodegenerative disorders. The current study was designed to evaluate the neuroprotective impact of Felodipine (10 mg/kg, orally) as a CCB on motor and biochemical dysfunction associated with experimentally induced PD using rotenone (2.5 mg/kg, IP) and to investigate the underlying mechanisms. Rotenone induced deleterious neuromotor outcomes, typical of those associated with PD. The striatum revealed increased oxidative burden and NO levels with decreased antioxidant capacity. Nrf2 content significantly decreased with the accumulation of α-synuclein and tau proteins in both the substantia nigra and striatum. These observations significantly improved with felodipine treatment. Of note, felodipine increased dopamine levels in the substantia nigra and striatum as confirmed by the suppression of inflammation and the significant reduction in striatal NF-κB and TNF-α contents. Moreover, felodipine enhanced mitophagy, as confirmed by a significant increase in mitochondrial Parkin and suppression of LC3a/b and SQSTM1/p62. In conclusion, felodipine restored dopamine synthesis, attenuated oxidative stress, inflammation, and mitochondrial dysfunction, and improved the mitophagy process resulting in improved PD-associated motor impairment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is characterized by oxidative stress and neuroinflammation as key pathological features. Emerging evidence suggests that nuclear factor erythroid 2 related factor 2-antioxidant response element (Nrf2-ARE), phosphatidylinositol 3‑kinase-protein kinase B (PI3K-Akt), c-Jun N-terminal kinase-extracellular signal-regulated kinase 1/2 (JNK-ERK1/2), and toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-kB) pathways play pivotal roles in PD pathogenesis. Orientin, a phenolic phytoconstituent, has demonstrated modulatory potential on these pathways in various experimental conditions other than PD. In this study, we aimed to evaluate the neuroprotective effects of Orientin against rotenone-induced neurodegeneration in SH-SY5Y cell lines and the Swiss albino mice model of PD. Orientin was administered at doses 10 and 20 µM in cell lines and 10 and 20 mg/kg in mice, and its effects on rotenone-induced neurodegeneration were investigated. Oxidative stress markers including mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as inflammatory markers including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were measured. The expression levels of genes related to Nrf2-ARE (Nrf2), PI3K/Akt (Akt), JNK-ERK1/2 (TNF-α), and TLR4/NF-kB (TNF-α) pathways were measured to understand the modulatory effect of Orientin on these pathways. Additionally, behavioral studies assessing locomotor activity, muscle coordination, and muscle rigidity were conducted with mice. Our results indicate that Orientin dose-dependently attenuated rotenone-induced changes in oxidative stress markers, inflammatory markers, gene expression levels, and behavioral parameters. Therefore, our study concludes that Orientin exhibits significant neuroprotective benefits against rotenone-induced PD by modulating Nrf2-ARE, PI3K-Akt, JNK-ERK1/2, and TLR4/NF-kB pathways., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2024. The Author(s).)

Background: A substantial body of evidence is drawing connections between Parkinson's disease (PD) and the phenomena of oxidative stress and mitochondrial dysfunction. Polyphyllin VI (PPVI), an active compound found in Rhizoma Paridis-commonly known as Chonglou (CL) in China, has been identified for its various pharmacological properties, including anti-tumor and anti-inflammatory effects., Objective: In the present study, an in vitro model of PD was established by treating SH-SY5Y cells with rotenone (ROT), to evaluate the potential neuroprotective effects of polyphyllin VI and its underlying mechanism., Methods: SH-SY5Y cells were treated with ROT to establish an in vitro model of PD. The effects of polyphyllin VI on cell viability were assessed using the resazurin assay. Cell morphology was examined using a microscope. The YO-PRO-1/PI was used to detect apoptosis. Mito-Tracker Red CMXRos, Mito-Tracker Green, and JC-1 were used to detect the effects of polyphyllin Ⅵ on mitochondrial viability, morphology, and function. Oxidative stress-related marker detection kits were used to identify the effects of polyphyllin VI on oxidative stress. Western blot analysis was employed to investigate the signaling pathways associated with neuroprotection., Results: PPVI increased ROT-induced SH-SY5Y cell viability and improved ROT-induced cellular morphological changes. PPVI ameliorated ROT-induced oxidative stress status, and attenuated mitochondrial function and morphological changes. PPVI may exert neuroprotective effects through FOXO3α/CREB1/DJ-1-related signaling pathways., Conclusion: These preliminary findings suggested that PPVI possesses neuroprotective attributes in vitro, and it may be a potential candidate for PD treatment. However, extensive research is necessary to fully understand the mechanisms of PPVI and its effectiveness both in vitro and in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is often accompanied by depression, which may appear before motor signs. Oleanolic acid (OA), a pentacyclic triterpenoid substance, have many pharmacological properties. However, its efficacy in treating PD-related chronic unpredictable stress (CUS) is unknown. Our study used behavioral, biochemical, and immunohistochemical techniques to assess how OA affected PDrelated CUS. Rotenone (1 mg/kg i.p. for first 21 days) was used to induce Parkinsonism, and modest psychological & environmental stresses generated CUS (from day 22 to day 43) in animals. The study included daily i.p. administration of OA (5, 10, and 20 mg/kg) from day 1 to day 57 in male swiss albino mice. Animals were evaluated for behavioral, biochemical parameters, neurotransmitters, and immunohistochemical expression following the treatment. Results of the study revealed that treatment with OA at all doses alleviated the core symptoms of CUS linked to PD and improved motor and non-motor function. OA therapy significantly lowered IL-1β, TNF-α ( p  < 0.01, < 0.01, < 0.001), IL-6 ( p  < 0.05, < 0.01, < 0.001), oxidative stress ( p  < 0.05, < 0.01, < 0.01), and elevated norepinephrine ( p  < 0.05, < 0.01, < 0.01), dopamine, and serotonin ( p  < 0.05, < 0.01, < 0.001) levels. Moreover, OA therapy substantially reduced α-synuclein ( p  < 0.05, < 0.01, < 0.01) aggregation and increased BDNF ( p  < 0.05, < 0.01, < 0.001) & Nrf-2 ( p  < 0.05, < 0.01, < 0.01) levels, which boosts neuronal dopamine survival. The study's findings indicated that OA ameliorates depressive-like behavior persuaded by CUS in PD, decreases neuroinflammation, and improves neurotransmitter concentration via activating Nrf2-BDNF-dopaminergic pathway.

Effects not related with the inhibition of complex I of the mitochondrial electron transport chain are studied in S. pombe, which lacks it. This study aims: First, the use of a strategy with S. pombe strains to investigate the toxicity, mechanisms of action, interactions and detoxication by efflux pumps. Second, to investigate the mechanisms of toxic action of rotenone. In the dose-response assessment, the yeast presented a good correlation with the toxicity in Daphnia magna for 15 chemicals. In the mechanistic study, the mph1Δ strain presented marked specificity to the interaction with microtubules by carbendazim. DNA damage caused by hydroxyurea, an inhibitor of deoxynucleotide synthesis, was identified with marked specificity with the rad3Δ strain. The sty1Δ strain was very sensitive to the oxidative and osmotic stress induced by hydrogen peroxide and potassium chloride, respectively, being more sensitive to oxidative stress than the pap1Δ strain. The protection by exclusion pumps was also evaluated. Rotenone presented low toxicity in S. pombe due to the lack of its main target, and the marked protection by the exclusion transporters Bfr1, Pmd1, Caf5 and Mfs1. Marked cellular stress was detected. Finally, the toxicity of rotenone could be potentiated by the fungicide carbendazim and the antimetabolite hydroxyurea. In conclusion, the use of S. pombe strains is a valid strategy to: a) assess global toxicity; b) investigate the main mechanisms of toxic action, particularly spindle and DNA interferences, and osmotic and oxidative stress not related to complex I inhibition; c) explore the detoxication by efflux pumps; and d) evaluate possible chemical interactions. Therefore, it should be useful for the investigation of adverse outcome pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is one of the most common neurodegenerative conditions. Alpha-synuclein deposition, Lewy bodies (LBs) formation, disruption of the autophagic machinery, apoptosis of substantia nigra dopaminergic neurons, oxidative stress, and neuroinflammation are all pathologic hallmarks of PD. The leaves of the stinging Nettle (Urtica dioica L.) have a long history as an herbal cure with antioxidant, anti-inflammatory, anti-cancer, immunomodulatory, and neuroprotective properties. The current study aims for the first time to investigate the role of Nettle supplementation on Rotenone-induced PD. Rats were divided into five groups; a Saline control, Nettle control (100 mg/kg/day), Rotenone control (2 mg/kg/day), Rotenone + Nettle (50 mg /kg/day), and Rotenone + Nettle (100 mg/kg). After four weeks, the rats were examined for behavioral tests. The midbrains were investigated for histopathological alteration and immunohistochemical reaction for Tyrosine hydroxylase in the dopaminergic neurons, α-synuclein for Lewy bodies, caspase 3 for apoptotic neurons, LC3 and P62 for autophagic activity. Midbrain homogenates were examined for oxidative stress markers. mRNA expression of TNFα and Il6; inflammatory markers, Bcl-2, BAX and Caspase 3; apoptosis markers, were detected in midbrains. The results showed that Nettle caused recovery of midbrain dopaminergic neurons, by inhibiting apoptosis, inflammation, and oxidative stress and by restoring the autophagic machinery with clearance of α-synuclein deposits. We can conclude that Nettle is a potentially effective adjuvant in the treatment of Parkinson's disease., Competing Interests: Conflict of Interest None declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Cajanus cajan (L) Millsp (Fabaceae) seed decoction is used by traditional healers in Nigeria as nerve tonic, hence, could be beneficial in the treatment of Parkinson's disease (PD), a progressive and debilitating neurodegenerative disease that imposes great burden on the healthcare system globally., Aim of the Study: This study aimed at investigating the neuroprotective effect of ethanol seed extract of Cajanus cajan (CC) in the treatment of rotenone-induced motor symptoms and non-motor symptoms associated with PD., Materials and Methods: To assess the protective action of CC on rotenone-induced motor- and non-motor symptoms of PD, mice were first pretreated with CC (50, 100 or 200 mg/kg, p.o.) an hour before oral administration of rotenone (1 mg/kg, p.o, 0.5% in carboxyl-methylcellulose) for 28 consecutive days and weekly behavioural tests including motor assessment (open field test (OFT), rotarod, pole and cylinder tests) and non-motor assessment (novel object recognition (NOR), Y-maze test (YM), forced swim and tail suspension, gastric emptying and intestinal fluid accumulation tests) were carried out. The animals were euthanized on day 28 followed by the collection of brain for assessment of oxidative stress, inflammatory markers and immunohistochemical analysis of the striatum (STR) and substantia nigra (SN). Phytochemicals earlier isolated from CC were docked with protein targets linked with PD pathology such as; catechol-O-methyltransferase (COMT), tyrosine hydroxylase (TH) and Leucine rich receptor kinase (LRRK)., Results: this study showed that CC significantly reduced rotenone-induced spontaneous motor impairment in OFT, pole, cylinder and rotarod tests in mice as well as significant improvement in non-motor features (significant reversal of rotenone-induced deficits discrimination index and spontaneous alternation behaviour in NORT and YM test, respectively, reduction in immobility time in forced swim/tail suspension test, gastrointestinal disturbance in intestinal transit time in mice. Moreso, rotenone-induced neurodegeneration, oxidative stress and neuroinflammation were significantly attenuated by CC administration. In addition, docking analysis showed significant binding affinity of CC phytochemicals with COMT, TH and LRRK2 receptors., Conclusion: Cajanus cajan seeds extract prevented both motor and non-motor features of Parkinson disease in mice through its antioxidant and anti-inflammatory effects. Hence, could be a potential phytotherapeutic adjunct in the management of Parkinson disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Leber's hereditary optic neuropathy (LHON) is driven by mtDNA mutations affecting Complex I presenting as progressive retinal ganglion cell dysfunction usually in the absence of extra-ophthalmic symptoms. There are no long-term neuroprotective agents for LHON. Oral nicotinamide provides a robust neuroprotective effect against mitochondrial and metabolic dysfunction in other retinal injuries. We explored the potential for nicotinamide to protect mitochondria in LHON by modelling the disease in mice through intravitreal injection of the Complex I inhibitor rotenone. Using MitoV mice expressing a mitochondrial-tagged YFP in retinal ganglion cells we assessed mitochondrial morphology through super-resolution imaging and digital reconstruction. Rotenone induced Complex I inhibition resulted in retinal ganglion cell wide mitochondrial loss and fragmentation. This was prevented by oral nicotinamide treatment. Mitochondrial ultrastructure was quantified by transition electron microscopy, demonstrating a loss of cristae density following rotenone injection, which was also prevented by nicotinamide treatment. These results demonstrate that nicotinamide protects mitochondria during Complex I dysfunction. Nicotinamide has the potential to be a useful treatment strategy for LHON to limit retinal ganglion cell degeneration., (© 2024. The Author(s).)

Peripheral autonomic nervous system (P-ANS) dysfunction is a critical non-motor phenotype of Parkinson's disease (PD). The majority of PD cases are sporadic and lack identified PD-associated genes involved. Epidemiological and animal model studies suggest an association with pesticides and other environmental toxins. However, the cellular mechanisms underlying toxin induced P-ANS dysfunctions remain unclear. Here, we mapped the global transcriptome changes in human induced pluripotent stem cell (iPSC) derived P-ANS sympathetic neurons during inhibition of the mitochondrial respiratory chain by the PD-related pesticide, rotenone. We revealed distinct transcriptome profiles between acute and chronic exposure to rotenone. In the acute stage, there was a down regulation of specific cation channel genes, known to mediate electrophysiological activity, while in the chronic stage, the human P-ANS neurons exhibited dysregulation of anti-apoptotic and Golgi apparatus-related pathways. Moreover, we identified the sodium voltage-gated channel subunit SCN3A/Nav1.3 as a potential biomarker in human P-ANS neurons associated with PD. Our analysis of the rotenone-altered coding and non-coding transcriptome of human P-ANS neurons may thus provide insight into the pathological signaling events in the sympathetic neurons during PD progression., Competing Interests: Declaration of competing interest The authors have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is the second most common neurodegenerative disorder, yet treatment options are limited. Clozapine (CLZ), an antipsychotic used for schizophrenia, has potential as a PD treatment. CLZ and its metabolite, Clozapine-N-Oxide (CNO), show neuroprotective effects on dopaminergic neurons, with mechanisms needing further investigation. This study aimed to confirm the neuroprotective effects of CLZ and CNO in a rotenone-induced mouse model and further explore the underlying mechanisms of CNO-afforded protection. Gait pattern and rotarod activity evaluations showed motor impairments in rotenone-exposed mice, with CLZ or CNO administration ameliorating behavioral deficits. Cell counts and biochemical analysis demonstrated CLZ and CNO's effectiveness in reducing rotenone-induced neurodegeneration of dopaminergic neurons in the nigrostriatal system in mice. Mechanistic investigations revealed that CNO suppressed rotenone-induced ferroptosis of dopaminergic neurons by rectifying iron imbalances, curtailing lipid peroxidation, and mitigating mitochondrial morphological changes. CNO also reversed autolysosome and ferritinophagic activation in rotenone-exposed mice. SH-SY5Y cell cultures validated these findings, indicating ferritinophage involvement, where CNO-afforded protection was diminished by ferritinophagy enhancers. Furthermore, knockdown of NCOA4, a crucial cargo receptor for ferritin degradation in ferritinophagy, hampered rotenone-induced ferroptosis and NCOA4 overexpression countered the anti-ferroptotic effects of CNO. Whereas, iron-chelating agents and ferroptosis enhancers had no effect on the anti-ferritinophagic effects of CNO in rotenone-treated cells. In summary, CNO shielded dopaminergic neurons in the rotenone-induced PD model by modulating NCOA4-mediated ferritinophagy, highlighting a potential therapeutic pathway for PD treatment. This research provided insights into the role of NCOA4 in ferroptosis and suggested new approaches for PD therapy., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The MTS cell viability test was used to screen a mini library of natural and synthetic 1,4-naphthoquinone derivatives (1,4-NQs) from marine sources. This screening identified two highly effective compounds, U-443 and U-573 , which showed potential in protecting Neuro-2a neuroblastoma cells from the toxic effects of rotenone in an in vitro model of neurotoxicity. The selected 1,4-NQs demonstrated the capability to reduce oxidative stress by decreasing the levels of reactive oxygen species (ROS) and nitric oxide (NO) in Neuro-2a neuroblastoma cells and RAW 264.7 macrophage cells and displayed significant antioxidant properties in mouse brain homogenate. Normal mitochondrial function was restored and the mitochondrial membrane potential was also regained by 1,4-NQs after exposure to neurotoxins. Furthermore, at low concentrations, these compounds were found to significantly reduce levels of proinflammatory cytokines TNF and IL-1β and notably inhibit the activity of cyclooxygenase-2 (COX-2) in RAW 264.7 macrophages. The results of docking studies showed that the 1,4-NQs were bound to the active site of COX-2, analogically to a known inhibitor of this enzyme, SC-558. Both substances significantly improved the behavioral changes in female CD1 mice with rotenone-induced early stage of Parkinson's disease (PD) in vivo. It is proposed that the 1,4-NQs, U-443 and U-573 , can protect neurons and microglia through their potent anti-ROS and anti-inflammatory activities.

Ethnopharmacological Relevance: Fructus Gardeniae, with the effects of discharging fire, eliminating vexation, reducing fever and causing diuresis, and cooling blood to remove apthogentic heat, could be used to treat Parkinson's disease (PD). Geniposide, as the main active ingredient of Fructus Gardeniae, has been shown to have neuroprotective effects in several rodent models. Rotenone, a commonly used neurotoxin, induced PD model progresses slowly, but simulates the pathological changes of PD's slow progression., Aim of the Study: Herein, we mainly investigated the neuroprotective effects of geniposide on rotenone-induced mouse model of PD and the underlined mechanism., Materials and Methods: C57BL/6 mice were treated with rotenone (30 mg/kg, p. o.) daily for 60 days. Geniposide (25 and 50 mg/kg, p. o.) were administered at alterative day 30 min before rotenone. On day 60, the challenging beam, spontaneous activity, and adhesive removal tests were performed to evaluate the motor activity. Dopamine, DOPAC and HVA levels were detected by UPLC-MS/MS methods. Dopaminergic neurodegeneration was assessed using immunohistochemistry staining. ROS production, MDA level and GSH: GSSG ratio were measured to analyze oxidative stress. Cleavage of PARP and caspase-3 were detected to assess neuronal apoptosis. The expression of Nrf2 and mTOR signaling were detected using Western blot., Results: Geniposide improved motor dysfunction, restored neurotransmitters levels, and attenuated dopaminergic neurodegeneration induced by rotenone in mice. Geniposide suppressed rotenone-induced neuronal oxidative damage associated with Nrf2 signaling, and neuronal apoptosis involving mTOR pathway., Conclusions: Geniposide may exert a neuroprotective effect in a mouse model of PD by rotenone, and this effect might be relevant to Nrf2 associated antioxidant signaling and mTOR involved anti-apoptosis pathway., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bumblebees are essential pollinators of both wildflowers and crops and face multiple anthropogenic stressors, particularly the utilization of pesticides. Rotenone is an extensively applied neurotoxic pesticide that possesses insecticidal activities against a wide range of pests. However, whether environmentally realistic exposure levels of rotenone can damage neurons in bumblebee brains is still uncertain. Using single-cell RNA-seq, we revealed that rotenone induced cell-specific responses in bumblebee brains, emphasizing the disruption of energy metabolism and mitochondrial dysfunction in glial cells. Correspondingly, the gene regulatory network associated with neurotransmission was also suppressed. Notably, rotenone could specially reduce the number of dopaminergic neurons, impairing bumblebee's ability to fly and crawl. We also found impaired intestinal motility in rotenone-treated bumblebees. Finally, we demonstrated that many differentially expressed genes in our snRNA-seq data overlapped with rotenone-induced Parkinson's disease risk genes, especially in glial cells. Although rotenone is widely used owing to its hypotoxicity, we found that environmentally realistic exposure levels of rotenone induced disturbed glial energetics and locomotor dysfunction in bumblebees, which may lead to an indirect decline in this essential pollinator., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)