MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

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.

Retinal damage promotes mitochondrial transfer in the visual system of a mouse model of Leber hereditary optic neuropathy.

Lenadogene nolparvovec is a gene therapy which has been developed to treat Leber hereditary optic neuropathy (LHON) caused by a point mutation in the mitochondrial NADH dehydrogenase 4 (ND4) gene. Clinical trials have demonstrated a significant improvement of visual acuity up to 5 years after treatment by lenadogene nolparvovec but, surprisingly, unilateral treatment resulted in bilateral improvement of vision. This contralateral effect - similarly observed with other gene therapy products in development for MT-ND4-LHON - is supported by the migration of viral vector genomes and their transcripts to the contralateral eye, as reported in animals, and post-mortem samples from two patients. In this study, we used an AAV2 encoding fluorescent proteins targeting mitochondria to investigate whether these organelles themselves could transfer from the treated eye to the fellow one. We found that mitochondria travel along the visual system (optic chiasm and primary visual cortex) and reach the contralateral eye (optic nerve and retina) in physiological conditions. We also observed that, in a rotenone-induced model of retinal damage mimicking LHON, mitochondrial transfer from the healthy to the damaged eye was accelerated and enhanced. Our results thus provide a further explanation for the contralateral beneficial effect observed during clinical studies with lenadogene nolparvovec.

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.

Orientin Modulates Nrf2-ARE, PI3K/Akt, JNK-ERK1/2, and TLR4/NF-kB Pathways to Produce Neuroprotective Benefits in Parkinson's Disease.

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 3­kinase-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-1ß (IL-1ß), 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.

Gut microbiota-induced CXCL1 elevation triggers early neuroinflammation in the substantia nigra of Parkinsonian mice.

Gut microbiota disturbance and systemic inflammation have been implicated in the degeneration of dopaminergic neurons in Parkinson's disease (PD). How the alteration of gut microbiota results in neuropathological events in PD remains elusive. In this study, we explored whether and how environmental insults caused early neuropathological events in the substantia nigra (SN) of a PD mouse model. Aged (12-month-old) mice were orally administered rotenone (6.25 mg·kg-1·d-1) 5 days per week for 2 months. We demonstrated that oral administration of rotenone to ageing mice was sufficient to establish a PD mouse model and that microglial activation and iron deposition selectively appeared in the SN of the mice prior to loss of motor coordination and dopaminergic neurons, and these events could be fully blocked by microglial elimination with a PLX5622-formulated diet. 16 S rDNA sequencing analysis showed that the gut microbiota in rotenone-treated mice was altered, and mice receiving faecal microbial transplantation (FMT) from ageing mice treated with rotenone for 2 months exhibited the same pathology in the SN. We demonstrated that C-X-C motif chemokine ligand-1 (CXCL1) was an essential molecule, as intravenous injection of CXCL1 mimicked almost all the pathology in serum and SN induced by oral rotenone and FMT. Using metabolomics and transcriptomics analyses, we identified the PPAR pathway as a key pathway involved in rotenone-induced neuronal damage. Inhibition of the PPARγ pathway was consistent in the above models, whereas its activation by linoleic acid (60 mg·kg-1·d-1, i.g. for 1 week) could block these pathological events in mice intravenously injected with CXCL1. Altogether, these results reveal that the altered gut microbiota resulted in neuroinflammation and iron deposition occurring early in the SN of ageing mice with oral administration of rotenone, much earlier than motor symptoms and dopaminergic neuron loss. We found that CXCL1 plays a crucial role in this process, possibly via PPARγ signalling inhibition. This study may pave the way for understanding the "brain-gut-microbiota" molecular regulatory networks in PD pathogenesis. The aged C57BL/6 male mice with rotenone intragastric administration showed altered gut microbiota, which caused systemic inflammation, PPARγ signalling inhibition and neuroinflammation, brain iron deposition and ferroptosis, and eventually dopaminergic neurodegeneration in PD.

Neuroprotective Effects of Probucol against Rotenone-Induced Toxicity via Suppression of Reactive Oxygen Species Production in SH-SY5Y Cells.

Probucol is a hyperlipidemic drug with antioxidant properties. It has been reported to prevent mitochondrial dysfunction, reduce oxidative stress, and suppress neurotoxicity in neurodegenerative disease models, including Parkinson's disease models. However, the molecular mechanisms underlying the neuroprotective effects of probucol have been not examined yet. Thus, in this study, we investigated whether probucol can alleviate the effects of a mitochondrial complex I inhibitor, rotenone, on a human neuroblastoma cell line (SH-SY5Y). We evaluated the cell viability and cytotoxicity and apoptosis rates of SH-SY5Y cells treated with rotenone and probucol or edaravone, a known free-radical scavenger. Subsequently, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels in the cells were evaluated to determine the effects of probucol on mitochondrial function. We found that rotenone caused cytotoxicity, cell apoptosis, and mitochondrial dysfunction, enhanced ROS generation, and impaired MMP. However, probucol could inhibit this rotenone-induced decrease in cell viability, MMP loss, intracellular ROS generation, and apoptosis. These results suggest that probucol exerts neuroprotective effects via MMP stabilization and the inhibition of ROS generation. Additionally, this effect of probucol was equal to or greater than and more persistent than that of edaravone. Thus, we believe probucol may be a promising drug for the treatment of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases.

Protection Activity of 1,4-Naphthoquinones in Rotenone-Induced Models of Neurotoxicity.

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-1ß 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.

Rotenone-induced cell apoptosis via endoplasmic reticulum stress and PERK-eIF2α-CHOP signalling pathways in TM3 cells.

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.

Effects of Chronic Exposure to Low Doses of Rotenone on Dopaminergic and Cholinergic Neurons in the CNS of Hemigrapsus sanguineus.

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.

The Effect of Neuronal CoQ10 Deficiency and Mitochondrial Dysfunction on a Rotenone-Induced Neuronal Cell Model of Parkinson's Disease.

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 Q10 (CoQ10) 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, CoQ10 status, and ATP synthesis. Additionally, significant increases were observed in intracellular and mitochondrial ROS production. Remarkably, CoQ10 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 CoQ10 supplementation.

Naringenin Nanocrystals Mitigate Rotenone Neurotoxicity in SH-SY5Y Cell Line by Modulating Mitophagy and Oxidative Stress.

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.

Hesperetin protects against rotenone-induced motor disability and neurotoxicity via the regulation of SIRT1/NLRP3 signaling.

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.

Microglia-specific knock-out of NF-κB/IKK2 increases the accumulation of misfolded α-synuclein through the inhibition of p62/sequestosome-1-dependent autophagy in the rotenone model of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder worldwide, with a greater prevalence in men than women. The etiology of PD is largely unknown, although environmental exposures and neuroinflammation are linked to protein misfolding and disease progression. Activated microglia are known to promote neuroinflammation in PD, but how environmental agents interact with specific innate immune signaling pathways in microglia to stimulate conversion to a neurotoxic phenotype is not well understood. To determine how nuclear factor kappa B (NF-κB) signaling dynamics in microglia modulate neuroinflammation and dopaminergic neurodegeneration, we generated mice deficient in NF-κB activation in microglia (CX3CR1-Cre::IKK2fl/fl ) and exposed them to 2.5 mg/kg/day of rotenone for 14 days, followed by a 14-day post-lesioning incubation period. We postulated that inhibition of NF-κB signaling in microglia would reduce overall inflammatory injury in lesioned mice. Subsequent analysis indicated decreased expression of the NF-κB-regulated autophagy gene, sequestosome 1 (p62), in microglia, which is required for targeting ubiquitinated α-synuclein (α-syn) for lysosomal degradation. Knock-out animals had increased accumulation of misfolded α-syn within microglia, despite an overall reduction in neurodegeneration. Interestingly, this occurred more prominently in males. These data suggest that microglia play key biological roles in the degradation and clearance of misfolded α-syn and this process works in concert with the innate immune response associated with neuroinflammation. Importantly, the accumulation of misfolded α-syn protein aggregates alone did not increase neurodegeneration following exposure to rotenone but required the NF-κB-dependent inflammatory response in microglia.

Changes in glial cells and neurotrophic factors due to rotenone-induced oxidative stress in Nrf2 knockout mice.

Glaucoma is one of the most common causes of blindness worldwide. It is thought to be a multifactorial disease with underlying mechanisms that include mitochondrial dysfunction and oxidative stress. Here, we used NF-E2 related factor 2 (Nrf2) knockout (KO) mice, which are vulnerable to oxidative stress, to examine a neuroprotective effect against oxidative stress due to rotenone, a mitochondrial complex I inhibitor. Wild-type (WT) and Nrf2 KO mice received an oral solution of rotenone for 30 days. We then extracted the retinas and performed immunohistochemistry and quantitative RT-PCR. We also prepared a primary Müller cell culture of samples from each mouse, added 30 µM rotenone, and then measured cell viability, cytotoxicity and CellRox absorbance. We also examined gene expression. We found a significant increase in the number of 8-OHdG-positive retinal ganglion cells (RGCs) after rotenone administration in both the WT and Nrf2 KO mice. There was no difference in the number of RNA-binding protein with multiple splicing (RBPMS)-positive RGCs in the WT and Nrf2 KO mice, but Nrf2 KO mice that were given rotenone had significantly less retinal gene expression of RBPMS than Nrf2 KO mice given a control. Moreover, there was significantly higher mRNA gene expression of vimentin and glial fibrillary acidic protein (GFAP) in Nrf2 KO mice that received rotenone than WT mice that received rotenone. A statistical analysis of the in vitro experiment showed that cell viability was lower, cytotoxicity was higher, and oxidative stress was higher in the Müller cells of the Nrf2 KO mice than the WT mice. Finally, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) were significantly higher in the Müller cells of the Nrf2 KO mice than the WT mice. These findings suggest that in Nrf2 KO mice under oxidative stress caused by rotenone, temporary neurotrophic factors are secreted from the Müller cells, conferring neuroprotection in these cells.

Intranasal Rotenone Induces Alpha-Synuclein Accumulation, Neuroinflammation and Dopaminergic Neurodegeneration in Middle-Aged Mice.

Accumulation of alpha-synuclein (α-syn) is central to the pathogenesis of Parkinson's disease (PD). Previous studies suggest that α-syn pathology may originate from the olfactory bulb (OB) or gut in response to an unknown pathogen and later progress to the different brain regions. Aging is viewed as the utmost threat to PD development. Therefore, studies depicting the role of age in α-syn accumulation and its progression in PD are important. In the present study, we gave intranasal rotenone microemulsion for 6 weeks in 12-month-old female BALB/c mice and found olfactory dysfunction after 4 and 6 weeks of rotenone administration. Interestingly, motor impairment was observed only after 6 weeks. The animals were sacrificed after 6 weeks to perform western blotting and immunohistochemical studies to detect α-syn pathology, neuroinflammation and neurodegeneration. We found α-syn accumulation in OB, striatum, substantia nigra (SN) and cortex. Importantly, we found significant glial cell activation and neurodegeneration in all the analysed regions which were absent in our previous published studies with 3 months old mice even after they were exposed to rotenone for 9 weeks indicating age is a crucial factor for α-syn induced neuroinflammation and neurodegeneration. We also observed increased iron accumulation in SN of rotenone-exposed aged mice. Moreover, inflammaging was observed in OB and striatum of 12-month-old BALB/c mice as compared to 3-month-old BALB/c mice. In conclusion, there is a difference in sensitivity between adult and aged mice in the development and progression of α-syn pathology and subsequent neurodegeneration, for which inflammaging might be the crucial probable mechanism.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis in a dopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

The Protective Effect of Lithium Against Rotenone may be Evolutionarily Conserved: Evidence from Eisenia fetida, a Primitive Animal with a Ganglionic Brain.

Chronic exposure to stress is a non-adaptive situation that is associated with mitochondrial dysfunction and the accumulation of reactive oxygen species (ROS), especially superoxide anion (SA). This accumulation of ROS produces damage-associated molecular patterns (DAMPs), which activate chronic inflammatory states and behavioral changes found in several mood disorders. In a previous study, we observed that an imbalance of SA triggered by rotenone (Ro) exposure caused evolutionarily conserved oxi-inflammatory disturbances and behavioral changes in Eisenia fetida earthworms. These results supported our hypothesis that SA imbalance triggered by Ro exposure could be attenuated by lithium carbonate (LC), which has anti-inflammatory properties. The initial protocol exposed earthworms to Ro (30 nM) and four different LC concentrations. LC at a concentration of 12.85 mg/L decreased SA and nitric oxide (NO) levels and was chosen to perform complementary assays: (1) neuromuscular damage evaluated by optical and scanning electron microscopy (SEM), (2) innate immune inefficiency by analysis of Eisenia spp. extracellular neutrophil traps (eNETs), and (3) behavioral changes. Gene expression was also evaluated involving mitochondrial (COII, ND1), inflammatory (EaTLR, AMP), and neuronal transmission (nAchR α5). LC attenuated the high melanized deposits in the circular musculature, fiber disarrangement, destruction of secretory glands, immune inefficiency, and impulsive behavior pattern triggered by Ro exposure. However, the effects of LC and Ro on gene expression were more heterogeneous. In summary, SA imbalance, potentially associated with mitochondrial dysfunction, appears to be an evolutionary component triggering oxidative, inflammatory, and behavioral changes observed in psychiatric disorders that are inhibited by LC exposure.

The Mechanism of SNHG8/Microrna-421-3p/Sorting Nexin 8 Axis on Dopaminergic Neurons in Substantia Nigra in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting the aging population. Particularly, long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in PD, while the role of lncRNA SNHG8 in PD remains to be further explored. C57BL/6 mice were induced by rotenone to establish a PD model in vivo, and then the dopaminergic (DA) neuronal damage and locomotor dysfunction in rotenone-treated mice were evaluated. Murine DA cell line MN9D was treated with rotenone to establish a cellular PD model in vitro. Then, the viability, apoptosis, mitochondrial dysfunction, endoplasmic reticulum stress, and autophagy in rotenone-treated MN9D cells were assessed. Expression levels of SNHG8, microRNA-421-3p (miR-421-3p), and sorting nexin 8 (SNX8) in the substantia nigra (SN) of PD mice and rotenone-treated MN9D cells were detected. The interaction between SNHG8 and miR-421-3p, and the targeting relationship between SNX8 and miR-421-3p were confirmed. SNHG8 and SNX8 expression levels were decreased while miR-421-3p expression level was increased in the SN of PD mice and rotenone-treated MN9D cells. Upregulated SNHG8 ameliorated dopaminergic neuron damage and locomotor dysfunction in PD mice. Meanwhile, upregulated SNHG8 enhanced viability, diminished apoptosis, and alleviated mitochondrial dysfunction, endoplasmic reticulum stress, and autophagy in rotenone-treated MN9D cells. Mechanistically, SNHG8 bound to miR-421-3p, and miR-421-3p targeted SNX8. Overexpressed SNHG8 downregulates miR-421-3p to alleviate rotenone-induced dopaminergic neuron injury in PD via upregulating SNX8.

1α,25-Dihydroxyvitamin D3 (VD3) Shows a Neuroprotective Action Against Rotenone Toxicity on PC12 Cells: An In Vitro Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by dopaminergic cell loss in the substantia nigra, and PD brains show neuroinflammation, oxidative stress, and mitochondrial dysfunction. The study evaluated the neuroprotective activity of 1α,25-dihydroxy vitamin D3 (VD3), on the rotenone (ROT)-induced cytotoxicity in PC12 cells. The viability parameters were assessed by the MTT and flow cytometry, on cells treated or not with VD3 and/or ROT. Besides, ROS production, cell death, mitochondrial transmembrane potential, reduced GSH, superoxide accumulation, molecular docking (TH and Keap1-Nrf2), and TH, Nrf2, NF-kB, and VD3 receptor protein contents by western blot were evaluated. VD3 was shown to improve the viability of ROT-exposed cells. Cells exposed to ROT showed increased production of ROS and superoxide, which decreased after VD3. ROT decrease in the mitochondrial transmembrane potential was prevented, after VD3 treatment and, VD3 was shown to interact with tyrosine hydroxylase (TH) and Nrf2. While ROT decreased TH, Nrf2, and NF-kB expressions, these effects were reversed by VD3. In addition, VD3 also increased VD3 receptor protein contents and values went back to those of controls after ROT exposure. VD3 protects PC12 cells against ROT damage, by decreasing oxidative stress and improving mitochondrial function. One target seems to be the TH molecule and possibly an indirect Nrf2 activation could also justify its neuroprotective actions on this PC12 cell model of PD.

Metabolic dysfunctions in the intranigral rotenone model of Parkinson's disease.

Parkinson disease (PD) is a chronic neurodegenerative disorder characterized by a progressive loss of dopamine neurons in the substantia nigra pars compacta (SNpc). In the last years, a growing interest to study the relationship between metabolic dysfunction and neurodegenerative disease like PD has emerged. This study aimed to evaluate the occurrence of possible changes in metabolic homeostasis due to intranigral rotenone administration, a neurotoxin that damages dopaminergic neurons leading to motor impairments mimicking those that happen in PD. Male Wistar rats were distributed into two groups: sham (n = 10) or rotenone (n = 10). Sham group received, bilaterally, within the SNpc, 1 µL of vehicle dimethyl-sulfoxide (DMSO) and the experimental group was bilaterally injected with 1 µL of rotenone (12 µg/µL). Twenty-four hours after the stereotaxic surgeries, the animals underwent the open field test followed by subsequent peripheral blood and cerebrospinal fluid (CSF) samples collection for biochemical testing. The results showed that rotenone was able to replicate the typical motor behavior impairment seen in the disease, i.e., decrease in locomotion (P = 0.05) and increase in immobility (P = 0.01) with a strong correlation (r = - 0.85; P < 0.0001) between them. In addition, it was demonstrated that this model is able to decrease plasmatic total-cholesterol (P = 0.04) and HDL-cholesterol (P = 0.007) potentially impacting peripheral metabolism. Hence, it was revealed a potential ability to reproduce relevant metabolic dysfunctions like hyperglycemia which could be explained by acute and systemic mitochondrial rotenone toxicity and SNpc nigral toxicity. Such mechanisms may still be responsible for the potential occurrence of CSF-hyperglycemia (d = 0.7). Since intranigral rotenone is an early phase model of PD, the present results open a new road for studies aiming to investigate metabolic changes in PD.