Low-dose Rotenone Research Articles

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson’s Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Inhibition of mitochondrial complex I leading to NAD+/NADH imbalance in type 2 diabetic patients who developed late stent thrombosis: Evidence from an integrative analysis of platelet bioenergetics and metabolomics

Type 2 diabetes mellitus (T2DM) is a strong indicator of late stent thrombosis (LST). Platelet bioenergetic dysfunction, although critical to the pathogenesis of diabetic macrovascular complications, remains uncharacterized in T2DM patients who developed LST. Here, we explored the mechanistic link between the alterations in platelet bioenergetics and LST in the setting of T2DM. Platelet bioenergetics, metabolomics, and their interactomes were analyzed in a nested case-control study including 15 T2DM patients who developed LST and 15 matched T2DM patients who did not develop LST (non-LST). Overall, we identified a bioenergetic alteration in T2DM patients with LST characterized by an imbalanced NAD+/NADH redox state resulting from deficient mitochondrial complex I (NADH: ubiquinone oxidoreductase) activity, which led to reduced ATP-linked and maximal mitochondrial respiration, increased glycolytic flux, and platelet hyperactivation compared with non-LST patients. Congruently, platelets from LST patients exhibited downregulation of tricarboxylic acid cycle and NAD+ biosynthetic pathways as well as upregulation of the proximal glycolytic pathway, a metabolomic change that was primarily attributed to compromised mitochondrial respiration rather than increased glycolytic flux as evidenced by the integrative analysis of bioenergetics and metabolomics. Importantly, both bioenergetic and metabolomic aberrancies in LST platelets could be recapitulated ex vivo by exposing the non-LST platelets to a low dose of rotenone, a complex I inhibitor. In contrast, normalization of the NAD+/NADH redox state, either by increasing NAD+ biosynthesis or by inhibiting NAD+ consumption, was able to improve mitochondrial respiration, inhibit mitochondrial oxidant generation, and consequently attenuate platelet aggregation in both LST platelets and non-LST platelets pretreated with low-dose rotenone. These data, for the first time, delineate the specific patterns of bioenergetic and metabolomic alterations for T2DM patients who suffer from LST, and establish the deficiency of complex I-derived NAD+ as a potential pathogenic mechanism in platelet abnormalities.

Rotenone aggravates PD-like pathology in A53T mutant human α-synuclein transgenic mice in an age-dependent manner.

Multiple factors such as genes, environment, and age are involved in developing Parkinson’s disease (PD) pathology. However, how various factors interact to cause PD remains unclear. Here, 3-month and 9-month-old hα-syn+⁣/− mice were treated with low-dose rotenone for 2 months to explore the mechanisms that underline the environment–gene–age interaction in the occurrence of PD. We have examined the behavior of mice and the PD-like pathologies of the brain and gut. The present results showed that impairments of the motor function and olfactory function were more serious in old hα-syn+/– mice with rotenone than that in young mice. The dopaminergic neuron loss in the SNc is more in old hα-syn+/– mice with rotenone than in young mice. Expression of hα-syn+/– is increased in the SNc of hα-syn+/– mice following rotenone treatment for 2 months. Furthermore, the number of activated microglia cells increased in SNc and accompanied the high expression of inflammatory cytokines, namely, TNF-α and IL-18 in the midbrain of old hα-syn+/– mice treated with rotenone. Meanwhile, we found that after treatment with rotenone, hα-syn positive particles deposited in the intestinal wall, intestinal microflora, and T lymphocyte subtypes of Peyer’s patches changed, and intestinal mucosal permeability increased. Moreover, these phenomena were age-dependent. These findings suggested that rotenone aggravated the PD-like pathologies and affected the brain and gut of human α-syn+/– transgenic mice in an age-dependent manner.

Time Course of Neurobehavioral Disruptions and Regional Brain Metabolism Changes in the Rotenone Mice Model of Parkinson's Disease.

Parkinson’s disease (PD) is characterized by slow progression with a long prodromal stage and the gradual evolution of both neuropsychological symptoms and subtle motor changes, preceding motor dysfunction. Thus, in order for animal models of PD to be valid, they should reproduce these characteristics of the disease. One of such models, in which neuropathology is induced by chronic injections of low doses of mitochondrial toxin rotenone, is well established in rats. However, data on this model adapted to mice remain controversial. We have designed the study to describe the timecourse of motor and non-motor symptoms during chronic subcutaneous administration of rotenone (4 mg/kg daily for 35 days) in C57BL/6 mice. We characterize the underlying neuropathological processes (dopaminergic neuron degeneration, regional brain metabolism, monoamine neurotransmitter and lipid peroxidation changes) at different timepoints: 1 day, 2 weeks and 5 weeks of daily rotenone exposure. Based on the behavioral data, we can describe three stages of pathology: cognitive changes from week 2 of rotenone exposure, subtle motor changes in week 3–4 and motor dysfunction starting roughly from week 4. Neuropathological changes in this model include a general decrease in COX activity in different areas of the brain (acute effect of rotenone) and a more specific decrease in midbrain (chronic effect), followed by significant neurodegeneration in SNpc but not VTA by the 5th week of rotenone exposure. However, we were unable to find changes in the level of monoamine neurotransmitters neither in the striatum nor in the cortex, nor in the level of lipid peroxidation in the brainstem. Thus, the gradual progression of pathology in this model is linked with metabolic changes, rather than with oxidative stress or tonic neurotransmitter release levels. Overall, this study supports the idea that a low-dose rotenone mouse model can also reproduce different stages of PD as well as rats.

Intranasal Exposure to Low-Dose Rotenone Induced Alpha-Synuclein Accumulation and Parkinson's Like Symptoms Without Loss of Dopaminergic Neurons.

Epidemiologically Parkinson's disease (PD) is associated with chronic ingestion or inhalation of environmental toxins leading to the development of motor symptoms. Though neurotoxin-based animal models played a major role in understanding diverse pathogenesis, they failed to identify the risk assessment due to uncommon route of toxin exposure. Towards this, the available neurotoxin-based intranasal (i.n.) PD models targeting olfactory bulb (OB) have demonstrated the dopaminergic (DAergic) neurodegeneration in both OB and substantia nigra (SN). Despite that, the studies detecting the alpha-synuclein (α-syn) accumulation in OB and its progression to other brain regions due to inhalation of environmental toxins are still lacking. Herein, we developed oil in water microemulsion of rotenone administered intranasally to the mice at a dose which is not detectable in blood, brain, and olfactory bulb by LCMS method. Our data reveals that 9weeks of rotenone exposure did not induce olfactory and motor dysfunction. Conversely, after 16weeks of washout period, rotenone treated mice showed both olfactory and motor impairment, along with α-syn accumulation in the OB and striatum without glial cell activation and loss of dopaminergic neurons. The results depict the progressive nature of the developed model and highlight the role of α-syn in PD like pathology or symptoms. Together, our findings suggest the adverse consequences of early exposure to the environmental toxins on the olfactory system for a shorter period with relevance to the development of synucleinopathy or Parkinson's disease in its later stage.

Chronic Systemic Exposure to Low-Dose Rotenone Induced Central and Peripheral Neuropathology and Motor Deficits in Mice: Reproducible Animal Model of Parkinson’s Disease

Epidemiological studies demonstrated that pesticide exposure, such as rotenone and paraquat, increases the risk of Parkinson’s disease (PD). Chronic systemic exposure to rotenone, a mitochondrial complex I inhibitor, could reproduce many features of PD. However, the adoption of the models is limiting because of variability in animal sensitivity and the inability of other investigators to consistently reproduce the PD neuropathology. In addition, most of rotenone models were produced in rats. Here, we tried to establish a high-reproducible rotenone model using C57BL/6J mice. The rotenone mouse model was produced by chronic systemic exposure to a low dose of rotenone (2.5 mg/kg/day) for 4 weeks by subcutaneous implantation of rotenone-filled osmotic mini pump. The rotenone-treated mice exhibited motor deficits assessed by open field, rotarod and cylinder test and gastrointestinal dysfunction. Rotenone treatment decreased the number of dopaminergic neuronal cells in the substantia nigra pars compacta (SNpc) and lesioned nerve terminal in the striatum. In addition, we observed significant reduction of cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) and the intestinal myenteric plexus. Moreover, α-synuclein was accumulated in neuronal soma in the SNpc, DMV and intestinal myenteric plexus in rotenone-treated mice. These data suggest that the low-dose rotenone mouse model could reproduce behavioral and central and peripheral neurodegenerative features of PD and be a useful model for investigation of PD pathogenesis.

Mitochondrial mass governs the extent of human T cell senescence.

The susceptibility of human CD4+ and CD8+ T cells to senesce differs, with CD8+ T cells acquiring an immunosenescent phenotype faster than the CD4+ T cell compartment. We show here that it is the inherent difference in mitochondrial content that drives this phenotype, with senescent human CD4+ T cells displaying a higher mitochondrial mass. The loss of mitochondria in the senescent human CD8+ T cells has knock‐on consequences for nutrient usage, metabolism and function. Senescent CD4+ T cells uptake more lipid and glucose than their CD8+ counterparts, leading to a greater metabolic versatility engaging either an oxidative or a glycolytic metabolism. The enhanced metabolic advantage of senescent CD4+ T cells allows for more proliferation and migration than observed in the senescent CD8+ subset. Mitochondrial dysfunction has been linked to both cellular senescence and aging; however, it is still unclear whether mitochondria play a causal role in senescence. Our data show that reducing mitochondrial function in human CD4+ T cells, through the addition of low‐dose rotenone, causes the generation of a CD4+ T cell with a CD8+‐like phenotype. Therefore, we wish to propose that it is the inherent metabolic stability that governs the susceptibility to an immunosenescent phenotype.

Earliest Mechanisms of Dopaminergic Neurons Sufferance in a Novel Slow Progressing Ex Vivo Model of Parkinson Disease in Rat Organotypic Cultures of Substantia Nigra.

The current treatments of Parkinson disease (PD) are ineffective mainly due to the poor understanding of the early events causing the decline of dopaminergic neurons (DOPAn). To overcome this problem, slow progressively degenerating models of PD allowing the study of the pre-clinical phase are crucial. We recreated in a short ex vivo time scale (96 h) all the features of human PD (needing dozens of years) by challenging organotypic culture of rat substantia nigra with low doses of rotenone. Thus, taking advantage of the existent knowledge, the model was used to perform a time-dependent comparative study of the principal possible causative molecular mechanisms undergoing DOPAn demise. Alteration in the redox state and inflammation started at 3 h, preceding the reduction in DOPAn number (pre-diagnosis phase). The number of DOPAn declined to levels compatible with diagnosis only at 12 h. The decline was accompanied by a persistent inflammation and redox imbalance. Significant microglia activation, apoptosis, a reduction in dopamine vesicle transporters, and the ubiquitination of misfolded protein clearance pathways were late (96 h, consequential) events. The work suggests inflammation and redox imbalance as simultaneous early mechanisms undergoing DOPAn sufferance, to be targeted for a causative treatment aimed to stop/delay PD.

A new mutation-independent approach to cancer therapy: Inhibiting oncogenic RAS and MYC, by targeting mitochondrial biogenesis.

Here, we used MCF7 cells as a model system to interrogate how MYC/RAS co-operativity contributes to metabolic flux and stemness in breast cancer cells. We compared the behavior of isogenic MCF7 cell lines transduced with c-Myc or H-Ras (G12V), either individually or in combination. Cancer stem cell (CSC) activity was measured using the mammosphere assay. c-Myc augmented both mammosphere formation and mitochondrial respiration, without any effects on glycolytic flux. In contrast, H-Ras (G12V) synergistically augmented both mammosphere formation and glycolysis, but only in combination with c-Myc, directly demonstrating MYC/RAS co-operativity. As c-Myc is known to exert its effects, in part, by stimulating mitochondrial biogenesis, we next examined the effects of another stimulus known to affect mitochondrial biogenesis, i.e. ROS production. To pharmacologically induce oxidative stress, we used Rotenone (a mitochondrial inhibitor) to target mitochondrial complex I. Treatment with Rotenone showed bi-phasic effects; low-dose Rotenone (1 to 2.5 nM) elevated mammosphere formation, while higher doses (10 to 100 nM) were inhibitory. Importantly, the stimulatory effects of Rotenone on CSC propagation were blocked using a mitochondrial-specific anti-oxidant, namely Mito-tempo. Thus, “mild” mitochondrial oxidative stress, originating at Complex I, was sufficient to pheno-copy the effects of c-Myc, effectively promoting CSC propagation. To validate the idea that mitochondrial biogenesis is required to stimulate CSC propagation, we employed Doxycycline, a well-established inhibitor of mitochondrial protein translation. Treatment with Doxycycline was indeed sufficient to block the stimulatory effects of H-Ras (G12V), c-Myc, and Rotenone on CSC propagation. As such, Doxycycline provides a strong rationale for designing new therapeutics to target mitochondrial biogenesis, suggesting a new “mutation-independent” approach to cancer therapy. In support of this notion, most currently successful anti-cancer agents therapeutically target “cell phenotypes”, such as increased cell proliferation, rather than specific genetic mutations. Remarkably, we demonstrated that Doxycycline inhibits the effects of diverse oncogenic stimuli, of both i) genetic (MYC/RAS) and ii) environmental (Rotenone) origins. Finally, we discuss the advantages of our “Proteomics-to-Genomics (PTG)” approach for in silico validation of new biomarkers and novel drug targets. In this context, we developed a new Myc-based Mito-Signature consisting of 3 mitochondrial genes (HSPD1; COX5B; TIMM44) for effectively predicting tumor recurrence (HR=4.69; p=2.4e-08) and distant metastasis (HR=4.94; p=2.8e-07), in ER(+) in breast cancer patients. This gene signature could serve as a new companion diagnostic for the early prediction of treatment failure in patients receiving hormonal therapy.

Exposure to low-dose rotenone precipitates synaptic plasticity alterations in PINK1 heterozygous knockout mice

Heterozygous mutations in the PINK1 gene are considered a susceptibility factor to develop early-onset Parkinson's disease (PD), as supported by dopamine hypometabolism in asymptomatic mutation carriers and subtle alterations of dopamine-dependent striatal synaptic plasticity in heterozygous PINK1 knockout (PINK1+/−) mice. The aim of the present study was to investigate whether exposure to low-dose rotenone of heterozygous PINK1+/− mice, compared to their wild-type PINK1+/+ littermates, could impact on dopamine-dependent striatal synaptic plasticity, in the absence of apparent structural alterations.Mice were exposed to a range of concentrations of rotenone (0.01–1mg/kg). Chronic treatment with concentrations of rotenone up to 0.8mg/kg did not cause manifest neuronal loss or changes in ATP levels both in the striatum or substantia nigra of PINK1+/− and PINK1+/+ mice. Moreover, rotenone (up to 0.8mg/kg) treatment did not induce mislocalization of the mitochondrial membrane protein Tom20 and release of cytochrome c in PINK1+/− striata. Accordingly, basic electrophysiological properties of nigral dopaminergic and striatal medium spiny neurons (MSNs) were normal. Despite the lack of gross alterations in neuronal viability in chronically-treated PINK1+/−, a complete loss of both long-term depression (LTD) and long-term potentiation (LTP) was recorded in MSNs from PINK1+/− mice treated with a low rotenone (0.1mg/kg) concentration. Even lower concentrations (0.01mg/kg) blocked LTP induction in heterozygous PINK1+/− MSNs compared to PINK1+/+ mice. Of interest, chronic pretreatment with the antioxidants alpha-tocopherol and Trolox, a water-soluble analog of vitamin E and powerful antioxidant, rescued synaptic plasticity impairment, confirming that, at the doses we utilized, rotenone did not induce irreversible alterations.In this model, chronic exposure to low-doses of rotenone was not sufficient to alter mitochondrial integrity and ATP production, but profoundly impaired the expression of long-term plasticity at corticostriatal synapses in PINK1 heterozygous knockout mice, suggesting that disruption of synaptic plasticity may represent an early feature of a pre-manifesting state of the disease, and a potential tool to test novel neuroprotective agents.

Investigation of tyrosine hydroxylase and BDNF in a low-dose rotenone model of Parkinson's disease

Tyrosine hydroxylase (TH, the rate limiting-enzyme in catecholamine synthesis) is regulated acutely via phosphorylation of 3 serine residues—Ser19, 31 and 40, and chronically via changes in TH protein levels. In this study, we aimed to investigate how TH is regulated in the brain, gut and adrenal gland as well as changes in mature brain-derived neurotrophic factor (mBDNF) and proBDNF levels in a low-dose (2mg/kg, 5 days/week for 4 weeks) rotenone model of Parkinson's disease (PD). Rearing behaviour decreased by week 3 in the rotenone group (p<0.01), with further decreases in rearing by week 4 (p<0.001); however, TH remained unchanged in the substantia nigra (SN) and striatum; TH levels were also unaltered in other catecholaminergic cell groups of the brainstem such as A1C1 neurons or locus coeruleus. In the olfactory bulb, TH protein decreased (2.5-fold, p<0.01) while Ser31 phosphorylation increased (1.4-fold, p<0.05) in the rotenone group. In contrast, TH protein was increased in the adrenal gland (2-fold, p<0.05) and colon (5-fold, p<0.05) of rotenone rats. mBDNF levels were not changed in the SN but were significantly reduced in plasma and significantly increased in the colon (2-fold, p<0.01) of rotenone-treated rats. This is the first study to assess TH and BDNF in the brain and periphery in the rotenone model before SN/striatum degeneration is evident. Together these results suggest that low-dose rotenone may have some potential to model the early stages of PD.

Hormetic effect of rotenone in primary human fibroblasts.

BackgroundRotenone inhibits the electron transfer from complex I to ubiquinone, in this way interfering with the electron transport chain in mitochondria. This chain of events induces increased levels of intracellular reactive oxygen species, which in turn can contribute to acceleration of telomere shortening and induction of DNA damage, ultimately resulting in aging. In this study, we investigated the effect of rotenone treatment in human fibroblast strains.ResultsFor the first time we here describe that rotenone treatment induced a hormetic effect in human fibroblast strains. We identified a number of genes which were commonly differentially regulated due to low dose rotenone treatment in fibroblasts independent of their cell origin. However, these genes were not among the most strongly differentially regulated genes in the fibroblast strains on treatment with rotenone. Thus, if there is a common hormesis regulation, it is superimposed by cell strain specific individual responses. We found the rotenone induced differential regulation of pathways common between the two fibroblast strains, being weaker than the pathways individually regulated in the single fibroblast cell strains. Furthermore, within the common pathways different genes were responsible for this different regulation. Thus, rotenone induced hormesis was related to a weak pathway signal, superimposed by a stronger individual cellular response, a situation as found for the differentially expressed genes.ConclusionWe found that the concept of hormesis also applies to in vitro aging of primary human fibroblasts. However, in depth analysis of the genes as well as the pathways differentially regulated due to rotenone treatment revealed cellular hormesis being related to weak signals which are superimposed by stronger individual cell-internal responses. This would explain that in general hormesis is a small effect. Our data indicate that the observed hormetic phenotype does not result from a specific strong well-defined gene or pathway regulation but from weak common cellular processes induced by low levels of reactive oxygen species. This conclusion also holds when comparing our results with those obtained for C. elegans in which the same low dose rotenone level induced a life span extending, thus hormetic effect.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0038-8) contains supplementary material, which is available to authorized users.

Inositol-Related Gene Knockouts Mimic Lithium’s Effect on Mitochondrial Function

The inositol-depletion hypothesis proposes that lithium attenuates phosphatidylinositol signaling. Knockout (KO) mice of two genes (IMPA1 or Slc5a3), each encoding for a protein related to inositol metabolism, were studied in comparison with lithium-treated mice. Since we previously demonstrated that these KO mice exhibit a lithium-like neurochemical and behavioral phenotype, here we searched for pathways that may mediate lithium's/the KO effects. We performed a DNA-microarray study searching for pathways affected both by chronic lithium treatment and by the KO of each of the genes. The data were analyzed using three different bioinformatics approaches. We found upregulation of mitochondria-related genes in frontal cortex of lithium-treated, IMPA1 and Slc5a3 KO mice. Three out of seven genes differentially expressed in all three models, Cox5a, Ndufs7, and Ndufab, all members of the mitochondrial electron transfer chain, have previously been associated with bipolar disorder and/or lithium treatment. Upregulation of the expression of these genes was verified by real-time PCR. To further support the link between mitochondrial function and lithium's effect on behavior, we determined the capacity of chronic low-dose rotenone, a mitochondrial respiratory chain complex I inhibitor, to alter lithium-induced behavior as measured by the forced-swim and the amphetamine-induced hyperlocomotion paradigms. Rontenone treatment counteracted lithium's effect on behavior, supporting the proposition suggested by the bioinformatics analysis for a mitochondrial function involvement in behavioral effects of lithium mediated by inositol metabolism alterations.The results provide support for the notion that mitochondrial dysfunction is linked to bipolar disorder and can be ameliorated by lithium. The phenotypic similarities between lithium-treated wild-type mice and the two KO models suggest that lithium may affect behavior by altering inositol metabolism.

Role of Metabotropic Glutamate Receptors in the Mechanisms of Experimental Parkinsonism Development

We studied the effects of metabotropic glutamate receptor 5 (mGluR5) antagonist 6-methyl-2-(phenylethynyl)-pyridine (MPEP) on the development of catalepsy and NO generation in the striatum of rats under conditions of long-term treatment with low doses of rotenone, a mitochondrial complex I inhibitor. In rats receiving single intraperitoneal injection of rotenone (1.5 mg/kg), NO concentration in the striatum did not differ from that in animals receiving sunflower oil. No signs of catalepsy were observed at these terms in both animal groups. It was demonstrated that long-term rotenone treatment induced catalepsy associated with enhanced NO production in the rat striatum. mGluR5 antagonist MPEP alleviated catalepsy caused by long-term rotenone treatment and prevented rotenone-induced stimulation of NO generation.

α-Synuclein Protects Neurons from Apoptosis Downstream of Free-Radical Production Through Modulation of the MAPK Signalling Pathway

α-Synuclein is a pre-synaptic chaperone and its accumulation contributes to differential cell loss in Parkinson's disease. Cytoplasmic expression of α-synuclein can directly modulate apoptotic pathways and contribute to cell survival, whereas induced over-expression of the protein causes oxidative stress through mitochondrial and cytosolic free-radical production. This study aimed to clarify the contribution of endogenous α-synuclein to oxidative stress and its association with cell death. Primary cortical neurons were derived from α-synuclein knock-out (Snca-/-) and wild-type (C57BL/6; WT) mice and treated with in vitro models of oxidative-stress, complex I inhibition and excitotoxicity. Mitochondrial free radical production was determined in isolated mitochondria derived from each mouse strain. Snca-/- derived cortical cultures were more susceptible (P<0.05) to oxidative-stress, but not excitotoxicity. This result was determined by significant increases in cell death (Propidium-Iodide staining) after 6h treatment in Snca-/- (45%±2.7SEM), relative to WT (33%±3.9SEM) cultures. α-Synuclein also confers significant (P<0.05) resistance to low-dose (5nM) rotenone toxicity, with a twofold reduction in cell death in WT, compared with Snca-/- cortical neurons. The expression of α-synuclein had no effect on cortical glutathione levels, or the production of reactive oxygen intermediates in isolated mitochondria. These data indicate that endogenous levels of α-synuclein confer resistance to oxidative stress downstream of free radical production and scavenging. The current data suggest that α-synuclein prevents cytochromec release and apoptosis through inhibition of the MAPK signalling pathway.

TNFR1-activated reactive oxidative species signals up-regulate osteogenic Msx2 programs in aortic myofibroblasts.

In LDLR(-/-) mice fed high-fat diabetogenic diets, osteogenic gene-regulatory programs are ectopically activated in vascular myofibroblasts and smooth muscle cells that promote arteriosclerotic calcium deposition. Msx2-Wnt signaling pathways previously identified as important for craniofacial skeletal development are induced in the vasculature by TNF, a prototypic cytokine mediator of the low-grade systemic inflammation of diabesity. To better understand this biology, we studied TNF actions on Msx2 in aortic myofibroblasts. TNF up-regulated Msx2 mRNA 4-fold within 3 h but did not regulate Msx1. Although IL-1β could also induce Msx2 expression, TNF-related apoptosis inducing ligand, receptor activator of nuclear factor-κB ligand, and IL-6 were inactive. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity and genetically induced Nox deficiency (p47phox(-/-)) reduced Msx2 induction, indicating contributions of reactive oxygen species (ROS) and redox signaling. Consistent with this, rotenone, an antagonist of mitochondrial complex I, inhibited TNF induction of Msx2 and Nox2, whereas pyruvate, an anapleurotic mitochondrial metabolic substrate, enhanced induction. Moreover, the glutathione peroxidase-mimetic ebselen abrogated this TNF response. Treatment of aortic myofibroblasts with hydrogen peroxide up-regulated Msx2 mRNA, promoter activity, and DNA-protein interactions. In vivo, SM22-TNF transgenic mice exhibit increased aortic Msx2 with no change in Msx1. Dosing SM22-TNF mice with either 20 ng/g Nox1 + 20 ng/g Nox2 antisense oligonucleotides or low-dose rotenone reduced arterial Msx2 expression. Aortic myofibroblasts from TNFR1(-/-) mice expressed levels of Msx2 that were 5% that of wild-type and were not inducible by TNF. Wnt7b and active β-catenin levels were also reduced. By contrast, TNF-inducible Msx2 expression was not reduced in TNFR2(-/-) cells. Finally, when cultured under mineralizing conditions, TNFR1(-/-) aortic myofibroblasts exhibited reduced calcification compared with wild-type and TNFR2(-/-) cells. Thus, ROS metabolism contributes to TNF induction of Msx2 and procalcific responses in myofibroblasts via TNFR1. Strategies that reduce vascular Nox- or mitochondrially activated ROS signals may prove useful in mitigating arteriosclerotic calcification.

Progressive loss of dopaminergic neurons induced by unilateral rotenone infusion into the medial forebrain bundle

Rotenone, a mitochondrial complex 1 inhibitor, causes oxidative damage via production of reactive oxygen species. We examined the pathophysiology of neuronal and glial cells of the nigrostriatal pathway following unilateral infusion of varying doses of rotenone into the substantia nigra or medial forebrain bundle of adult male Sprague–Dawley rats, sacrificed 14 and 60 days after infusion. Immunofluorescence techniques were used to qualitatively and quantitatively assay dopaminergic neurons, their projections, glial cells, synapses, and oxidative stress. Rotenone infusion into the substantia nigra at all concentrations caused extensive damage and tissue necrosis, therefore of limited relevance for producing a Parkinson disease model. Infusion of 0.5 μg of rotenone targeting the medial forebrain bundle induced oxidative stress in dopaminergic neurons causing ongoing cell stress as defined by an elevation of stress granule and oxidative stress markers. This treatment resulted in the loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra ( p ≤ 0.01) and loss of tyrosine hydroxylase immunoreactive nerve fibres and synaptic specialisations in the striatum ( p ≤ 0.01). The infusion of 0.5 μg of rotenone also caused an increase in astrocytes and microglial cells in the substantia nigra in comparison to control ( p ≤ 0.01). We examined the time-dependent reduction of tyrosine hydroxylase-positive nerve fibres and cell bodies in the striatum and substantia nigra respectively, with a progressive reduction evident 60 days after infusion ( p ≤ 0.01, p ≤ 0.05). Dopaminergic axons exposed to low-dose rotenone undergo oxidative stress, with a resultant ongoing loss of dopaminergic neurons, providing an animal model relevant to Parkinson disease.

Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation

Demyelination occurs in multiple inherited mitochondrial diseases. We studied which genes were induced as a consequence of differentiation in rodent and human oligodendroglia. Cholesterol, myelin and mitochondrial genes were significantly increased with oligodendroglial differentiation. Mitochondrial DNA content per cell and acetyl CoA-related transcripts increased significantly; thus, the large buildup of cholesterol necessary for myelination appears to require mitochondrial production of acetyl-CoA. Oligodendroglia were treated with low doses of the mitochondrial inhibitor rotenone to test the dependence of differentiation on mitochondrial function. Undifferentiated cells were resistant to rotenone, whereas differentiating cells were much more sensitive. Very low doses of rotenone that did not affect viability or ATP synthesis still inhibited differentiation, as measured by reduced levels of the myelin transcripts 2′,3′-Cyclic Nucleotide-3′-Phosphodiesterase and Myelin Basic Protein. Thus, mitochondrial transcripts and mtDNA are amplified during oligodendroglial differentiation, and differentiating oligodendroglia are especially sensitive to mitochondrial inhibition, suggesting mechanisms for demyelination observed in mitochondrial disease.

Vascular Pathology in Male Lewis Rats following Short-Term, Low-Dose Rotenone Administration

The long-term administration of low doses of rotenone has been used to produce a model of Parkinson disease (PD) in rats. However, only about 50% of similarly treated rats develop the PD-like syndrome, with many dying during the first few days of treatment. The lesions in male Lewis rats that became moribund or died after short-term, low-dose rotenone administration are described. Dosed rats had fibrinoid change and acute hemorrhage involving small arteries and arterioles of the brain and lungs. The thalamus, hypothalamus, and medulla oblongata were most frequently and severely affected. Blood vessels in the brain of some male Lewis rats appeared acutely susceptible to the effects of rotenone. Understanding the selective nature of the fibrinoid change and hemorrhage might explain how rotenone produces PD-like signs and lesions in rats, and it might also provide the basis for a model of intraparenchymal hemorrhagic cerebrovascular disease (i.e., hemorrhagic strokes) in humans.

Effects of chronic low dose rotenone treatment on human microglial cells

BackgroundExposure to toxins/chemicals is considered to be a significant risk factor in the pathogenesis of Parkinson's disease (PD); one putative chemical is the naturally occurring herbicide rotenone that is now used widely in establishing PD models. We, and others, have shown that chronic low dose rotenone treatment induces excessive accumulation of Reactive Oxygen Species (ROS), inclusion body formation and apoptosis in dopaminergic neurons of animal and human origin. Some studies have also suggested that microglia enhance the rotenone induced neurotoxicity. While the effects of rotenone on neurons are well established, there is little or no information available on the effect of rotenone on microglial cells, and especially cells of human origin. The aim of the present study was to investigate the effects of chronic low dose rotenone treatment on human microglial CHME-5 cells.MethodsWe have shown previously that rotenone induced inclusion body formation in human dopaminergic SH-SY5Y cells and therefore used these cells as a control for inclusion body formation in this study. SH-SY5Y and CHME-5 cells were treated with 5 nM rotenone for four weeks. At the end of week 4, both cell types were analysed for the presence of inclusion bodies, superoxide dismutases and cell activation (only in CHME-5 cells) using Haematoxylin and Eosin staining, immunocytochemical and western blotting methods. Levels of active caspases and ROS (both extra and intra cellular) were measured using biochemical methods.ConclusionThe results suggest that chronic low dose rotenone treatment activates human microglia (cell line) in a manner similar to microglia of animal origin as shown by others. However human microglia release excessive amounts of ROS extracellularly, do not show excessive amounts of intracellular ROS and active caspases and most importantly do not show any protein aggregation or inclusion body formation. Human microglia appear to be resistant to rotenone (chronic, low dose) induced damage.