N27 Cells Research Articles

Allosteric Activation of SIRT1 by 2,4-Dihydroxy-azaflavanone Averts MPP+-Mediated Dysfunction in Mitochondrial Biogenesis and Bioenergetics: Implications for Parkinson's Disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder that affects dopamine neurons of the substantia nigra pars compacta (SNpc), resulting in motor dysfunction. Among the pathways examined, mitochondria and α-synuclein were found to play a major role in the disease progression. Hence, several attempts are being made to restore mitochondrial bioenergetics or protein aggregation pathways as disease-modifying strategies. Our earlier studies reported the protective effect of 2,4-dihydroxy-azaflavanone (azaflavanone) in a transgenic Drosophila fly model of PD. In the present study, we found that azaflavanone acts as an allosteric activator of SIRT1 in both cell-free and cell-based systems and the effects were more pronounced as compared to resveratrol. Also, azaflavanone appears to interact selectively with SIRT1 as other SIRTs such as SIRT3 and SIRT6 did not exhibit any gross changes in cellular thermal shift assay (CETSA). Molecular docking studies depicted a higher docking score with azaflavanone than with resveratrol. Further, N27 cells treated with azaflavanone exhibited a dose-dependent increase in the mitotracker staining, mtDNA/nuclear DNA ratio, and also mitochondrial bioenergetics. The observed effects appear to be due to the activation of SIRT1, as evidenced by an increase in the expression of PGC-1α and TFAM, which are the downstream targets of SIRT1. Lastly, the Parkinsonian mimic MPP+-induced disturbance in the mitochondrial membrane potential, mitochondrial bioenergetics, and biogenesis were ameliorated by azaflavanone. Overall, our findings indicate that azaflavanone, being an antioxidant and an allosteric activator of SIRT1, is a promising compound for ameliorating the pathophysiology of PD.

Expression of G2019S LRRK2 in Rat Primary Astrocytes Mediates Neurotoxicity and Alters the Dopamine Synthesis Pathway in N27 Cells via Astrocytic Proinflammatory Cytokines and Neurotrophic Factors.

Astrocytes in the brain contribute to various essential functions, including maintenance of the neuronal framework, survival, communication, metabolic processes, and neurotransmitter levels. Leucine-rich repeat kinase 2 (LRRK2) is associated with the pathogenesis of Parkinson's disease (PD). LRRK2 is expressed in neurons, microglia, and astrocytes and plays diverse roles in these cell types. We aimed to determine the effects of mutant human G2019S-LRRK2 (GS-hLRRK2) in rat primary astrocytes (rASTROs). Transfection with GS-hLRRK2 significantly decreased cell viability compared to transfection with the vector and wild-type human LRRK2 (WT-hLRRK2). GS-hLRRK2 expression significantly reduced the levels of nerve growth factor and increased the levels of proinflammatory cytokines (interleukin-1β and tumor necrosis factor α) compared to the vector and WT-hLRRK2 expression. Furthermore, GS-hLRRK2 expression in rASTROs promoted astrogliosis, which was characterized by increased expression of glial fibrillary acidic protein and vimentin. Treatment with the conditioned medium of G2019S LRRK2-expressing rASTROs decreased N27 cell viability compared to treatment with that of WT-hLRRK2-expressing rASTROs. Consequently, the regulation of the dopamine synthesis pathway was affected in N27 cells, thereby leading to altered levels of tyrosine hydroxylase, dopamine transporter, Nurr1, and dopamine release. Overall, the G2019S LRRK2 mutation disrupted astrocyte function, thereby aggravating PD progression.

Mito-metformin protects against mitochondrial dysfunction and dopaminergic neuronal degeneration by activating upstream PKD1 signaling in cell culture and MitoPark animal models of Parkinson's disease.

Impaired mitochondrial function and biogenesis have strongly been implicated in the pathogenesis of Parkinson's disease (PD). Thus, identifying the key signaling mechanisms regulating mitochondrial biogenesis is crucial to developing new treatment strategies for PD. We previously reported that protein kinase D1 (PKD1) activation protects against neuronal cell death in PD models by regulating mitochondrial biogenesis. To further harness the translational drug discovery potential of targeting PKD1-mediated neuroprotective signaling, we synthesized mito-metformin (Mito-Met), a mitochondria-targeted analog derived from conjugating the anti-diabetic drug metformin with a triphenylphosphonium functional group, and then evaluated the preclinical efficacy of Mito-Met in cell culture and MitoPark animal models of PD. Mito-Met (100-300 nM) significantly activated PKD1 phosphorylation, as well as downstream Akt and AMPKα phosphorylation, more potently than metformin, in N27 dopaminergic neuronal cells. Furthermore, treatment with Mito-Met upregulated the mRNA and protein expression of mitochondrial transcription factor A (TFAM) implying that Mito-Met can promote mitochondrial biogenesis. Interestingly, Mito-Met significantly increased mitochondrial bioenergetics capacity in N27 dopaminergic cells. Mito-Met also reduced mitochondrial fragmentation induced by the Parkinsonian neurotoxicant MPP+ in N27 cells and protected against MPP+-induced TH-positive neurite loss in primary neurons. More importantly, Mito-Met treatment (10 mg/kg, oral gavage for 8 week) significantly improved motor deficits and reduced striatal dopamine depletion in MitoPark mice. Taken together, our results demonstrate that Mito-Met possesses profound neuroprotective effects in both in vitro and in vivo models of PD, suggesting that pharmacological activation of PKD1 signaling could be a novel neuroprotective translational strategy in PD and other related neurocognitive diseases.

The Protection of EGCG Against 6-OHDA-Induced Oxidative Damage by Regulating PPARγ and Nrf2/HO-1 Signaling.

6-Hydroxydopamine (6-OHDA) is a classic neurotoxin that has been widely used in Parkinson's disease research. 6-OHDA can increase intracellular reactive oxygen species (ROS) and can cause cell damage, which can be attenuated with (-)-Epigallocatechin-3-gallate (EGCG) treatment. However, the mechanism by which EGCG alters the 6-OHDA toxicity remains unclear; In this study, we found 6-OHDA (25 μM) alone increased intracellular ROS concentration in N27 cells, which was attenuated by pretreating with EGCG (100 μM). We evaluated the intracellular oxidative damage by determining the level of thiobarbituric acid reactive substances (TBARS) and protein carbonyl content. 6-OHDA significantly increased TBARS by 82.7% (P < .05) and protein carbonyl content by 47.8 (P < .05), compared to the control. Pretreatment of EGCG decreased TBARS and protein carbonyls by 36.4% (P < .001) and 27.7% (P < .05), respectively, compared to 6-OHDA alone treatment. Antioxidant effect was tested with E2-related factor 2 (Nrf2), heme oxygenase-1(HO-1) and peroxisome-proliferator activator receptor γ (PPARγ) expression. 6-OHDA increased Nrf2 expression by 69.6% (P < .001), HO-1 by 173.3% (P < .001), and PPARγ by 122.7% (P < .001), compared with untreatment. EGCG pretreatment stabilized these alterations induced by 6-OHDA. Our results suggested that the neurotoxicity of 6-OHDA in N27 cells was associated with ROS pathway, whereas pretreatment of EGCG suppressed the ROS generation and deactivated the Nrf2/HO-1 and PPARγ expression.

STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease

IntroductionParkinson's disease (PD) is the most prevalent disorder of the basal ganglia, propagated by the degeneration of axon terminals within the striatum and subsequent loss of dopaminergic neurons in the substantia nigra (SN). Exposure of environmental neurotoxins and mutations of several mitochondrial and proteasomal genes are primarily responsible. MethodsTo determine whether signal transducer and activator of transcription 3 (STAT3) could protect dopaminergic neurons against degeneration, we first screened it in the in vitro capacity using immortalized rat dopaminergic N27 cells under 6-OHDA neurotoxicity. We then evaluated the effectiveness of constitutively active (ca) STAT3 as a neuroprotective agent on N27 cells in a 6-hydroxydopamine (6-OHDA) induced rat model of PD and compared it to control animals or animals where AAV/caRheb was expressed in SN. Behavioral outcomes were assessed using rotational and cylinder assays and mitochondrial function using reactive oxygen species (ROS) levels. ResultsUsing flow cytometry, the in vitro analysis determined caSTAT3 significantly decreased dopaminergic neuronal death under 6-OHDA treatment conditions. Importantly, in vivo overexpression of caSTAT3 in SN dopaminergic neurons using AAV-mediated expression demonstrated significant neuroprotection of dopaminergic neurons following 6-OHDA. Both caSTAT3 and caRheb + caSTAT3 co-injection into substantia nigra reduced D-amphetamine-induced rotational behavior and increased ipsilateral forelimb function when compared to control animals. In addition, caSTAT3 decreased mitochondrial ROS production following 6-OHDA induced neurotoxicity. ConclusioncaSTAT3 confers resistance against ROS production in mitochondria of susceptible SN dopaminergic neurons potentially offering a new avenue for treatment against PD.

Extracellular Vesicles and Their Renin-Angiotensin Cargo as a Link between Metabolic Syndrome and Parkinson's Disease.

Several studies showed an association between metabolic syndrome (MetS) and Parkinson's disease (PD). The linking mechanisms remain unclear. MetS promotes low-grade peripheral oxidative stress and inflammation and dysregulation of the adipose renin-angiotensin system (RAS). Interestingly, brain RAS dysregulation is involved in the progression of dopaminergic degeneration and PD. Circulating extracellular vesicles (EVs) from MetS fat tissue can cross the brain-blood barrier and may act as linking signals. We isolated and characterized EVs from MetS and control rats and analyzed their mRNA and protein cargo using RT-PCR and the ExoView R200 platform, respectively. Furthermore, cultures of the N27 dopaminergic cell line and the C6 astrocytic cell line were treated with EVs from MetS rats. EVs were highly increased in MetS rat serum, which was inhibited by treatment of the rats with the angiotensin type-1-receptor blocker candesartan. Furthermore, EVs from MetS rats showed increased pro-oxidative/pro-inflammatory and decreased anti-oxidative/anti-inflammatory RAS components, which were inhibited in candesartan-treated MetS rats. In cultures, EVs from MetS rats increased N27 cell death and modulated C6 cell function, upregulating markers of neuroinflammation and oxidative stress, which were inhibited by the pre-treatment of cultures with candesartan. The results from rat models suggest EVs and their RAS cargo as a mechanism linking Mets and PD.

Synthesis, In Vitro Biological Evaluation of Antiproliferative and Neuroprotective Effects and In Silico Studies of Novel 16E-Arylidene-5α,6α-epoxyepiandrosterone Derivatives

Steroids constitute an important class of pharmacologically active molecules, playing key roles in human physiology. Within this group, 16E-arylideneandrostane derivatives have been reported as potent anti-cancer agents for the treatment of leukemia, breast and prostate cancers, and brain tumors. Additionally, 5α,6α-epoxycholesterol is an oxysterol with several biological activities, including regulation of cell proliferation and cholesterol homeostasis. Interestingly, pregnenolone derivatives combining these two modifications were described as potential neuroprotective agents. In this research, novel 16E-arylidene-5α,6α-epoxyepiandrosterone derivatives were synthesized from dehydroepiandrosterone by aldol condensation with different aldehydes followed by a diastereoselective 5α,6α-epoxidation. Their cytotoxicity was evaluated on tumoral and non-tumoral cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, the assessment of the neuroprotective activity of these derivatives was performed in a dopaminergic neuronal cell line (N27), at basal conditions, and in the presence of the neurotoxin 6-hydroxydopamine (6-OHDA). Interestingly, some of these steroids had selective cytotoxic effects in tumoral cell lines, with an IC50 of 3.47 µM for the 2,3-dichlorophenyl derivative in the breast cancer cell line (MCF-7). The effects of this functionalized epoxide on cell proliferation (Ki67 staining), cell necrosis (propidium iodide staining), as well as the analysis of the nuclear area and near neighbor distance in MCF-7 cells, were analyzed. From this set of biological studies, strong evidence of the activation of apoptosis was found. In contrast, no significant neuroprotection against 6-OHDA-induced neurotoxicity was observed for the less cytotoxic steroids in N27 cells. Lastly, molecular docking simulations were achieved to verify the potential affinity of these compounds against important targets of steroidal drugs (androgen receptor, estrogen receptor α, and 5α-reductase type 2, 17α-hydroxylase-17,20-lyase and aromatase enzymes). This in silico study predicted a strong affinity between most novel steroidal derivatives and 5α-reductase and 17α-hydroxylase-17,20-lyase enzymes.

Overexpressed miRNA-nov-1 promotes manganese-induced apoptosis in N27 cells by regulating Dhrs3 to activate mTOR signaling pathway

Environmental and occupational chronic manganese exposure can cause neurotoxicity and apoptosis. Moreover, microRNAs (miRNAs) are extensively involved in the process of neuronal apoptosis. Therefore, it is crucial to study the mechanism of miRNA in manganese-induced neuronal apoptosis and to find potential targets. In the present study, we found that the expression of miRNA-nov-1 was increased after N27 cells were exposed to MnCl2. Then, seven different cell groups were constructed by lentiviral infection of cells, and the overexpression of miRNA-nov-1 promoted the apoptosis process of N27 cells. Further studies showed a negative regulatory relationship between miRNA-nov-1 and dehydrogenase/reductase 3 (Dhrs3). The up-regulation of miRNA-nov-1 reduced the protein level of Dhrs3 in N27 cells exposed to manganese, increased the expression of a caspase-3 protein, activated the rapamycin (mTOR) signaling pathway, and increased cell apoptosis. Furthermore, we found that the expression of the Caspase-3 protein was decreased after the low expression of miRNA-nov-1, the mTOR signaling pathway was inhibited, and reduced cell apoptosis. However, these effects were reversed by the knockdown of Dhrs3. Taken together, these results suggested that overexpression of miRNA-nov-1 can promote manganese-induced apoptosis in N27 cells by activating the mTOR signaling pathway and negatively regulating Dhrs3.

Receptor-Independent Anti-Ferroptotic Activity of TrkB Modulators.

Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer's disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current study investigated whether modulators of TrkB signalling affect ferroptosis. Cell viability, C11 BODIPY, and cell-free oxidation assays were used to observe the impact of TrkB modulators, and an immunoblot assay was used to detect TrkB expression. TrkB modulators such as agonist BDNF, antagonist ANA-12, and inhibitor K252a did not affect RSL3-induced ferroptosis sensitivity in primary cortical neurons expressing detectable TrkB receptors. Several other modulators of the TrkB receptor, including agonist 7,8-DHF, activator phenelzine sulphate, and inhibitor GNF-5837, conferred protection against a range of ferroptosis inducers in several immortalised neuronal and non-neuronal cell lines, such as N27 and HT-1080 cells. We found these immortalised cell lines lack detectable TrkB receptor expression, so the anti-ferroptotic activity of these TrkB modulators was most likely due to their inherent radical-trapping antioxidant properties, which should be considered when interpreting their experimental findings. These modulators or their variants could be potential anti-ferroptotic therapeutics for various diseases.

Minute-sensitive real-time monitoring of neural cells through printed graphene microelectrodes

Real-time and high-throughput cytometric monitoring of neural cells exposed to injury mechanisms is invaluable for in-vitro studies. Electrical impedance spectroscopy via microelectrode arrays is a label-free technique for monitoring of neural growth and their detachment upon death. In this method, the interface material plays a vital role to provide desirable attachment cues for the cell network. Thus, here we demonstrate the electrohydrodynamic patterning of aqueous graphene for microelectrode fabrication. We investigated whether the wrinkled surface morphology of the electrodes fabricated by this deposition method expands their electroactive surface area and thus enables a rapid response time. The nano-scale quality of the graphene lattice is characterized by Raman spectroscopy and Transmittance electron microscopy. N27 rat dopaminergic neural cells were cultured on the chips and the surface morphology of the microelectrodes during cellular growth was investigated by Scanning electrode spectroscopy. Attachment of the neural population on the graphene microelectrodes was parametrized and the change in the impedance spectrum of this cell population was quantified at 10 Hz to 10 kHz frequencies along with the change in TUBB3 gene expression. The viability test of the cell population on the biosensor demonstrated no significant difference in comparison to the control, and a cell density of 2289 cell/mm2 was achieved. As a proof of concept, the confluent N27 cell population was exposed to UV and its cytotoxic impact on neural detachment and lift-off was monitored. The multiplexed detection of cellular activity was reported with a temporal resolution of one minute.

The Role of Lipid Rafts and Membrane Androgen Receptors in Androgen's Neurotoxic Effects.

Sex differences have been observed in multiple oxidative stress–associated neurodegenerative diseases. Androgens, such as testosterone, can exacerbate oxidative stress through a membrane androgen receptor (mAR), AR45, localized to lipid rafts in the plasma membrane. The goal of this study is to determine if interfering with mAR localization to cholesterol-rich lipid rafts decreases androgen induced neurotoxicity under oxidative stress environments. We hypothesize that cholesterol-rich caveolar lipid rafts are necessary for androgens to induce oxidative stress generation in neurons via the mAR localized within the plasma membrane. Nystatin was used to sequester cholesterol and thus decrease cholesterol-rich caveolar lipid rafts in a neuronal cell line (N27 cells). Nystatin was applied prior to testosterone exposure in oxidatively stressed N27 cells. Cell viability, endocytosis, and protein analysis of oxidative stress, apoptosis, and mAR localization were conducted. Our results show that the loss of lipid rafts via cholesterol sequestering blocked androgen-induced oxidative stress in cells by decreasing the localization of mAR to caveolar lipid rafts.

Stable expression of the human dopamine transporter in N27 cells as an in vitro model for dopamine cell trafficking and metabolism

Dopamine (DA) metabolism and cell trafficking are critical for the proper functioning of DA neurons. Disruption of these DA processes can yield toxic products and is implicated in neurological conditions including Parkinson's disease (PD). To investigate pathogenic mechanisms involving DA neurons, in vitro models that recapitulate DA metabolism and trafficking in vivo are crucial. N27 cells are a widely used model for PD; however, these cells exhibit little expression of the DA transporter (DAT) confounding studies of DA uptake and metabolism. This lack of adequate DAT expression calls into question the use of this cell line as a model to study DA cell trafficking and metabolism. To overcome this problem, we stably expressed the human DAT (hDAT) in N27 cells to develop cells that we named N27-BCD. This approach allows for characterization of toxicants that may alter DA metabolism, trafficking, and/or interactions with DAT. N27-BCD cells are more sensitive to the neurotoxins 1-methyl-4-phenylpyridinium (MPTP/MPP+) and 6-hydroxydopamine (6-OHDA). N27-BCD cells allowed for clear observation of DA metabolism, whereas N27 cells did not. Here, we propose that stable expression of hDAT in N27 cells yields a useful model of DA neurons to study the impact of altered DA cell trafficking and metabolism.

Alpha-Synuclein Preformed Fibrils Induce Cellular Senescence in Parkinson's Disease Models.

Emerging evidence indicates that cellular senescence could be a critical inducing factor for aging-associated neurodegenerative disorders. However, the involvement of cellular senescence remains unclear in Parkinson’s disease (PD). To determine this, we assessed the effects of α-synuclein preformed fibrils (α-syn PFF) or 1-methyl-4-phenylpyridinium (MPP+) on changes in cellular senescence markers, employing α-syn PFF treated-dopaminergic N27 cells, primary cortical neurons, astrocytes and microglia and α-syn PFF-injected mouse brain tissues, as well as human PD patient brains. Our results demonstrate that α-syn PFF-induced toxicity reduces the levels of Lamin B1 and HMGB1, both established markers of cellular senescence, in correlation with an increase in the levels of p21, a cell cycle-arrester and senescence marker, in both reactive astrocytes and microglia in mouse brains. Using Western blot and immunohistochemistry, we found these cellular senescence markers in reactive astrocytes as indicated by enlarged cell bodies within GFAP-positive cells and Iba1-positive activated microglia in α-syn PFF injected mouse brains. These results indicate that PFF-induced pathology could lead to astrocyte and/or microglia senescence in PD brains, which may contribute to neuropathology in this model. Targeting senescent cells using senolytics could therefore constitute a viable therapeutic option for the treatment of PD.

Filaricidal activity of Daniellia oliveri and Psorospermum febrifugum extracts

BackgroundDrugs currently used for controlling onchocerciasis and lymphatic filariasis (LF) are mainly microfilaricidal, with minimal or no effect on the adult worms. For efficient management of these diseases, it is necessary to search for new drugs with macrofilaricidal activities that can be used singly or in combination with existing ones. Daniellia oliveri and Psorospermum febrifugum are two plants commonly used in the local management of these infections in Bambui, a township in the North West Region of Cameroon, but there is currently no documented scientific evidence to support their claimed anthelmintic efficacy and safety. The aim of this study was to provide evidence in support of the search for means to eliminate these diseases by screening extracts and chromatographic fractions isolated from these plants for efficacy against the parasitic roundworms Onchocerca ochengi and Brugia pahangi.MethodsThe viability of O. ochengi adult worms was assessed using the MTT/formazan assay. Fully confluent monkey kidney epithelial cells (LLC-MK2) served as the feeder layer for the O. ochengi microfilariae (mfs) assays. Viability of the mfs was assessed by microscopic examination for mean motility scoring (relative to the negative control) every 24 h post addition of an extract. The Worminator system was used to test the effects of the extracts on adult B. pahangi motility, and mean motility units were determined for each worm. Cytotoxicity of the active extracts on N27 cells was assessed using the MTS assay.ResultsExtracts from D. oliveri and P. febrifugum were effective against the adult roundworms O. ochengi and B. pahangi. Interestingly, extracts showing macrofilaricidal activities against O. ochengi also showed activity against O. ochengi mfs. The hexane stem bark extract of D. oliveri (DOBHEX) was more selective for adult O. ochengi than for mfs, with a half maximal and 100% inhibitory concentration (IC50 and IC100, respectively) against adult O. ochengi of 13.9 and 31.3 μg/ml, respectively. The in vitro cytotoxicity of all active extracts on N27 cells showed selective toxicity for parasites (selectivity index > 1). Bioassay-guided fractionation of the extracts yielded fractions with activity against adult B. pahangi, thus confirming the presence of bioactive principles in the plant extracts.ConclusionsOur study supports the use of D. oliveri and P. febrifugum in the traditional treatment of onchocerciasis and LF. The further purification of active extracts from these plants could yield lead compounds for filarial drug discovery and development.Graphical

Organochlorine Pesticide Dieldrin Suppresses Cellular Interferon-Related Antiviral Gene Expression.

Organochlorine pesticides (OCPs) are persistent pollutants linked to diverse adverse health outcomes. Environmental exposure to OCPs has been suggested to negatively impact the immune system but their effects on cellular antiviral responses remain unknown. Transcriptomic analysis of N27 rat dopaminergic neuronal cells unexpectedly detected high level expression of genes in the interferon (IFN)-related antiviral response pathways including the IFN-induced protein with tetratricopeptide repeats 1 and 2 (Ifit1/2) and the MX Dynamin Like GTPases Mx1 and Mx2. Interestingly, treatment of N27 cells with dieldrin markedly downregulated the expression of many of these genes. Dieldrin exterted a similar effect in inhibiting IFIT2 and MX1 gene expression in human SH-SY5Y neuronal cells induced by an RNA viral mimic, polyinosinic: polycytidylic acid (poly I:C) and IFIT2/3 gene expression in human pulmonary epithelial cells exposed to human influenza H1N1 virus. Mechanistically, dieldrin induced a rapid rise in levels of intracellular reactive oxygen species (iROS) and a decrease in intracellular glutathione (GSH) levels in SH-SY5Y cells. Treatment with N-acetylcysteine, an antioxidant and GSH biosynthesis precursor, effectively blocked both dieldrin-induced increases in iROS and its inhibition of poly I:C-induced upregulation of IFIT and MX gene expression, suggesting a role for intracellular oxidative status in dieldrin's modulation of antiviral gene expression. This study demonstrates that dieldrin modulates key genes of the cellular innate immune responses that are normally involved in the host's cellular defense against viral infections. Our findings have potential relevance to understanding the organismal effects of environmentally persistent organochlorine contaminants on the mammalian cellular immune system.

Mitochondrial and transcriptome responses in rat dopaminergic neuronal cells following exposure to the insecticide fipronil

The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 μM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 μM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 μM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 μM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 μM fipronil treatments, but there was a ∼40–60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 μM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 μM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 μM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson’s disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents.

On chip detection of glial cell-derived neurotrophic factor secreted from dopaminergic cells under magnetic stimulation

Glial cell-derived neurotrophic factor (GDNF) is a small protein potently promoting the survival of dopaminergic and motor neurons. GDNF can be secreted from different types of cells including the dopaminergic neural cell line, N27. N27 cells, a rat dopaminergic neural cell line, is regarded as a suitable in vitro model for Parkinson's disease (PD) research. For PD treatment, transcranial magnetic stimulation (TMS), a noninvasive therapeutic method, showed beneficial clinical effects, but the mechanism for its benefit is not understood. Because GDNF is a potent neurotrophic factor, it is of great value to evaluate if GDNF secretion from N27 cells can be affected by magnetic stimulation (MS). However, the current methods for detecting GDNF are time-consuming and expensive. In this paper we outline the detection of GDNF secretion from N27 cells by ultrasensitive nanopore thin film sensors (nanosensor) for the first time. As low as 2 pg/mL GDNF can be readily detected by the nanosensor. Furthermore, we show that MS can promote GDNF secretion from N27 cells. Specifically, the GDNF concentration in N27 cell-conditioned media under MS treatment shows statistically significant increase up to 2-fold after 5 days in vitro in comparison with the control. This nanosensor along with the in vitro PD model N27 cells provides a low-cost, easy-to-use, sensitive approach for studying potential cell biological mechanisms of the clinical benefits of MS on PD.

Investigating the Effects of Epoxy Fatty Acids against LPS and Pesticide Induced Toxicity

Neuroinflammation is a hallmark of many neurodegenerative diseases including Parkinson’s Disease (PD) and Alzheimer’s Disease (AD), and mitigating neuroinflammation could avert the progression of these disorders. While it is commonly known that inflammation is propagated by COX metabolites of arachidonic acid (ARA) called prostaglandins, it is less known that metabolites of ARA produced by CYP450 called epoxyeicosatrienoic acids (EETs) have been shown to have an anti‐inflammatory and anti‐apoptotic effect in many disease models. Previous studies have demonstrated that increasing the biological availability of EETs by inhibiting soluble epoxide hydrolases (sEH) reduces MPTP or paraquat induced neuroinflammation and prevents the loss of dopaminergic neurons caused by these toxicants. However, the mechanisms underlying such effect is still unknown. In this study, we have treated primary rat astrocyte mixtures with LPS to determine how EETs can affect regulation of growth factors such as its BDNF, GDNF, and VEGF‐A/B. In addition, primary microglia culture was treated with LPS to determine whether microglial activation can be mitigated through the addition of EETs. Finally, N27 dopaminergic neurons were treated with a pesticide rotenone, and the effect of EETs on the survival of dopaminergic neurons were assessed. We have found that EETs and/or sEH inhibition can affect all cells in ways that are be neuroprotective against neuroinflammatory disorders such as Parkinson’s Disease, with reduced inlammatory marker, increased growth factor signaling, and increased neural viability. Future studies may investigate how inhibition of sEH can be used to combat PD in human patients.Support or Funding InformationThis work was supported by the National Institute of Environmental Health Sciences (NIEHS) Grant R01 ES002710, NIEHS Superfund Research Program P42 ES004699, and NIEHS T32 ES007059 (J.A).EETs and sEH inhibitors cause reduction in rotenone induced loss of N27 cell viability.Figure 1EETs and sEH inhibitor along with LPS stimulate growth factor signaling and reduce LPS induced IL‐1b induction.Figure 2

Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment

BackgroundThe role of sex hormones on cellular function is unclear. Studies show androgens and estrogens are protective in the CNS, whereas other studies found no effects or damaging effects. Furthermore, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Alzheimer’s disease, depression, and Parkinson’s disease. The goal of this study is to examine the relationship between sex hormones (i.e., androgens and estrogens) and oxidative stress on cell viability.MethodsN27 and PC12 neuronal and C6 glial phenotypic cell lines were used. N27 cells are female rat derived, whereas PC12 cells and C6 cells are male rat derived. These cells express estrogen receptors and the membrane-associated androgen receptor variant, AR45, but not the full-length androgen receptor. N27, PC12, and C6 cells were exposed to sex hormones either before or after an oxidative stressor to examine neuroprotective and neurotoxic properties, respectively. Estrogen receptor and androgen receptor inhibitors were used to determine the mechanisms mediating hormone-oxidative stress interactions on cell viability. Since the presence of AR45 in the human brain tissue was unknown, we examined the postmortem brain tissue from men and women for AR45 protein expression.ResultsNeither androgens nor estrogens were protective against subsequent oxidative stress insults in glial cells. However, these hormones exhibited neuroprotective properties in neuronal N27 and PC12 cells via the estrogen receptor. Interestingly, a window of opportunity exists for sex hormone neuroprotection, wherein temporary hormone deprivation blocked neuroprotection by sex hormones. However, if sex hormones are applied following an oxidative stressor, they exacerbated oxidative stress-induced cell loss in neuronal and glial cells.ConclusionsSex hormone action on cell viability is dependent on the cellular environment. In healthy neuronal cells, sex hormones are protective against oxidative stress insults via the estrogen receptor, regardless of sex chromosome complement (XX, XY). However, in unhealthy (e.g., high oxidative stress) cells, sex hormones exacerbated oxidative stress-induced cell loss, regardless of cell type or sex chromosome complement. The non-genomic AR45 receptor, which is present in humans, mediated androgen’s damaging effects, but it is unknown which receptor mediated estrogen’s damaging effects. These differential effects of sex hormones that are dependent on the cellular environment, receptor profile, and cell type may mediate the observed sex differences in oxidative stress-associated CNS disorders.

Pulmonary function and functional capacity in patients with mucopolysaccharidosis

Dysfunction of protein handling has been implicated in many neurodegenerative diseases and inhibition of the ubiquitin-proteasome system (UPS) has been linked to the formation of protein aggregates and proteinopathies in such diseases. While proteasomal inhibition could trigger an array of downstream protein handling changes including up-regulation of heat shock proteins (HSPs), induction of molecular chaperones, activation of the ER stress/unfolded protein response (UPR), autophagy and aggresome formation, little is known of the relationship of proteasomal inhibition to the sequence of activation of these diverse protein handling systems. In this study we utilized the reversible proteasome inhibitor MG132 and examined the activity of several major protein handling systems in the immortalized dopaminergic neuronal N27 cell line. In the early phase (up to 6 h after proteasomal inhibition), MG132 induced time-dependent proteasomal inhibition which resulted in stimulation of the UPR, increased autophagic flux and stimulated heat shock protein response as determined by increased levels of phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), C/EBP homologous protein (CHOP)/GADD153, turnover of autophagy related microtubule-associated protein 1 light chain 3 (LC3) and increased levels of Hsp70 respectively. After prolonged proteasomal inhibition induced by MG132, we observed the formation of vimentin-caged aggresome-like inclusion bodies. A recovery study after MG132-induced proteasomal inhibition indicated that the autophagy-lysosomal pathway participated in the clearance of aggresomes. Our data characterizes the relationship between proteasome inhibition and activation of other protein handling systems. These data also indicated that the induction of alternate protein handling systems and their temporal relationships may be important factors that determine the extent of accumulation of misfolded proteins in cells as a result of proteasome inhibition.