Neuronal Cell Culture Model Research Articles

Abstract 152: Development of a Chronic Angiotensin II (AngII)-stimulated Neuronal Cell Culture Model

Dysregulation of AngII-mediated intra-neuronal signaling contributes to the pathogenesis of hypertension. Previous studies using AngII-sensitive neuronal cell culture models suggest that AngII induces chronic changes in the expression of various cell signaling proteins, including its own receptors. However, many of these studies stimulated cultured neurons with a single administration of exogenous AngII and harvested neurons 3-24 hours later to investigate changes in mRNA and protein levels. One limitation of these “chronic” AngII stimulation studies is the lack of evidence indicating the stability of AngII in neuronal cell culture media. Here, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Further, we hypothesized that AngII receptors are differentially expressed depending on the chronicity of stimulation. AngII (100nM) was added to catecholaminergic (CATH.a) neurons in culture, media was collected 15 min - 24 hr later, and AngII levels were determined by liquid chromatography-tandem mass spectrometry. AngII was rapidly metabolized with media levels of AngII returning to near baseline within 3 hr of administration (baseline: 2.2 nM AngII; 15 min: 50.6 nM; 30 min: 42.9 nM; 1hr: 28.8 nM ; 3hr: 4.0 nM, n = 3-4). To begin investigating the relevance of this observation, we measured mRNA levels of AngII type 1 (AT1R) and type 2 (AT2R) receptors using real time quantitative RT-PCR in CATH.a neurons 24 hr after a single administration of AngII (100nM) or repeated administration every 3 hr. While a single administration of AngII did not alter AT1R mRNA levels, replenishing media every 3 hr with fresh AngII did induce a 20% decrease. In addition, fresh AngII every 3 hr resulted in a 41% decrease in AT2R mRNA levels; whereas, a single dose of AngII increased AT2R mRNA by 19%. These data indicate that AngII is quickly metabolized in neuronal cell culture media, and that supplementing fresh AngII every 3 hr to maintain elevated levels chronically yields different changes in AngII receptor mRNA levels as compared to a single administration. In conclusion, we suggest that to accurately model hypertension, where AngII is chronically elevated, the metabolism of AngII in a given cell culture model should first be determined.

Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna Delivery

Optimising non-viral vectors for neuronal siRNA delivery presents a significant challenge. Here, we investigate a co-formulation, consisting of two amphiphilic cyclodextrins (CDs), one cationic and the other PEGylated, which were blended together for siRNA delivery to a neuronal cell culture model. Co-formulated CD-siRNA complexes were characterised in terms of size, charge and morphology. Stability in salt and serum was also examined. Uptake was determined by flow cytometry and toxicity was measured by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Incorporation of a PEGylated CD in the formulation had significant effects on the physical and biological properties of CD.siRNA complexes. Co-formulated complexes exhibited a lower surface charge and greater stability in a high salt environment. However, the inclusion of the PEGylated CD also dramatically reduced gene silencing efficiency due to its effects on neuronal uptake. The co-formulation strategy for cationic and PEGylated CDs improved the stability of the CD.siRNA delivery systems, although knockdown efficiency was impaired. Future work will focus on the addition of targeting ligands to the co-formulated complexes to restore transfection capabilities.

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.

Angiotensin II (AngII) is rapidly metabolized in neuronal cell culture media as determined by liquid chromatography‐tandem mass spectrometry (LC MS/MS)

Previous reports using AngII‐sensitive neuronal cell culture models suggest that AngII induces chronic changes in various cell signaling proteins (e.g. kinases & transcription factors). Many of these studies stimulated cultured neurons with a single administration of exogenous AngII and harvested neurons 1–3 days later to investigate changes in protein levels. One limitation of such “chronic” AngII stimulation studies is the lack of evidence indicating the length of time exogenous AngII remains in neuronal cell culture media. Herein, using LC MS/MS, we tested the hypothesis that exogenous AngII is quickly metabolized to other angiotensin peptides in neuronal cell culture media. AngII (104.62pg/μl) was added to cultured CATH.a neurons and media was collected 15 min – 24 hr later for LC MS/MS analysis. Media levels of AngII rapidly and significantly decreased over time (52.8pg/μl AngII at 15 min; 4.2pg/ul at 3 hr; 0.37pg/μl at 24 hr, p<0.05, n = 3–4). At early time points (15 min – 3 hr), levels of AngIII and Ang1–7, two peptides generated from AngII, increased with a subsequent decrease at later times. Levels of AngI and AngIV did not significantly change. These studies provide new insight into the half‐life and metabolism of AngII in neuronal cell culture media and suggest previous studies using a single administration of AngII to study “chronic” AngII intra‐neuronal signaling pathways be reevaluated. NIH R01HL103942

Hydrogen peroxide (H2O2) is not increased in angiotensin II (AngII)‐stimulated neurons overexpressing superoxide dismutase (SOD)

Previous studies have shown that SOD overexpression in neurons inhibits AngII intra‐neuronal signaling; thus, suggesting that superoxide radicals (O2•−) are key signaling intermediates driving AngII‐induced neuronal activation. However, considering H2O2 is a direct product of O2•− dismutation by SOD, an alternative interpretation is that increased H2O2 mediates the inhibition of AngII signaling in SOD overexpressing neurons. We aimed to determine if H2O2 levels are indeed increased in SOD overexpressing neurons stimulated with AngII. To query this, we used a neuronal cell culture model expressing HyPer, a H2O2‐sensitive fluorescent protein. To determine the sensitivity of HyPer to H2O2, neurons were stimulated with increasing concentrations of H2O2 (1–100 μM) and changes in HyPer fluorescence were observed by real‐time confocal imaging. Robust increases in HyPer fluorescence were detected after treatment of ≥5 μM, but not after 1 μM H2O2. In contrast, no changes in HyPer fluorescence were observed after AngII (100 nM) stimulation in control or SOD overexpressing neurons. These data suggest that AngII does not increase H2O2 levels in SOD overexpressing neurons, or does so at concentrations below the level of HyPer detection. These data further support the hypothesis that SOD overexpression inhibits AngII intra‐neuronal signaling by decreasing O2•− levels, but not by increasing H2O2 levels. AHA 0930204N.

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury, lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.

Accelerated Fibril Formation of α-Synuclein by an IF-Inserted F36V Mutant

FK506 binding proteins (FKBPs) are molecular chaperones with the cis-trans peptidyl-prolyl isomerase (PPIase) activity, that are used to help nascent proteins fold correctly. 1 Some FKBPs, including FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, have been shown to possess PPIase activity, and have also exhibited the potential to modulate alpha-synuclein (α-Syn) aggregation, which plays a crucial role in Parkinson’s Disease (PD). 2 Among these, FKBP12 is the most potent for stimulating the aggregation of α-Syn, as has been demonstrated in vitro 3 as well as in a neuronal cell culture model. 4 These findings led us to develop better FKBP12-derived chaperones that can more effectively regulate the aggregation of α-Syn as targets for PD treatment. With an aim to construct a more powerful molecular chaperone with a higher PPIase activity, we engineered the F36V system, which is similar to that reported by the Schmid group. 5,6 In a previous study, we successfully constructed an F36VIF hybrid by replacing the flap of F36V with an insertion-in-flap(IF) domain from sensitive-to-lysis D (SlyD) protein of Escherichia coli (E. coli). 7 The IF that was inserted was the 61- amino acid domain starting with AYG and ending with LKF that protrudes from a loop of the FKBP domain near the PPIase catalytic site, which is indicative of its hydrophobicity. The F36V mutant imparted a better model to fit ligands via this protruding structural part than FKBP12. 8 By exploiting the structural contact between them, the Wandless group established a regulation system in which the stability of a target protein fused to some mutants of F36V depends on the presence of a small molecule, such as Shield-1; 9 when fused with a destabilizing domain (DD), the target protein is believed be degraded either via a proteasome pathway or by another process that has not yet been identified. Applying the cleaning-up concept that uses a DD domain to degrade pre-formed aggregated proteins, which are found in many neurodegenerative diseases (NDDs), we targeted the development of an IF-domain-inserted F36V system to regulate amyloid fibril formation. Using this system, we envision that the regulation of amyloid fibril formation could be switched on and off by a small molecule. In our previous study, we reported that F36VIF with the His6-tag gene was obtained by site-directed mutagenesis from FKBPIF/pET28a plasmid. 5 The resulting F36VIF protein was successfully expressed in BL21(DE3) in the presence of IPTG and further separated using Ni 2+ -affinity chromatography. The chymotrypsin-coupled assays 10 showed that the PPIase activity of F36VIF (202% of control) was remarkably higher than that of F36V itself (128% of control). 6 It is worth noting that the PPIaes activity of F36VIF

Altered localization, abnormal modification and loss of function of Sigma receptor-1 in amyotrophic lateral sclerosis

Intracellular accumulations of mutant, misfolded proteins are major pathological hallmarks of amyotrophic lateral sclerosis (ALS) and related disorders. Recently, mutations in Sigma receptor 1 (SigR1) have been found to cause a form of ALS and frontotemporal lobar degeneration (FTLD). Our goal was to pinpoint alterations and modifications of SigR1 in ALS and to determine how these changes contribute to the pathogenesis of ALS. In the present study, we found that levels of the SigR1 protein were reduced in lumbar ALS patient spinal cord. SigR1 was abnormally accumulated in enlarged C-terminals and endoplasmic reticulum (ER) structures of alpha motor neurons. These accumulations co-localized with the 20s proteasome subunit. SigR1 accumulations were also observed in SOD1 transgenic mice, cultured ALS-8 patient's fibroblasts with the P56S-VAPB mutation and in neuronal cell culture models. Along with the accumulation of SigR1 and several other proteins involved in protein quality control, severe disturbances in the unfolded protein response and impairment of protein degradation pathways were detected in the above-mentioned cell culture systems. Furthermore, shRNA knockdown of SigR1 lead to deranged calcium signaling and caused abnormalities in ER and Golgi structures in cultured NSC-34 cells. Finally, pharmacological activation of SigR1 induced the clearance of mutant protein aggregates in these cells. Our results support the notion that SigR1 is abnormally modified and contributes to the pathogenesis of ALS.

In vitro MS‐based proteomic analysis and absolute quantification of neuronal‐glial injury biomarkers in cell culture system

MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.

Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips

Tauopathies including Alzheimer’s disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer’s disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner.Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer’s disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy.Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates.

O3‐04‐03: Identification of a signal transduction mechanism of amyloid‐β/PrPC interaction

Recently, the cellular prion protein (PrPC) has been reported to be an essential co-factor in mediating the neurotoxic effect of oligomeric Aβ. However, the signaling mechanisms by which oligomeric Aβ induces neurotoxicity through PrPC remain elusive. In the previous study, the link between PrPC and NMDA receptor has been demonstrated and, indeed, we and others found that the inhibition of NMDA receptor can prevent neuronal cell death induced by Aβ. However, it is unclear whether PrPC is a key element for NMDA receptor activation by Aβ. Therefore, in this study, we investigated the role of PrPC in oligomeric Aβ-mediated NMDA receptor activation. We used neuronal cell culture models and organotypic hippocampal slice culture to test the physical interaction between PrPC and NMDA receptor and to measure NMDA receptor activity regulated by PrPC/Aβ oligomer interaction. We found that oligomeric Aβ activated NMDA receptor subunit 2B (NR2B) which was mediated by Fyn. In addition, we confirmed that PrPC, Fyn and NR2B are physically bound to each other and form the signal transduction complex for Aβ. Here, we report that the molecular interaction among PrPC, Fyn and NR2B is critical in oligomeric Aβ-induced neurotoxic signaling through PrPC. As the activation of NMDA receptor has been linked to neurodegeneration and aberrant processing of amyloid-β precursor proteins, the signal transduction mechanism identified in this study might provide an important connection to the neurodegeneration, a characteristic of disease including Alzheimer disease.

P4‐032: Investigating the downstream targets of memantine in primary neuronal cell culture models

P4‐032: Investigating the downstream targets of memantine in primary neuronal cell culture models

Dysequilibrium of neuronal proliferation and apoptosis in a pharmacological animal model of psychosis

Growing evidence implicates that abnormal stem cell proliferation and neurodegenerative mechanisms may be involved in the pathogenesis of neuropsychiatric disorders including schizophrenia. Here, we studied the underlying pathomechanisms of psychosis. We are employing a translational approach combining in vivo data with supplementary data from an adult neuronal stem cell-derived cell culture model by generating a large number of analytes in our specimens following a multiplexing strategy. In the animal model the NMDA receptor was chronically antagonized by MK-801 at ultralow doses. As a result of this, we were able to demonstrate a roughly twofold increased density of PCNA positive cells in the germinal zone of the dentate gyrus indicating enhanced neuroproliferative activity. In vitro stem cell experiments additionally pointed to this direction showing an increase both in proliferation and neuronal differentiation after MK-801 treatment. These alterations were partially prevented by coapplication of the dopamine receptor antagonist haloperidol. In addition, apoptotic activity assessed by immunohistochemical demonstration of cleaved caspase-3 stainings was unaffected by MK-801 treatment. These observations were largely supported by microarray gene expression analysis, which permits high-throughput multiplexed assessment of expression data from a comprehensive set of genes and showed parallels with data from human post mortem studies. In conclusion, our data support the notion, that abnormal proliferation due to anti-apoptotic mechanisms may represent a factor in the pathogenesis of psychosis. Thus, research on the exact interplay between glutamatergic neurotransmission and neuronal proliferation deserves more attention. This dual in vivo and in vitro strategy described here may prove as a suitable model for addressing complex neuropsychiatric diseases especially when taking advantage of the potential of multiplex technologies not only in diagnostics but also in basic research.

Early induction of c-Myc is associated with neuronal cell death

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases.

Comparative Analysis of Different Peptidyl-Prolyl Isomerases Reveals FK506-binding Protein 12 as the Most Potent Enhancer of α-Synuclein Aggregation

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.

Rotenone and Paraquat Linked to Parkinson’s Disease: Human Exposure Study Supports Years of Animal Studies

A growing body of evidence suggests pesticides may play a role in Parkinson’s disease (PD) in humans. Self-reported PD has been associated with lifetime use of pesticides, and animal studies have suggested that the pesticides paraquat and rotenone can cause oxidative stress and mitochondrial dysfunction, respectively—posited mechanisms of action in PD—as well as symptoms in rodents similar to human PD. Now, researchers have linked human exposure to paraquat and rotenone with PD [EHP 119(6):866–872; Tanner et al.]. Their study is the first analysis of pesticides classified by presumed mechanism of action rather than by intended use or chemical class. The researchers assessed lifetime use of pesticides as reported by participants in the Agricultural Health Study, a prospective study of private pesticide applicators (mostly farmers) and their spouses in Iowa and North Carolina. In the nested case–control study, neurologists specializing in movement disorders identified 110 people with PD and 358 controls without who were frequency matched by age, sex, and state. The researchers collected detailed information about use of 31 different pesticides as well as covariate information including lifelong smoking and family history of PD. Each pesticide was classified according to its mechanism of action as either an oxidative stressor or a mitochondrial complex I inhibitor. For participants with PD, the researchers considered pesticide use that occurred before their PD diagnosis. For controls, they considered pesticide use that occurred before the median age of PD diagnosis for cases in the corresponding age-, sex-, and state-specific groups. Among the mitochondrial complex I inhibitors studied, the researchers found the strongest association between PD and use of rotenone. Among oxidative stressors, they found the strongest association between PD and use of paraquat. Participants with PD were 2.5 times more likely than controls to have reported use of rotenone or paraquat. The results may have far-reaching implications, considering the widespread use of these pesticides. Paraquat remains one of the most widely used herbicides worldwide, and rotenone was used ubiquitously before most uses were voluntarily stopped in the United States in 2007. Other agents associated with mitochondrial complex I inhibition, such as permethrin, remain in common use. The authors point out several limitations of the study, including the fact that participants reported exposure to many pesticides, and effects of other agents can’t be excluded. However, the associations between PD and paraquat and rotenone remained after adjustment for overall pesticide use. It also was impossible to measure pesticide exposure, only estimate it retrospectively. Future investigations of combinations of pesticides and of other mechanistic groups will be important, as will mechanistic studies that model exposure conditions similar to those occurring in humans.

Neuroprotective Effect of Resveratrol Against Methamphetamine-Induced Dopaminergic Apoptotic Cell Death in a Cell Culture Model of Neurotoxicity

A growing body of evidence suggests that oxidative stress-mediated cell death signaling mechanisms may exert neurotoxic effects of methamphetamine (MA)-induced dopaminergic neuronal loss. However, the means by which oxidative stress induced by MA causes neurodegeneration remains unclear. In recent years, resveratrol has garnered considerable attention owing to its antioxidant, anti-inflammatory, anti-aging, and neuroprotective properties. In the present study, we sought to investigate the neuroprotective effects of resveratrol against apoptotic cell death in a mesencephalic dopaminergic neuronal cell culture model of MA neurotoxicity. MA treatment in the N27 dopaminergic neuronal cell model produced a time-dependent activation of the apoptotic cascade involving caspase-3 and DNA fragmentation. We found that the caspase-3 activation preceded DNA fragmentation. Notably, treatment with resveratrol almost completely attenuated MA-induced caspase-3 activity, but only partially reduced apoptotic cell death. We conclude that the neuroprotective effect of resveratrol is at least in part mediated by suppression of caspase-3 dependent cell death pathways. Collectively, our results demonstrate that resveratrol can attenuate MA-induced apoptotic cell death and suggest that resveratrol or its analogs may have therapeutic benefits in mitigating MA-induced dopaminergic neurodegeneration.

Neuroprotective and Neurorescue Effects of a Novel Polymeric Nanoparticle Formulation of Curcumin (NanoCurc™) in the Neuronal Cell Culture and Animal Model: Implications for Alzheimer's disease

Alzheimer's disease (AD) is characterized by deposition of amyloid-β (Aβ) plaques within the brain parenchyma followed by synaptic loss and neuronal death. Deposited Aβ reacts with activated microglia to produce reactive oxygen species (ROS) and cytochemokines, which lead to severe neuroinflammation. Curcumin is a yellow polyphenol compound found in turmeric, a widely used culinary ingredient that possesses anti-inflammatory and anti-cancer properties and may show efficacy as a potential therapeutic agent in several neuro-inflammatory diseases including AD. However, poor aqueous solubility and sub-optimal systemic absorption from the gastrointestinal tract may represent factors contributing to its failure in clinical trials. To increase curcumin's bioavailability, a polymeric nanoparticle encapsulated curcumin (NanoCurc™) was formulated which is completely water soluble. NanoCurc™ treatment protects neuronally differentiated human SK-N-SH cells from ROS (H2O2) mediated insults. NanoCurc™ also rescues differentiated human SK-N-SH cells, which were previously insulted with H2O2. In vivo, intraperitoneal (IP) NanoCurc™ injection at a dose of 25mg/kg twice daily in athymic mice resulted in significant curcumin levels in the brain (0.32 μg/g). Biochemical study of NanoCurc™-treated athymic mice revealed decreased levels of H2O2 as well as caspase 3 and caspase 7 activities in the brain, accompanied by increased glutathione (GSH) concentrations. Increased free to oxidized glutathione (GSH:GSSH) ratio in athymic mice brain versus controls also indicated a favorable redox intracellular environment. Taken together, these results suggest that NanoCurc™ represents an optimized formulation worthy of assessing the therapeutic value of curcumin in AD.

Cytoprotective effects of the volatile anesthetic sevoflurane are highly dependent on timing and duration of sevoflurane conditioning: Findings from a human, in-vitro hypoxia model

Using animal models, volatile anesthetics have been recognized for their neuroprotective effects. Nevertheless, there is still disagreement about the optimal duration and timing of conditioning with the volatile anesthetic sevoflurane in the human system. In the study presented, we employed a human neuronal cell culture model to investigate the effects of hypoxia and to evaluate potential cytoprotective properties of different sevoflurane conditioning strategies. Sevoflurane was applied to human IMR-32 cells in which hypoxic conditions were induced for 2 h using our recently described two-enzyme model (Zitta et al., Eur. J. Pharmacol., 2010). Cellular effects of hypoxia and sevoflurane conditioning were evaluated by lactate dehydrogenase (LDH) measurements, brightfield microscopy, ELISAs, cytometric bead arrays, Westernblotting and RT-PCR. Hypoxia increased the release of LDH into the culture medium after 24 h (normoxia: 0.15 ± 0.02 a.u; hypoxia: 0.69 ± 0.08 a.u, P < 0.001) and expression of hypoxia associated genes HIF-1alpha, VEGF, catalase. Cytoprotective effects were observed in cultures that received sevoflurane for 30 min before hypoxia (preconditioning: 0.41 ± 0.07 a.u., P < 0.01) and for 30 min during the hypoxic period (intraconditioning: 0.20 ± 0.01 a.u., P < 0.001). Application of sevoflurane after the hypoxic insult did not lead to cytoprotection (postconditioning: 0.73 ± 0.12 a.u., P > 0.05). Conditioning with sevoflurane for a total of 3 h before, during and after hypoxia, however, resulted in an enhanced release of LDH (periconditioning: 0.97 ± 0.10 a.u., P < 0.01) and additional cell damage. Hypoxia and sevoflurane intraconditioning were associated with changes in erk1/2 phosphorylation (T202/Y204) and HIF-1alpha protein levels, whereas phosphorylation of akt (S473) was not significantly altered. Our results suggest short pre- and intraconditioning with sevoflurane as most potent strategies to reduce hypoxia induced neuronal cell damage.

Alpha-Synuclein Loss in Spinal Muscular Atrophy

Spinal muscular atrophy, the most prevalent hereditary motor neuron disease, is caused by mutations in the survival motor neuron (SMN) 1 gene. A significant reduction in the encoded SMN protein leads to the degeneration of motor neurons. However, the molecular events leading to this process are not well understood. The present study uses a previously developed neuronal cell culture model of spinal muscular atrophy for a multiplex transcriptome analysis. Furthermore, gene expression analysis was performed on in vitro cell cultures, as well as tissue samples of spinal muscular atrophy patients and transgenic mice. RNA and subsequent Western blot protein analyses suggest that low SMN levels are associated with significantly lower alpha-synuclein expression. Examination of two genes related to vesicular transport showed a similar though less dramatic decrease in expression. The 140-amino acid protein alpha-synuclein, dominant mutations of which have previously been associated with an autosomal dominant form of Parkinson's disease, is strongly expressed in select neurons of the brain. Although not well understood, the physiologic functions of alpha-synuclein have been linked to synaptic vesicular neurotransmitter release and neuroprotection, suggesting a possible contribution to Smn-deficient motor neuron pathology. Furthermore, alpha-synuclein may be a genetic modifier or biomarker of spinal muscular atrophy.