Neuroscience

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OPTIMIZATION OF TWO METHODS FOR ASSESSING CELL VIABILITY AND CYTOPROTECTION IN C6 ASTROCYTES
(2014-03) Kubelka, Nicholas K.; Rybalchenko, Nataliya; Singh, Meharvan
Purpose: The purpose of this study was to determine the best method for measuring cell viability in the rat C6 astrocyte cell model in response to two cytotoxic insults, hydrogen peroxide (H2O2) and iodoacetic acid (IAA). Methods: Two assays were evaluated: calcein-AM assay for detecting live cell number, and a flow cytometry-based assay to assess live versus dead populations. Cells were treated with 0, 10, 20 or 50 μM H2O2 (2 hours) or IAA (3 hours). The calcein-AM assay was evaluated in a 96-well plate format. Flow cytometry results were obtained using a C6 Accuri model Flow Cytometer, and cells were stained with both calcein-AM (peak emission range 485-535 nm, fluorescence channel 1 (FL1)) and ethidium homodimer (peak emission range 610-30 nm, fluorescence channel 3 (FL3)). Results: Treated cells displayed a concentration dependent decrease in cell viability (calculated EC50 = 18μM for H2O2, 23 μM for IAA). Cells treated for 3 hours with IAA showed a concentration dependent transition from high FL1, low FL3 fluorescence to low FL1, high FL3 fluorescence, indicating a transition from high to low viability. Conclusions: This study describes two methods for cell viability detection: calcein fluorescence by high-throughput analysis and simultaneous calcein and ethidium homodimer staining for flow cytometry cell gating for individual cell analysis. Having successfully utilized both the 96-well assay and the flow cytometry protocols to assess cell viability, we plan to extend the current studies by assessing how brain-active steroids protect glia from insults relevant to brain aging and certain neurodegenerative diseases.
INDUCTION OF APOPTOSIS VIA TESTOSTERONE IN OXIDATIVELY DAMAGED DOPAMINERGIC CELLS
(2014-03) Simmons, Olivia C.; Holmes, Shaletha S.; Cunningham, Rebecca
Following an episode of stroke, reduction of blood supply to brain cells can lead to conditions of oxidative stress (OS) in brain cells, or neurons, specifically in the dopaminergic neurons. The loss of dopaminergic neurons manifests itself as Parkinson’s disease (PD). Classically, men have a higher incidence of developing PD post-stroke than females. This suggests a role of testosterone (T) in the development of PD after induction of OS in neurons. We postulate that T in OS-induced states will activate the pathway of apoptosis, or programmed cell death, via an enzyme called caspase to induce death of dopaminergic brain cells, and thus symptoms of PD. To test our hypothesis, we first pre-treated dopaminergic cell lines with hydrogen peroxide (H2O2) to simulate stroke-induced OS. The cells were then treated with differing concentrations of T (0, 1, 10, 100 nM), representing the physiologic ranges of T in humans. Expressions of pro-caspase-3 and pro-caspase-9, the uncleaved precursors to caspase-3 & caspase-9, respectively, in the cells were quantified using Western Blot analysis. Statistical significance of our findings was reported using ANOVA and Fisher’s post hoc analysis with SAS software. Our experiments showed a trend of decreased expression of pro-caspase-9, and a significant decrease in pro-caspase-3 expression in the H+T treatment conditions as compared to the control conditions. These results point to the apoptotic cell death pathway via caspase-3 and caspase-9 as the mechanism by which increased T levels lead to PD in stroke patients. Purpose (a): Following ischemic stroke, reduction of blood supply to brain cells can lead to conditions of oxidative stress (OS) in neurons, specifically in the dopaminergic neurons of the substantia nigra (SN). The loss of dopaminergic neurons manifests itself as Parkinson’s disease (PD). Classically, men have a higher incidence of developing PD post-stroke than females. This suggests a role of testosterone (T) in the development of PD after induction of OS in neurons. We postulate that T in OS-induced states will activate the caspase pathway of apoptosis to induce dopaminergic cell death, and thus symptoms of PD. Methods (b): To test our hypothesis, we first pre-treated N27 dopaminergic cell lines with hydrogen peroxide (H2O2) to simulate stroke-induced OS. The cells were then treated with differing concentrations of T (0, 1, 10, 100 nM), representing the physiologic ranges of T in humans. Expressions of pro-caspase-3 and pro-caspase-9, the uncleaved precursors to caspase-3 & caspase-9, respectively, in the cells were quantified using Western Blot analysis. Statistical significance of our findings was reported using ANOVA and Fisher’s post hoc analysis with SAS software and p <0.05 as significant. Results (c): Our experiments showed a trend of decreased expression of pro-caspase-9, and a significant decrease in pro-caspase-3 expression in the H+T treatment conditions as compared to the control conditions. Conclusions (d): These results point to the apoptotic pathway via caspase-3 and caspase-9 as the mechanism by which increased T levels lead to PD in stroke patients.
TUMOR NECROSIS FACTOR-α CONFERS CYTOTOXICITY IN ASTROCYTES UNDER OXIDATIVE STRESS VIA INHIBITION OF NF-κB SIGNALING
(2014-03) Singhal, Ashutosh; Ghorpade, Anuja
Oxidative stress and inflammation together recognized as central feature of stroke and other neurodisorders. In acute ischemic stroke, formation of H2O2 causes brain injury, which appear to be exacerbated by inflammation such as IL-1β or TNF-α produced after reperfusion. However, evidences also show that TNF-α helps in recovery and repair. Therefore, role of TNF-α is unclear. Further, it is unknown how our brain cells i.e. astrocytes are affected when oxidative stress and inflammation coexist. Here we examined the effects of H2O2 on cell survival in cultured human astrocytes co-stimulated with TNF-α or IL-1β. Data showed H2O2-treatment significantly increased astrocytes death; however, IL-1β or TNF-α-alone did not. Interestingly, co-treatment of TNF-α, but not IL-1β with non-toxic dose of H2O2 significantly increased astrocyte cell death. The toxicity of co-treatment of TNF-α and H2O2 was significantly higher than respective dose of H2O2-alone. Investigations of mechanisms revealed that H2O2 inhibited TNF-α-induced translocation of a transcription factor NF-κB to the nucleus in astrocytes thereby inhibiting cellular defense and/or survival pathways. H2O2 also decreased other TNF-a receptor associated proteins, RIP1, IκB kinases activation, thereby inhibited IκB-α degradation and NF-κB nuclear translocation. This supports the evidence of H2O2 as a modulator of pro-inflammatory signaling and explains the increased sensitivity of astrocytes during brain injury. These data also signify need to design strategy to combat oxidative stress during neuroinflammation and repair. Purpose (a): Oxidative stress and inflammation together recognized as central feature of both acute and chronic neurological disorders. In acute ischemic stroke formation of H2O2 causes brain injury, which appear to be exacerbated by IL-1β or TNF-α produced after reperfusion. However, evidences also show that TNF-α helps in recovery and repair. Therefore, role of TNF-α is unclear. Further, it is unknown how astrocytes are affected when oxidative stress and inflammation coexist. Methods (b): Here we examined the effects of H2O2 on cell survival and NF-kB dynamics in cultured human astrocytes co-stimulated with TNF-α or IL-1β. Results (c): Data showed H2O2-treatment significantly increased apoptosis in astrocytes in dose-dependent manner; however, IL-1β or TNF-α-alone did not. Interestingly, co-treatment of TNF-α, but not IL-1β with non-toxic dose of H2O2 significantly increased apoptosis in astrocytes. The toxicity of co-treatment of TNF-α and H2O2 was significantly higher than respective dose of H2O2-alone. Investigations of mechanisms revealed that H2O2 inhibited TNF-α-induced translocation of NF-kB to the nucleus in astrocytes thereby inhibiting cellular defense and/or survival pathways. H2O2 decreased TNFR1 associated protein, RIP1 level, necessary for IkB kinases activation, thereby inhibited IkB-a degradation and NF-kB nuclear translocation. The real time PCR analysis of oxidative stress pathway showed H2O2 decrease of antioxidant machinery of astrocytes. Investigation of apoptosis pathway showed that H2O2 increased the expression of TRAILR1/R2, Fas and FADD, which lead TNF-α-induced caspase-dependent apoptosis. Conclusions (d): This study supports the evidence of H2O2 as a modulator of pro-inflammatory signaling and explains the increased sensitivity of astrocytes during brain injury. These data also signify need to design strategy to combat oxidative stress during neuroinflammation and repair.
THE SIGMA 1 RECEPTOR SELECTIVE LIGAND LS-1-1-137 ATTENUATES THE 2,5-DIMETHOXYIODOAMPHETAMINE - INDUCED HEAD TWITCH RESPONSE.
(2014-03) Malik, Maninder; Rangel-Barajas, Claudia; Mach, Robert; Luedtke, Robert R.
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) is known to cause hallucinations in humans and the administration of DOI in rodents has been reported to induce head twitches (Canal and Morgan 2012). DOI has been proposed as an animal model to identify drugs for the treatment of Tourette syndrome (TS) and other neuropsychiatric disorders. In this study, we used DOI-induced head twitch response (HTR) as our experimental model to screen antipsychotic properties of novel compounds. Our study results indicate that LS-1-137 may represent a novel antipsychotic drug. Purpose (a): 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) is known to cause hallucinations in humans and the administration of DOI in rodents has been reported to induce head twitches (Canal and Morgan 2012). DOI has been proposed as an animal model to identify drugs for the treatment of Tourette syndrome (TS) and other neuropsychiatric disorders. The exact mechanisms leading to head twitches in mice remain elusive. Several receptors, neural pathways and intracellular proteins have been implicated in the modulation of the HTR. However, no studies have investigated a possible role of sigma receptors in DOI-dependent HTR in mice. In this study we report the possible involvement of the sigma 1 receptor in the murine DOI dependent HTR in male DBA/2J mice and the ability of the novel sigma 1 vs. sigma 2 receptor selective compound, LS-1-137, to modulate that response. Methods (b): A filtration-binding assay was used to characterize the binding properties of novel sigma compound at D2 like dopamine, muscarinic, serotonin 2A and 2C and at sigma receptors. In this study, male DBA/2J mice were used. On the day of testing, mice were weighed and placed individually in an open ended Plexiglas cylinder with a clean paper towel floor, in a dimly lighted room. Animals were allowed to habituate to the cylinder for 15 minutes prior to the injection of vehicle (5% DMSO in sterile deionized water) or test drug intraperitoneal (i.p.). Five minutes later the DOI was administered to the animal by i.p. injection and the mouse was returned to the cylinder. A head twitch response was defined as a rapid left to right (or right to left) movement of the head, without the involvement of the limbs. Two observers counted the number of head twitches by visual examination and the number of head twitches was recorded at 5-minute intervals. The data points are presented as the mean values obtained by two observers. To evaluate the drug's effect on motor performance and coordination, a Rotarod test was performed. Results (c): LS-1-137 exhibits high affinity binding at sigma 1 receptors (Ki = 3.2 nM) and is 80-fold selective at sigma 1 versus sigma 2 receptors. It also binds with low affinity at D2-like (D2, D3 and D4) dopamine, muscarinic and serotonin (2A and 2C) receptors. In the present studies RHM-1-86, a sigma 2 receptor selective antagonist, was not able to attenuate the HTR. The sigma 1 receptor agonists PRE-084 and PPCC were also not able to inhibit the HTR. However, LS-1-137 and the sigma 1 antagonists haloperidol and BD1047 significantly inhibited the DOI-dependent HTR in DBA/2J mice. Furthermore, rotarod studies indicate that LS-1-137 does not compromise agility or muscular coordination in DBA/2J mice within a dose range capable of attenuating the effects of DOI. Conclusions (d): These observations implicate sigma 1 receptors in mediating the DOI-dependent HTR and suggest that LS-1-137 may have antipsychotic properties in vivo.
CHARACTERIZATION OF COCAINE-CONDITIONED LOCOMOTOR RESPONSES BY MODULATION OF ENVIRONMENTAL CONTEXT AND NEURAL PLASTICITY-SIGNALING PATHWAYS
(2014-03) Nguyen, Jacques D.; Forster, Michael J.
Rodent models are commonly used for the study of substance abuse and addiction. The objective of this study was to characterize the cocaine-conditioned locomotor response, a behavioral phenomenon observed in mice, following an acute injection of cocaine, and to determine its mechanism of action for potential therapeutic targeting. Compounds known to modulate neural plasticity were evaluated for their ability to affect the acquisition and expression of the conditioned behavior. Purpose (a): In rodents, increase in locomotion is a hallmark effect of psychostimulant exposure and conditioning that is associated with activation of mesocorticolimbic dopamine signals mediating reinforcing/rewarding actions. The objective of this study was to characterize the cocaine-conditioned locomotor response following an acute injection of cocaine, specifically the modulating roles of environmental context and plasticity-associated signals. Methods (b): Cocaine (40mg/kg) was administered to different groups of Swiss-Webster, C57Bl/6, or DBA2 mice via intraperitoneal injection (i.p.), in either a locomotor activity testing apparatus or the home cage, 2 hours following an activity test under saline. Mice placed in the testing chambers were given 30 minutes to explore freely and locomotion was monitored using a Digiscan photocell apparatus. A conditioned effect of cocaine was inferred by an increase in horizontal activity counts relative to home cage cocaine controls during a test in the same apparatus on the following day. Compounds known to modulate neural plasticity-associated signaling cascades were evaluated for their ability to affect the acquisition and expression of cocaine-conditioned locomotor response, using a two-day protocol. Mice were administered haloperidol (0.05-1 mg/kg), dizocilpine (0.01-0.25mg/kg), nifedipine (0.1-10 mg/kg), cycloheximide (2.5-10mg/kg), or vehicle, prior to placement into the activity chambers on the test day for expression or prior to acquisition day. Results (c): Haloperidol (0.25-1 mg/kg) inhibited expression of the cocaine-conditioned locomotion, though failed to alter acquisition of the behavioral response. Dizocilpine (0.05-0.25 mg/kg) attenuated acquisition and exacerbated expression. Nifedipine had no effect on the conditioned locomotor response. Cycloheximide (2.5-10 mg/kg) attenuated acquisition of the conditioned response. Conclusions (d): These findings suggest that plasticity-dependent signaling pathways mediate associations of context following acute cocaine exposure and are necessary for the acquisition and expression of the cocaine-conditioned locomotor response.
USE OF RECOMBINANT TISSUE PLASMA ACTIVATOR (RT-PA) IN SILENT AORTIC DISSECTION PRESENTING AS AN ISCHEMIC STROKE
(2014-03) Cheung, Ryan J.; Mantilla, Emmanuel C. Jr.; Smith-Barbaro, Peggy
The use of thrombolytic therapy in the management of acute ischemic stroke has increased since the National Institute of Neurological Disorders and Stroke (NINDS) established its efficacy in improving clinical outcome at three months in patients when treated within a three-hour window. However, a controversy exists in its use in patients with aortic dissection presenting with neurologic symptoms, mimicking an acute ischemic type of stroke, due to risk for further hemorrhage and interference with hemostasis. This is a case of a 60-year-old female who presented with an acute left sided hemiplegia, and was treated with recombinant tissue plasma activator (rt-PA) after inclusion criteria was met. MRI showed moderate embolic left middle cerebral artery infarct and a computed tomography angiography (CTA) performed post thrombolytic therapy showed a Stanford type A aortic dissection. Following rt-PA administration, the patient improved neurologically the next day, and underwent surgery for repair of the dissection. Despite the neurologic and clinical improvement after thrombolysis, the patient was not able to tolerate the surgery and expired a day after the operation. Nonetheless, this case still provides evidence that rt-PA is an appropriate an effective treatment in the case of an ischemic stroke secondary to an aortic dissection. Purpose (a): This is a case study demonstrating the use of recombinant tissue plasma activator (rt-PA) in the treatment of an ischemic stroke secondary to a silent aortic dissection. In light of recent studies on clinical outcomes, there is debate about the use of rt-PA to treat a questionable stroke with a high suspicion for aortic dissection. This purpose of this case is to show that such time-sensitive, potentially life-saving treatment can be delivered without any negative side effects towards an aortic dissection such as intracranial bleeding or an aortic rupture. This case provides further evidence that rt-PA should not be delayed as the clinical benefits of reducing stroke morbidity and mortality outweighs the potential risks. Methods (b): This case describes a 60 year old patient presenting to the emergency department with an ischemic stroke and treated with rt-PA. The patient initially presented with right sided gaze preference and left-sided hemiplegia. In further reassessing the patient, a Stanford Type A aortic dissection with right carotid artery involvement was discovered upon CTA. The decision was made to attempt a surgical repair of the dissection. During the surgery, the vessel defect was corrected, but the patient sustained right heart failure refractory to vasopressors. Following surgery, the patient was transferred to the ICU, intubated, and in critical condition. Despite being maintained on heparin, a repeat head CT showed no transformation from an ischemic to a hemorrhagic stroke. The patient died two days later due to complications from the surgery. Results (c): During the initial assessment of the ischemic stroke, no signs of hemorrhage were present so the decision to give rt-PA was made. Following administration of rt-PA, the patient subsequently improved; there were no gaze asymmetry and left sided movements were noted. The patient did not show any signs of intracranial bleeding with rt-PA and heparin therapy. It is also important to note that administration of rt-PA did not appear to worsen the aortic dissection. Conclusions (d): The results of this case study suggest that rt-PA, when indicated, is an appropriate and effective treatment in the case of ischemic stroke secondary to an aortic dissection. However, surgical corrections of aortic dissection in these cases do carry a high mortality rate, so clinical judgment must be carefully exercised for each individual case.
METHYLENE BLUE PROTECTS ASTROCYTES FROM HYPOXIA-REOXYGENATION INJURY BY IMPROVING CELLULAR BIOENERGETICS
(2014-03) Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yang, Shaohua
In ischemic stroke, which is caused by a clot that blocks a brain blood vessel, a portion of the brain is deprived of oxygen and nutrients that are carried in the blood. When this clot is removed either by time or drugs, the affected region is flooded with an overabundance of nutrients and oxygen to a degree that is larger than the cells in the region can process and this unbalances the system and causes further damage. In our current study, we are testing a chemical compound called Methylene Blue to see if it can improve the cells’ ability to handle this excess of nutrients and oxygen available to them at the time of clot dissolution and thereby improve their survival after ischemic stroke. Purpose (a): Ischemic Stroke inflicts a double blow to the affected brain region by characteristically presenting a period of acute ischemia during which the cells are completely deprived of valuable nutrients (Glucose & oxygen) resulting in cell death, however as the blood flow is restored (Spontaneously/surgically) the surviving cells are exposed to an overwhelming levels of glucose and oxygen resulting in reperfusion injury which further aggravates the cellular injury inflicted by ischemia. Methylene Blue (MB) is a heterocyclic aromatic compound shown to function as an alternative electron carrier and improve glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen in the brain. In our current study we aim to delineate if MB is protective in astrocyte against hypoxia-reoxygenation injury and determine its underlying mechanism. Methods (b): Primary astrocytes cultures isolated from day old C57BL6 were used in the current study. Protective role of Methylene blue (MB) in primary astrocyte cultures was evaluated in an in vitro model of cellular hypoxia (0.1% O2, 6h) and re-oxygenation (24h). The effect of MB on glucose uptake was determined by using the 2-NBDG assay. Oxygen sensitive dye Tris (2,2′-bipyridyl) dichloro Ruthenium(II) hexahydrate and Fluorescence Life Time Imaging (FLTI) was used to determine the effect of MB on intracellular oxygen concentration. ATP assay was used to determine the effect of MB on cellular energy status. Results (c): Results from cell viability assay showed that MB treatment significantly protected astrocytes from hypoxia-reoxygenation induced cell death. MB treatment significantly increased cellular glucose uptake in primary astrocyte cultures. FLTI showed that MB significantly increased intracellular oxygen concentration in primary astrocytes. Astrocytes treated with MB also had significantly higher ATP concentration compared to non-treated cells. Conclusions (d): Methylene Blue protects astrocytes against hypoxia-reoxygenation injury by improving astrocyte bioenergetics.
ROLE OF ALCOHOL IN PRIMARY HUMAN ASTROCYTES
(2014-03) Pandey, Richa; Ghorpade, Anuja
Purpose (a): The aim of this study was to investigate alcohol-mediated activation of human astrocytes and subsquential alterations in their inflammatory functions. Methods (b): Primary human astrocytes were incubated with or without alcohol at doses of 25, 50, 100 mM for 3, 5, and 7 days. Cells and culture supernatants were collected. Astrocyte morphology was examined by immunocytochemical staining for glial fibrillary acidic protein (GFAP). Expressions of TIMP-1 and pro-inflammatory cytokines including CXCL8 and CCL2 were measured by ELISA. Cell metabolic activity, proliferation, and apoptosis were analyzed by MTT assay, BrdU cell proliferation assay, and Cell Death Detection ELISAplus assay, respectively. Results (c): Alcohol exposure altered the morphology of astrocytes to a reactive phenotype as determined by GFAP immunostaining. Alcohol significantly upregulated TIMP-1 levels in dose-dependent manner (P< 0.05), with a peak at day 5 post-treatment. Moreover, alcohol treatment significantly upregulated CCL2 (P< 0.05) while CXCL8 was significantly downregulated in a dose as well as time-dependent manner (P< 0.05). In addition, alcohol exposure significantly decreased astrocytes viability (P< 0.05) and proliferation as measured by MTT and BrdU incorporation assay respectively, and significantly increased apoptosis (P< 0.05) in parallel experiments. Conclusions (d): In summary, our results suggest that alcohol may alter astrocyte inflammatory mediators and/or regulate astrocyte functions.
METHYLENE BLUE INHIBITS PROLIFERATION AND MAINTAINS SELF-RENEWAL OF RAT NEURAL STEM/PROGENITOR CELLS
(2014-03) Xie, Luokun; Roy Choudhury, Gourav; Park, Yong; Liu, Ran; Zhang, Chun-Li; Yorio, Thomas; Jin, Kunlin; Yang, Shaohua
Neural progenitor cells (NPCs)are important for neurogenesis and brain damage repair. To elucidate the effect of methylene blue on NPCs, we studied the self-renewal and proliferation of NPCs both in vitro and in vivo. We found that methylene blue restrains NPC proliferation and enahnces NPC self-renewal. Purpose (a): Neural stem cell-based treatment holds a new therapeutic opportunity for treating neurodegenerative disorders. While methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models, its potential effects on neural stem/progenitor cells (NSPCs) has not been addressed. Methylene blue can easily penetrate the blood brain barrier to access the brain parenchyma. Thus, its effects on NSPCs, whether positive or negative, need to be elucidated. Methods (b): We used in both in vitro culture model and in vivo study to test the effects of methylene blue on the proliferation, self-renewal and differentiation of NSPCs. Neurospheres were generated in vitro and were treated with methylene blue. NSPC proliferation was evaluated by Ki67 staining and propidium iodide staining. NSPC self-renewal was determined by serial passage assay. Real-time PCR was applied to test the expression of neural differentiation markers in NSPCs. To address the signal pathway responsible for the methylene blue-induced changes on NSPCs, expression of cyclins and mTORC1 activation were determined by real-time PCR and Western blot, respectively. The effects of methylene blue on NSPC proliferation was also confirmed by intracerebroventricular infusion of methylene blue in rats followed by BrdU and Nestin staining. Results (c): Methylene blue inhibits porliferation of rat NSPCs in the in vitro culture. Methylene blue treatment decreased most cyclin expression. In addition, methylene blue enhanced the self-renewal capacity of NSPCs, demonstrated by more neurosphere growth and inhibited differentiation marker expression in NSPCs. However, methylene blue did not impair committed neuronal differentiation. The change of cyclin expression is associated with the change of mTOR expression in methylene blue-treated NSPCs. Methylene blue repressed transcription of mTOR rather than enhance mTOR degradation. Consistent with in vitro data, methylene blue inhibited neural stem cell division in the subventricular zone, but did not influenced neuronal development in a short term. Conclusions (d): Our findings indicate that methylene blue could delay NSPCs senescence by enhancing NSPCs self-renewal capacity. However, the long-term effects of methylene blue on the in vivo NSPC pool needs further investigation. The impact of methylene blue on NSPCs should be taken into account in future therapy with methylene blue, either for the peripheral diseases or for the CNS disorders.
OVEREXPRESSION OF THE POU DOMAIN TRANSCRIPTION FACTOR, BRN3B CAUSES NEURITE OUTGROWTH IN CULTURED PC 12 CELLS UNDER CONDITION OF OXYGEN GLUCOSE DEPRIVATION
(2014-03) Phatak, Nitasha R.; Krishnamoorthy, Raghu R.
Loss of vision is either due to the injury or diseases of the eye like glaucoma. In glaucoma, retinal ganglion cells and optic nerve are affected, leading to decrease in vision. so in order to maintain or improve the vision in this condition, We need to find out different modalities of treatment from conventional glaucoma treatment. The protein, we are working has promising effect on retinal ganglion cell survival and outgrowth. We will be using neuronal cell line as a model for retinal ganglion cells. Oxygen glucose deprivation insult will mimic glaucomatous conditions. Purpose (a): Brn3b is a POU domain transcription factor shown to play a key role in regulating retinal ganglion cell axon outgrowth during development. Hypoxia is a contributing factor in many neurodegenerative diseases including glaucoma. The purpose of this study was to determine if overexpression of Brn3b could promote neurite outgrowth in cultured PC 12 cells during conditions of oxygen glucose deprivation (OGD). Methods (b): Rat Pheochromocytoma cells ( PC 12) were grown on poly-D-lysine coated 100 mm dishes and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b) or pCMV6-Empty (empty vector). Following 6 h of transfection, cells were maintained overnight in a differentiating medium containing NGF (100ng/ml). Subsequently, the cells were transferred to glucose free DMEM and maintained for 2 h in 0.5% O2 and 5% CO2 (for hypoxia) in an Invivo2 200 hypoxia chamber. For the normoxia controls, PC12 cells overexpressing Brn3b or Empty vector were maintained in differentiating medium for 2 h in 5% CO2 and 95% air in a standard incubator. Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43, TUBA-1 protein expression by immunoblot analysis. In another set of experiments, PC 12 cells were seeded on Poly-D-Lysine coated 25mm cover slips and transfected with either pCMV6-Brn3b or pCMV6 -Empty and maintained in differentiating medium for 4 days. The cells were subjected to either hypoxia (2h) or normoxia. Brn3b, GAP43 and TUBA-1 expression were analyzed using immunocytochemistry. Morphological changes in PC 12 cells transfected with Brn3b were studied by using LSM 510 confocal microscopy. Results (c): Immunoblot analysis confirmed overexpression of Brn3b in PC12 cells transfected with Brn3b cDNA in normoxic as well as in OGD conditions. Interestingly, a marked upregulation of GAP-43 and ac-TUBA expression was observed in Brn3b overexpressing cells under conditions of both normoxia and OGD. Overexpression of transcription factor Brn3b in PC12 cells produced a statistically significant increase in maximum neurite length and number of neurites per cell under conditions of both normoxia and OGD. A marked increase in immunostaining for Brn3b and neurite-specific GAP-43, TUBA-1 were also observed in PC12 cells overexpressing Brn3b in condition of normoxia and OGD. Conclusions (d): The POU domain transcription factor, Brn3b, could promote neurite outgrowth in PC12 cells under conditions of normoxia and as well as OGD.
THE EFFECTS OF ANDROGENS ON CASPASE-1 MEDIATED SIGNALING IN OXIDATIVE STRESSED DOPAMINE NEURONS
(2014-03) Holmes, Shaletha S.; Su, Chang; Singh, Meharvan; Cunningham, Rebecca
Parkinson’s disease (PD), a neurodegenerative disorder characterized by oxidative stress and the loss of dopamine neurons in the midbrain, affects the aging population. In fact, males have a higher risk for PD than females. While the mechanisms remain elusive, one possibility may be that androgens, such as testosterone, play a potential role. Our studies suggest that androgens can increase the expression of Caspase-1, an enzyme whose activity increases with oxidative stress and can result in mitochondrial collapse, ubiquitination, alpha-synuclein–positive lewy body accumulation, inflammation and apoptosis. Therefore, we hypothesize that in oxidative stress conditions, androgens suppress KLF4, a negative regulator of caspase-1, resulting in the increase of Caspase-1 to promote toxic protein accumulation, inflammation, and apoptotic neuronal cell death. This study is related to the role of testosterone in high levels of oxidative stress associated with aging. Purpose (a): Oxidative stress and an extensive loss of dopamine neurons in the nigrostriatal pathway are hallmarks of Parkinson’s disease (PD), a neurodegenerative disorder affecting millions of people. Males have a higher risk for PD than females. While the mechanisms remain elusive, one possibility may be that androgens, such as testosterone, play a potential role. Our studies suggest that androgens can increase the expression of Caspase-1, an enzyme whose activity increases with oxidative stress and can result in mitochondrial collapse, ubiquitination, alpha-synuclein–positive lewy body accumulation, inflammation and apoptosis. We hypothesize that in oxidative stress conditions, androgens suppress KLF4, a negative regulator of caspase-1, resulting in overexpression of Caspase-1 leading to toxic protein accumulation, inflammation, and apoptosis. Methods (b): We exposed a dopaminergic cell line (N27 cells) to a sublethal concentration of the pro-oxidant, tert-butyl hydrogen peroxide (H2O2) for 24 hrs and assessed cell viability in the presence or absence of testosterone. Results (c): Physiologically relevant concentrations of testosterone (0, 1, 10, 100 nM) failed to compromise cell viability in non-oxidatively stressed cells1. In contrast, testosterone did promote cell death in the H2O2 pre-treated cells. In H202 treated cells, testosterone increased caspase-1 expression and activation, as evidenced by an increase in cleaved caspase-1. In addition, KLF4 expression was decreased by testosterone in H2O2 treated cells. The role of KLF4 as a negative regulator of caspase-1 was confirmed in experiments showing that siRNA-mediated knockdown of KLF4 increased caspase-1 levels in H2O2 treated cells. Testosterone increased H202 mediated expression of COX2 signaling, a protein associated with inflammation. Also, testosterone decreased H2O2-induced ubiquitin expression resulting in the accumulation of toxic proteins. Further, testosterone increased H202 induced in apoptosis. Conclusions (d): Overall, these results indicate that androgens such as testosterone exert negative effects under oxidative stress conditions through the suppression of KLF4 and activation of caspase-1 signaling pathways leading to cell death. Thus, supporting a role for androgens for the gender bias observed in PD.
SRC-KINASE MEDIATES ANGIOTENSIN II INDUCED POTENTIATION IN TRPV4 AGONIST EVOKED CALCIUM TRANSIENTS IN HYPOTHALAMIC IMMORTALIZED NEURONAL CELL LINE 4B
(2014-03) Saxena, Ashwini; Bachelor, Martha E.; Carreno, Flavia R.; Cunningham, J. Thomas
We have previously demonstrated that in bile duct ligated rats, an animal model of inappropriate vasopressin (AVP) release, TRPV4 protein expression and membrane trafficking is increased in AVP neurons. Here, we used an in vitro approach with an immortalized AVP expressing neuroendocrine cell line (4B neurons) to investigate the possible regulation of TRPV4 by angiotensin II (Ang II). We characterized the presence of TRPV4 mRNA and protein in 4B cells. Ang II (100nM;1 hr) treatment significantly increased TRPV4 abundance (p. Purpose (a): ØInappropriate Vasopressin (AVP) release causes dilutional hyponatremia associated with heart and liver failure. Although the central molecular mechanisms that mediate inappropriate AVP release are not clear, plasma angiotensin II (Ang II) has been implicated as a factor in the pathogenesis of dilutional hyponatremia. Our previous studies using a rodent model of liver failure, have shown that increased TRPV4 expression in vasopressinergic neurons and elevated circulating AVP were blunted by normalization of the renin angiotensin system (RAS). Effects of circulating Ang II on neural networks may mediate cellular adaptations associated with changes in TRPV4 expression and/or sorting ØBased on our in vivo studies we speculate that modulation of transient receptor potential vanilloid (TRPV4) channels by means of changes in its membrane sorting could alter its gating, and thus contribute to changes in neural excitability that would be consistent with increased AVP release in rats with liver failure. To examine the effects of AngII treatment upon TRPV4 we utilized the rat hypothalamic AVP expressing neuronal cell line 4B. Methods (b): We used Western Blots to detect changes in TRPV4 protein in membrane fraction after drug treatments. In addition, we used calcium sensitive dye Fura 2-AM to detect changes in intracellular calcium after administration of a selective TRPV4 agonist - GSK 1016790A. Results (c): We characterized the presence of TRPV4 mRNA and protein in 4B cells. After Ang II (100nM;1 hr) treatment significantly increased TRPV4 levels in crude membrane fractions (p<0.001) and tyrosine phosphorylation of TRPV4 (p<0.001). Using calcium sensitive dye Fura-2AM, we noted that Ang II treated cells exhibited increased calcium transients in response to TRPV4 agonist, GSK1016790A (20nM, p<0.05). This increase was blocked by the Losartan (Ang II receptor antagonist) and Src-kinase inhibitor, PP2, but not by its analog PP3. Conclusions (d): Our data indicate that Ang II may facilitate TRPV4 trafficking and alter the phosphorylation status of TRPV4 through Src-kinases.
THE GLIAL RECEPTOR MECHANISM OF THE SYNTHESIS AND RELEASE OF BRAIN-DERIVED NEUROTROPHIC FACTOR WHEN EXPOSED TO PROGESTERONE IS MEDIATED VIA THE CLASSICAL PROGESTERONE RECEPTOR
(2014-03) Lee, Jay J.; Su, Chang; Singh, Meharvan
Purpose (a): Progesterone (P4) is cytoprotective in various experimental models, but our understanding of the mechanisms involved is still incomplete. It has been implicated that brain-derived neurotrophic factor (BDNF) signaling is an important mediator of P4’s protective actions. This experiment looked into a potential receptor mechanism by which progesterone can mediate BDNF synthesis, which is via the classical progesterone receptor (PR). C6 glial cells are neuronal cells that normally lack classical PR, and so it is an ideal model to look at the classical PR response to progesterone with relation to BDNF. The hypothesis is that cells that have the classical PR will synthesize more BDNF relative to cells that do not have the classical PR. Methods (b): In this experiment, a set of C6 glial cell cultures were transfected with classical PR genes. After allowing for growth, both the transfected and non-transfected C6 cells were exposed to progesterone and the concentration of BDNF in the cell lysis was determined via ELISA. Results (c): The results revealed that for the C6 cells that were not transfected with the classical PR, there was no significant change in the level of BDNF release regardless of the progesterone treatment (vehicle, 30 pg/mL BDNF; 0.1 nM P4, 29 pg/mL BDNF; 1 nM P4, 29 pg/mL BDNF; 10 nM P4, 31 pg/mL BDNF; 100 nM P4, 30 pg/mL BDNF; 1000 nM P4, 29 pg/mL BDNF). On the other hand, the C6 cells that were transfected with the classical PR showed an increase in BDNF production with the treatment of progesterone. The transfected C6 cells that were not treated with progesterone but rather with vehicle DMSO showed a BDNF release of 37 pg/mL. The transfected C6 cells that were treated with progesterone at concentrations of 10 nM and 100 nM showed a BDNF release of 61 pg/mL and 50 pg/mL respectively (P < 0.01). Conclusions (d): Overall, these results show that one receptor mechanism in neuronal cells that results in BDNF synthesis is via the classical progesterone receptor. The presence of the classical PR in C6 glial cells resulted in a statistically significant difference in the synthesis of BDNF relative to those that lack PR. Identifying and elucidating the mechanism by which progesterone confers neuroprotective benefits may aid in the development of new or novel treatments and/or drugs aimed at the prevention or treatment of many neurodegenerative diseases such as Alzheimer’s disease.
ISOLATION OF PRIMARY ASTROCYTES FROM HUMAN BRAIN TISSUE AND ASSESSMENT OF PROTOTYPICAL INFLAMMATORY RESPONSES FOR NEURODEGENERATIVE RESEARCH
(2014-03) Borgmann, Kathleen R.; Tang, Lin; Ghorpade, Anuja
Purpose (a): A common link in CNS disease is inflammation and the contribution of astrocyte inflammatory responses to neurodegeneration remains a focus of investigation. Non-human glial models may be limited in providing data that extrapolate directly to human neurodegenerative diseases, thus much remains to be learned in the genetically relevant context of primary human astroglial cultures. Methods (b): Here we describe the isolation and purification of primary human astrocytes from fetal brain in detail. We expand this protocol to include the assessment of astrocyte responses to inflammation through changes in cell morphology and expression of astrocyte specific markers, mitochondrial pore opening and activity, proinflammatory chemokine secretion and glutamate uptake. Results (c): Pure cultures were uniform in size and shape, and at least 95% positive for astrocyte markers. Mitochondrial pore staining revealed punctate calcein staining, which was decreased during inflammation. Upon treatment with a prototypical mediator of astrocyte inflammatory responses, interleukin (IL)-1beta, astrocyte processes became constricted; indicating a reactive astrocytic state, chemokine secretion increased significantly and the ability of astrocytes to clear glutamate was significantly impaired. Untreated cultures that demonstrated reactive phenotypes or those that failed to attain reactive states upon IL-1beta-treatment were excluded. Conclusions (d): These parameters established a framework to assess the overall purity, health, responsiveness to inflammation and thus the suitability of the culture for experimental use of primary human astrocyte cultures for neurodegenerative research.
MODULATION OF CHICKEN ASIC1 BY 2-GUANIDINE-4-METHYLQUINAZOLINE (GMQ) IN THE ABSENCE AND PRESENCE OF PSALMOTOXIN-1
(2014-03) Smith, Rachel N.; Gonzales, Eric B.
Ion channels are an important means of communication between the outside and inside of the cell. One family of ion channels, the acid-sensing ion channels (ASICs) are activated by a drop in pH surrounding the cell. Their activation allows the movement of ions from the outside and inside compartment of the cell, and specifically with ASICs, this exchange of ions can be involved many pathophysiological conditions. However, one shortcoming is that there remains a lack of specific agonists (channel activators) or antagonists (channel inactivators) for the different ASIC subtypes. Recent studies have indicated that there are natural venom toxins derived from organisms like sea anemones and tarantulas that can interact with ASICs. Additionally, compounds containing a guanidinium group, like the synthetic compound GMQ, can enhance channel activation in some of the ASIC subtypes. We sought to determine if the combination of these natural venom toxins and guanidine compounds can simultaneously interact with ASICs using a cellular model that contains no ASIC subtypes. We record the channel activity following the addition of the compounds both individually and simultaneously. Understanding the interaction of these compounds with the ASIC subtypes will provide the foundation for the design of novel drugs to influence the activity of these channels. Purpose (a): Acid-sensing ion channels (ASICs) are trimeric, sodium-selective channels that sense changes in extracellular acidity and are part of the epithelium sodium channel/degenerin (ENaC/DEG) family of ion channels. ASICs are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds, such as amiloride and 2-guanidine-4-methylquinazoline (GMQ). The nonproton ligand GMQ has been shown to stimulate ASIC3 by expanding the pH range of the ASIC window current, but decreased the sensitivity of other ASIC subtypes to protons. The effect of GMQ on chicken ASIC1 (cASIC1), which has been used to elucidate the protein crystal structures, is unknown. Furthermore, cASIC1 exhibits unique channel gating properties, including the spider toxin ASIC1a Psalmotoxin-1 (PcTx1) induced activation. Methods (b): We sought to elucidate the interaction of GMQ, PcTx1, and cASIC1 using whole-cell and outside-out patch clamp electrophysiology to provide additional insight into the nonproton ligand interaction with a structurally characterized ASIC construct. Results (c): Our studies revealed GMQ increases the cASIC1 proton sensitivity, as observed by a leftward shift in the proton activation curve. When alone, the nonproton ligand failed to activate cASIC1. Additionally, we observed GMQ concentration-dependent enhancement of the cASIC1 PcTx1 persistent current. Conclusions (d): Our data suggests that GMQ may have multiple sites of action on cASIC1 and may act as a “molecular wedge” that forces the desensitized ASIC into an open state. We anticipate that the revelation of GMQ stimulation in the cASIC1 subtype will warrant further investigations into nonproton ligand sensitivity in other ASIC subtypes and provide the foundation for the design of novel ligands that exploit the nonproton ligand site to influence ASIC activity.
ADENO-ASSOCIATED VIRUS CONSTRUCT ENABLES DIFFERENTIATION OF VASOPRESSIN AND OXYTOCIN NEUROPEPTIDE-EXPRESSING MAGNOCELLULAR NEURONS IN THE HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RAT
(2014-03) Knapp, Blayne A.; Little, Joel; Cunningham, Tom
Adeno-associated viral (AAV) vectors are useful tools for transfecting specific cell populations through the use of cell-type specific promoters. Recently, promoters that are specific for either vasopressin (AVP) or oxytocin (OXT) magnocellular neurosecretory cells (MNCs) have been designed that can be used with AAVs to selectively drive gene expression in these cells. The goal of this study was to validate this approach and determine whether it can cause the selective transfection of AVP versus OXT MNCs in the supraoptic nucleus of the hypothalamus (SON). In these studies, an AAV2 vector with an AVP promoter and GFP (p2.OVPI.EGFP) was stereotaxically injected into the SON of adult male Sprague-Dawley rats (226 - 250g bw) during isoflurane anesthesia. After 14 days, the rats were each anesthetized with inactin (100 mg/kg ip) and their brains where prepared for immunofluorescence. Two separate sections of coronal sections containing the SON were processed for either AVP or OXT immunohistochemistry using a Cy3 conjugated secondary antibody. Colocalization of GFP with either AVP or OXT immunofluorescence was determined by light microscopy. Our results indicate the colocalization of GFP and AVP in MNCs of the SON (89% GFP-AVP double labeling, n=3), and not GFP and OXT (0.08% GFP-OXT double labeling, n=3). Given this demonstration of successful vector transduction, we can conclude that the AAV2 vector is selective to AVP expressing MNCs, enabling us to distinguish AVP versus OXT MNCs in the SON. This capability will permit differentiation of neuronal types and their respective properties during later electrophysiological studies. R56 HL62569. Purpose (a): The goal of this study was to validate this approach and determine whether it can cause the selective transfection of AVP versus OXT MNCs in the supraoptic nucleus of the hypothalamus (SON). Methods (b): In these studies, an AAV2 vector with an AVP promoter and GFP (p2.OVPI.EGFP) was stereotaxically injected into the SON of adult male Sprague-Dawley rats (226-250g bw) during isoflurane anesthesia. After 14 days, the rats were each anesthetized with inactin (100 mg/kg ip) and their brains where prepared for immunofluorescence. Two separate sections of coronal sections containing the SON were processed for either AVP or OXT immunohistochemistry using a Cy3 conjugated secondary antibody. Colocalization of GFP with either AVP or OXT immunofluorescence was determined by light microscopy. Results (c): Our results indicate the colocalization of GFP and AVP in MNCs of the SON (89% GFP-AVP double labeling, n=3), and not GFP and OXT (0.08% GFP-OXT double labeling, n=3). Conclusions (d): Given this demonstration of successful vector transduction, we can conclude that the AAV2 vector is selective to AVP expressing MNCs, enabling us to distinguish AVP versus OXT MNCs in the SON. This capability will permit differentiation of neuronal types and their respective properties during later electrophysiological studies.

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