Neuroscience

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21741

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Treatment with Losartan Improves Baroreflexes in Obese Zucker Rats Coincident with Enhanced Response of Nucleus Tractus Solitarius (NTS) to Increased Arterial Pressure
(2015-03) Chaudhary, Parul; Schreihofer, Ann; Dohi, Akiko
Obese Zucker rats (OZR) have impaired baroreflex-mediated responses to acute rises in mean arterial pressure (MAP) coincident with the development of hypertension. In addition, the NTS becomes less responsive, with reduced phenylephrine (PE)-induced c-Fos expression and smaller physiological responses to glutamate in the NTS. Here, we determined if treatment with the angiotensin- AT1 receptor antagonist, losartan (LOS) improves baroreflexes and the PE-induced c-Fos expression in NTS of OZR. Baroreflex-mediated changes in HR to PE-evoked rises in MAP (40 mmHg) were measured in conscious rats implanted with femoral catheters. Rises in MAP were sustained with PE, and then rats were perfused after 90 min to examine NTS c-Fos expression. Treatment with LOS (for 5 wks) normalized MAP in OZR vs. LZR (untreated: 128±2 vs. 115±3 mmHg, with LOS: 111±3 vs. 112±2 mmHg; at 14.1 wks). LOS treatment enhanced PE-induced reductions in HR in OZR but not LZR. (untreated: 43±10 vs. -101±9 -bpm; with LOS: . -75±12 vs. -112±11bpm). Although LOS improved PE-induced reductions in HR in OZR, responses in LOS-treated OZR were still smaller vs. LOS-treated LZR, suggesting a partial restoration of baroreflexes. PE-induced c-Fos expression in NTS was less in untreated OZR vs. LZR (30±5 vs. 50±6 counts). Treatment with LOS enhanced PE-induced c-Fos expression in OZR (54±11 counts), but not LZR (58±11 counts), rendering comparable PE-induced c-Fos expression in LOS-treated OZR and LZR. These data suggest that LOS improves hypertension, baroreflexes, and NTS function in OZR. Further study is needed to determine whether improved function by LOS is explained by normalization of MAP in OZR.
Concomitant Mutations of Sodium Channels SCN8A and SCN2A Cause Dravet Syndrome Phenotype
(2015-03) Rao, Chethan K.; Basinger, Alice; Perry, M. Scott
Purpose: To report a unique case of Dravet Syndrome (DS) and investigate the relationship between inherited SCN2A and de novo SCN8A mutations in our patient; to determine whether a de novo SCN8A mutation can cause DS; to review the genetic causes of DS phenotype and the need for thorough genetic evaluation for SCN1A-negative DS cases Methods: In this observational, retrospective case study, we focused on patient records analysis including whole-exome sequencing, EEG, and MRI. Results: This study found reason to believe that the de novo SCN8A mutation acted as a phenotypic modifier of the inherited SCN2A mutation, resulting in DS. The study also found a significant need for genetic evaluation beyond SCN1A mutations. In addition, Stiripentol was found to be an effective treatment for DS in this patient. Conclusions: Based on our findings and those of relevant studies, our patient’s de novo SCN8A mutation likely acted as a phenotypic modifier of the inherited SCN2A mutation potentially through a “two-hit” mechanism. The vast array of genetic etiologies of DS warrants thorough genetic testing beyond the most common SCN1A mutation. Stiripentol, though currently an investigative drug, shows promise as an effective therapeutic agent in our patient and potentially other cases of non-SCN1A DS.
Pgrmc1/KLF4 Signaling Mediates the Neuron-Glia Crosstalk As A Neuroprotective Mechanism
(2015-03) Nguyen, Trinh; Sun, Fen; Su, Chang; Singh, Meharvan
We have recently found that Pgrmc1,a novel membrane-associated progesterone receptor,mediates P4-triggered BDNF release specifically from glia.To date,downstream signaling transduction consequent to Pgrmc1 activation has not been revealed.Here we provide evidence that P4 elicits a Pgrmc1/ERK5/KLF4 signaling cascade,which in turn,orchestrates glia-neuron communication via a BDNF (Brain-derived neurotrophic factor)-mediated intercellular crosstalk. We show that P4 triggered a significant release of mature BDNF from glia,and this effect was abolished by RNAi-mediated knock-down of Pgrmc1 or KLF4. Treatment of neuronal cultures with conditioned media from P4-treated astrocytes (P4-CM) induced a robust increase of synaptic marker expression,while blocking neurotrophin signaling can attenuate this effect,supporting that glia-derived BDNF induced synaptogenesis in neurons. In addition,P4-CM from glia significantly protected neurons against oxidative stress.Interestingly,over-expression of KLF4 in neurons resulted in an increase of TrkB / p75 ratio,supporting that neuronal activation of the KLF4 pathway “prepares” the neurons to interpret the glia-derived mature BDNF signaling as favorable to survival.Finally, we determined that the levels of Pgrmc1, KLF4 and BDNF expression were decreased in the hippocampi of aged mice,as well as in the 5´FAD mouse model of AD when compared to age-matched controls,suggesting that both “normal” and “pathological” aging (i.e., Alzheimer's disease) may diminish the sensitivity of the brain to the protective effects of P4 through down-regulating the Pgrmc1/KLF4/BDNF signaling system.
Chronic Intermittent Hypoxia increases oxidative stress and inflammation
(2015-03) Snyder, Brina; Shell, Brent; Cunningham, J. Thomas; Cunningham, Rebecca
Background: Inflammation has been linked with sleep apnea. Sleep apnea is a common comorbidity associated with neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease. Furthermore, neurodegenerative diseases have also been linked with inflammation. A possible mechanism underlying increased inflammation in these disorders is oxidative stress, a hallmark of neurodegeneration. To examine the role of oxidative stress on inflammation, we used chronic intermittent hypoxia (CIH), an established model for the hypoxemia associated with sleep apnea. CIH consists of recurring events of low oxygen followed by reoxygenation. Statement of Hypothesis: We hypothesize that CIH causes oxidative stress, which induces inflammation. Materials and methods: To test this hypothesis, plasma from adult male rats subjected to 7 days of CIH (3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day) or normoxia (room air) were tested for AOPP, an indicator of oxidative stress, and circulating inflammatory markers (such as IL-1b, IL-10, IL-4, IL-6). Additionally, a group of rats was administered a neurotropic AAV with shRNA for AT1a receptors in their forebrains and instrumented with telemetry for blood pressure recording prior to CIH treatment to determine the effects of angiotensin on CIH hypertension and oxidative stress. Significant results: Our results showed that CIH significantly increased circulating oxidative stress and inflammation. Interestingly, IL-1b, IL-2, and TNF-a inflammatory markers were associated with oxidative stress, unlike IL-10, IL-4, and IL-6 inflammatory markers. These markers were positively associated with IL-1b. Knockdown of angiotensin 1 receptors in the forebrain blocked the diurnal hypertension and CIH induced oxidative stress, indicating the involvement of CIH hypertension and central angiotensin receptors in CIH induced oxidative stress. Conclusions: These results indicate that both neurons and macrophages contribute to CIH induced oxidative stress and inflammation and that CIH oxidative stress and inflammation is dependent on central angiotensin receptors and CIH hypertension.
A Reliable Method for MRI Volumetric Assessment of Hippocampal Resection Following Anterior Temporal Lobectomy
(2015-03) Wilson, Jacob; Donahue, MD, David
Purpose: Surgical management of temporal lobe epilepsy in children is an established management of temporal lobe epilepsy (TLE). Surgery for TLE ranges from standard anterior temporal lobectomy (ATL) to selective resections which seek to spare temporal neocortex. Cortical dysplasia (CD) represents the most frequent non-neoplastic cause of TLE in children. Extent of resection (ER) of cortical dysplastic processes has been correlated with seizure control. The goal of this study was to design and test a standardized protocol based on anatomical boundaries for pre-operative (pre-op) and post-operative (post-op) imaging in order to measure hippocampal ER. Methods: Medical records and neuroimaging of a 2 year old developmentally delayed female TLE patient with CD in her right temporal lobe were reviewed. Seizure semiology, electrodiagnostics, and imaging studies indicated independent bilateral temporal dysfunction with the most severe deficit of activity in the right temporal lobe. Pathological analysis of temporal neocortex and mesial structures disclosed evidence of cortical dysplasia in all specimens. Pre-op MRI studies were performed using a Seimens Verio 3T unit generating 3 sequences: 1) T1 Magnetization Prepared Rapid Gradient Echo 3D with 1.0mm slice thickness, 2) T2 Turbo Spin Echo (TSE) Axial with 4.0mm slice thickness and, 3) T2 TSE Coronal with 3.0mm slice thickness. Post-op MRI studies were performed using a Seimens Espress 1.5T unit generating 3 sequences: 1) T1 Spin Echo (SE) Axial with 5.0mm slice thickness, 2) T2 TSE Axial with a 5.0mm slice thickness and, 3) T2 TSE Coronal with a 3.0mm slice thickness. Studies were co-registered via Hermes, a product of Hermes Medical Solutions, which rendered multiple image studies into one file. The resulting 3D brain map was then loaded into Amide, a free image viewing tool for registering and analyzing medical data image sets, which allowed a slice by slice evaluation of both pre-op and post-op images. After hippocampal boundaries were identified on each coronal slice, a region of interest (ROI) was created outlining hippocampal area. Amide calculated ROI areas for each slice and summed ROI areas to yield hippocampal volume. Results: Analysis of ROI yielded pre-op hippocampal volume of 2.691 cm3 vs. post-op volume of 0.259 cm3. ATL in our patient removed 96.25% of the hippocampus, leaving 3.75% of hippocampal tail remaining. Analysis completed after construction of protocol used to determine hippocampal anatomical borders. Conclusions: This project successfully designed and tested a standardized protocol based on anatomical boundaries for pre-op and post-op imaging in order to measure hippocampal volume ER. This protocol will allow for measurement of ER in future studies of TLE in pediatric populations.
Alcohol Regulates HIV-1-Mediated Astrocyte Inflammatory Responses Via cPLA2 Signaling Pathway
(2015-03) Pandey, Richa; Ghorpade, Anuja
Alcohol (EtOH) abuse and HIV-1 remain significant public health problems. Globally, drinkers have approximately 70-77% higher risk of HIV-infection than non-drinkers. The prevalence of alcohol abuse among HIV-positive individuals has been estimated to be between 29-60% in the United States. Many studies showed that neurodegeneration in alcohol abusers include exacerbated neuroinflammation and oxidative damage. However, how EtOH regulates HIV-1-induced astrocyte neuroinflammation is unknown. Thus, we explored mechanism(s) involved in alcohol-mediated activation of human astrocytes with HIV-1 and subsquent alterations in their inflammatory functions. Alcohol exposure altered the morphology of astrocytes, proinflammatory responses and induced cytotoxicity in a dose-dependent manner. Time-depended changes were also evaluated. Alcohol and HIV-1 co-treatment decreased cell viability and proliferation, while increasing apoptosis and mitochondrial depolarization. Alcohol and HIV-1 together increased the levels of proinflammatory molecules, IL-1b, TNF-a, CXCL8, TIMP-1 and more importantly, arachidonic acid, known to be downstream of cPLA2. Consistent with this observation, phospho-cPLA2 levels were augmented in HIV-1 and EtOH co-treatment as compared to HIV-1 or EtOH alone. COX2 was upregulated as measured by real time PCR and western blot, whereas co-treatment of HIV-1 and EtOH decreased CYP2E1 levels as compared to EtOH alone. In summary, our results demonstrate that EtOH-mediated astrocyte inflammation and cytotoxicity in context of HAND occurs via cPLA2 signaling.
Acid-sensing ion channel modulation by nonproton ligands: the influence of divalent cations
(2015-03) Smith, Rachel N.; Gonzales, Eric B.
1. The acid-sensing ion channel (ASIC) is emerging as a potential mediator for a variety of pathologies, such as stroke, pain, and mental health diseases. Despite their involvement in multiple age-related pathologies, the ability to selectively target the ASIC subtypes remains unidentified. As their name suggests, ASICs are activated by an increase in extracellular protons, however other ASIC ligands include natural venom toxins, guanidine containing compounds, and calcium. Nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate the peripheral ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in the structural determinant chicken ASIC1, despite having no direct modulation effect on the channel. We previously showed that cASIC1 could be activated by GMQ following a based on a partially activated channel state. Interestingly, low calcium concentrations cause the peripherally located ASIC3 subtype to be partially activated. We proposed that direct nonproton ligand activation of ASIC3 is possible due to the actions of the transmembrane domains (TMD) where calcium sensitivity resides. Additionally, the introduction of ASIC3 TMDs into a GMQ insensitive ASIC subtype (cASIC1) will reveal nonproton ligand sensitivity. 2. Chimeric receptors combining the extracellular, transmembrane, and intracellular domains of rat ASIC3 and chicken ASIC1 were generated to individually isolate the calcium and nonproton ligand effects on channel activation. Each chimeric receptor was assessed for function using whole cell patch clamp electrophysiology. 3. We confirmed that rASIC3 is activated and held open when extracellular calcium concentrations are reduced with minimal proton influence (pH 8.0). Low-calcium-activation of rASIC3 is further enhanced by the addition of GMQ in a concentration dependent manner. These effects are absent in cASIC1. The chimera termed cASIC1 (rASIC3-TM/ITC) is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium, although its sensitivity to GMQ is reduced. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3. 4. This data suggests that the ASIC3 TM domains dictate NPLSD influence on channel activity. Fully understanding how we can interrupt or enhance channel activation will allow us to preferentially target these ion channels, potentially leading to the promotion of developing novel therapeutics to interact with ASICs.
The role of DNA methylation in Glucocorticoid Receptor-mediated repression of Corticotropin releasing hormone (CRH) gene.
(2015-03) Bhave, Shreyas A.; Uht, Rosalie
Glucocorticoid receptor(GR)-mediated repression of corticotropin releasing hormone gene(crh) is an important component of a negative feedback which controls hypothalamic pituitary adrenal(HPA) axis. Failure of this negative feedback is often the cause of HPA axis dysregulation. The molecular mechanisms that lead to this failure are still unclear. The dysregulation of HPA axis is associated with many neuropsychiatric disorders such as depression, cognitive impairment, neuro-degeneration and mood disorders. Here we investigate the molecular mechanism by which GR maintains the repressed levels of crh. DNA methylation of crh promoter plays an important role in gene regulation. Sharma et al. have shown that, Dexamethasone(Dex)- a GR ligand treatment increases the methylation of CpG dinucleotides present in the crh promoter in IVB rat hypothalamic cells. Also,according to same study, Dex increases the recruitment of methyl CpG binding protein 2(MeCP2) to the crh proximal promoter region. Here we tested the hypothesis that whether DNA methyalation and MeCP2 are required for maintaining the GR mediated repressed levels of crh. We found that, in IVB cells inhibition of DNA methylation by 5-Aza-2-deoxycytidine (5-AzaDC) increased the crh expression in dose dependent manner. We also found that 5-AzaDC at 0.5uM concentration significantly inhibited the crh promoter methylation. Further we observed that Dex fails to maintain the repressed levels of crh in cells pretreated with 5-AzaDC. We used RT-qPCR for gene expression analysis. We also analyzed the role of MeCP2 in GR mediated repression of crh. Our data indicated that siRNA mediated knock down of MeCP2 protein leads to increased basal levels of crh expression. Also, Dex fails to repress the crh in absence of MeCP2 protein. Taken together our results indicate that DNA methylation and MeCP2 are required for maintaining the GR mediated repressed levels of crh.
The Effects of Androgens on COX2 signaling in Oxidatively Stressed Dopamine Neurons
(2015-03) Holmes, Shaletha S.; Nazarali, Rizwan; Cunningham, Rebecca
Hypothesis: We hypothesize that under oxidative stress conditions, the androgen, testosterone, will increase COX2 induced alpha-synuclein expression, leading to apoptosis in dopamine neurons. Materials and Methods: To test our hypothesis, we exposed a dopaminergic cell line (N27 cells) to a sublethal concentration of the pro-oxidant, tert-butyl hydrogen peroxide (H2O2) for 24 hours and assessed the role of testosterone on COX2 signaling. Results: Under low oxidative stress conditions, COX2 protein levels are low and alpha-synuclein expression and apoptosis are absent. However, under oxidative stress conditions, COX2, alpha synuclein, and apoptosis were increased, and these factors were exacerbated by testosterone. Conclusion: Our data shows that androgens may mediate the gender differences observed in PD by activating COX2 mediated inflammation and oxidative stress in dopamine neurons.
Effects of an acid-sensing ion channel modulator on inflammatory pain
(2015-03) Izurieta Munoz, Haydee S.; Sumien, Nathalie
Purpose The objective of this study was to evaluate GL-001 as an anti-nociceptive compound in an animal model of thermal and inflammatory pain. Acid-sensing ion channels (ASICs) are membrane-bound ion channels that are sensitive to protons and non-proton ligands. In particular, the ASIC3 channel subtype is highly sensitive to decreases in extracellular pH and is found predominantly in peripheral sensory neurons, making it a potential modulator of pain sensation. Non-proton ligand activators and blockers of ASIC3 have been documented to affect nociception in both humans and animal models. As the compound GL-001 shares structural similarity with the non-proton ligands of ASIC3, the hypothesis is that GL-001 will interact with the channel in a similar manner leading to decreased nociception. Materials and methods Sixty male and female C57BL/6J mice (2-3 months old) were fed either a control diet or the control diet supplemented with 6.25g of GL-001 per kg of diet for one week prior to and throughout the nociceptive tests. To assess thermal hyperalgesia, the distal portion of the tail of the mice was dipped in a water bath set at 52°C and the latency to withdraw their tails was used as a measure of pain sensitivity. To induce inflammatory pain, the mice were injected with 4% formalin in their right hindpaw, and their nociceptive behavior (licking the injected paw) was recorded and timed. Statistical analyses (t-test for the thermal hyperalgesia, and ANOVAs for the formalin test) were performed with alpha set at 0.05. Results Preliminary results indicated that neither sex nor treatment affected the latency to withdraw the tail in the thermal hyperalgesia test, even though the treated mice took 10% longer latencies than the controls. For the formalin test, males and females seemed to respond differently to the stimulus. The female mice treated with GL-001 seemed to recover faster than the controls, which was supported by main effects of treatment in the last 30 minutes of the test. Conclusion Our preliminary data suggest a potential effect of GL-001 on nociception, especially related to inflammation. Further, females seemed to be more responsive to GL-001 than males. Further studies will be required to determine the preferred dose of GL-001 for beneficial effects, and identify which ASIC subtypes might be involved in its mechanism of action.
Mechanisms by which estradiol (E2) suppress neuronal cox-2 gene expression
(2015-03) Stacey, Winfred; Uht, Rosalie
Purpose: Data from culture and animal models indicate that 17β-estradiol (E2) deprivation increases susceptibility to neurodegenerative and neuropsychiatric diseases. E2 plays a pivotal role in attenuating inflammatory response in the brain by suppressing expression of proinflammatory genes; however, the molecular mechanisms by which E2 suppress neuronal pro-inflammatory genes are not well established. The pro-inflammatory cyclooxygenase-2 gene (cox-2) is selectively expressed in neuronal populations of the amygdala, hippocampus and cortex. Upregulation of cox-2 expression has been implicated in cascades of deleterious effects that promote neuronal injury and dysfunction. The goal of this study is to elucidate the molecular mechanisms by which E2 suppress cox-2 expression in a neuronal context. Methods: To characterize the effect of E2 on cox 2 in a neuronal system, we used the AR-5 immortalized rat neuronal cell line. This cell line was developed by Kasckow et al. from rat embryonic amygdala cells. We first determined that the cell line constitutively expresses COX-2 protein by Western blots and immunocytochemistry and mRNA and hnRNA by RT-qPCR. To assess whether the E2 effect was mediated by ER-alpha (ERα) and/or ER-beta (ERβ), we treated the cells with E2, Diarylpropionitril (DPN), and propyl-pyrazole-triol (PPT). COX-2 mRNA and hnRNA levels were analyzed by RT-qPCR following treatments with the ligands. Results: Twenty-four hours of E2 exposure reduces neuronal COX-2 mRNA and hnRNA levels. E2 and DPN treatment led to suppression of COX-2 mRNA and hnRNA after 24hrs. In distinction, PPT had no effect. Conclusions: Collectively, the data indicate that E2 suppresses neuronal cox-2 expression through ERβ.
The Role of Sleep Apnea Induced Oxidative Stress in Stroke Pathogenesis and Recovery
(2015-03) Bullock, Monica; Schreihofer, Derek; Cunningham, Rebecca
Purpose: Obstructive sleep apnea (OSA) is a common, but under-diagnosed comorbidity among patients with a number of age-related disorders, including stroke, Alzheimer’s disease, and Parkinson’s disease. Many of the risk factors for sleep apnea, such as obesity, are modifiable and treatment of sleep apnea itself can limit its systemic effects. Because of these facts, understanding the role of OSA in more serious diseases may promote awareness and early diagnosis, thus preventing serious adverse health outcomes. Given the knowledge that sleep apnea increases oxidative stress, in order to investigate the effects of sleep apnea on the pathogenesis of and recovery from stroke, we used chronic intermittent hypoxia (CIH) as an animal model of sleep apnea in rats. Methods: 12 rats underwent behavioral testing and were then randomly assigned to chambers with either a constant normoxic environment or one that simulates the chronic intermittent hypoxia of sleep apnea. Cerebral ischemia was induced in rats by occlusion of the middle cerebral artery. After a day of recovery, cognitive impairment, oxidative stress, and the size of the ischemic lesion was measured. Results: The experiment showed that CIH increased the size of the stroke lesion in the brain. In this setting, CIH did not appear to alter circulating oxidative stress protein measures or acute stroke behaviors. Conclusion: Based on these results, sleep apnea co-morbidity can have deleterious effects on stroke outcomes.
Astrocyte AEG-1 regulation of Wnt/β-catenin signaling in HAND
(2015-03) Nooka, Shruthi; Ghorpade, Anuja
Purpose: Glial induced chronic inflammation contributes to the pathogenesis of HIV-1-associated neurocognitive disorder (HAND), but the molecular mechanisms of inflammatory regulation have not yet been fully understood. Astrocyte elevated gene (AEG-1), an HIV-1 and tumor necrosis factor (TNF)-α inducible gene, is associated with multiple signaling cascades such as nuclear factor (NF)-κB, Wnt/β-catenin during tumor progression. Recently, upregulation of Wnt signaling proteins was observed in spinal cord dorsal horn of HIV-1 patients. In addition, Wnt signaling regulates pro-inflammatory cytokines expression in several inflammatory diseases including rheumatoid arthritis, psoriasis. However, the relationship between AEG-1 and Wnt signaling pathway in HIV-1-associated neuropathogenesis has not been studied. Hereby, we proposed that astrocyte AEG-1 induces inflammation via classical Wnt signaling in HAND. Methods: Cultured human astrocytes were treated with HIV-1DJV, interleukin (IL)-1β and TNF-α. Astrocytes were nucleofected with AEG-1 and β-catenin specific siRNA. Changes in AEG-1, β-catenin and lymphoid enhancing factor (LEF)-1 levels were determined by RT-PCR, western blot analysis and immunocytochemistry. Co-immunoprecipitation (Co-IP) studies were performed to examine AEG-1 interacting proteins and NF-κB dynamics. Pro-inflammatory molecules such as CCL2 and CXCL8 levels were measured by ELISA. Results: HIV-1DJV in combination with IL-1β and TNF-α significantly upregulated AEG-1, β-catenin and LEF-1 mRNA levels. Nuclear translocation of β-catenin decreased significantly in siAEG-1 transfected astrocytes. Further, Co-IP studies showed AEG-1 interacting with β-catenin and LEF-1, and upon activation with pro-inflammatory stimuli, interaction increased in both the cytoplasm and nucleus. Both AEG-1 and β-catenin also interacted with NF-κB, suggesting a common denominator in regulating inflammation. AEG-1 and β-catenin transient knockdown followed by IL-1β treatment altered NF-κB mediated pro-inflammatory cytokines production. Conclusion: In summary, HAND-relevant stimuli upregulated classical Wnt signaling and increased interactions between AEG-1, β-catenin and NF-κB, suggesting AEG-1 mediated Wnt signaling regulation of NF-κB activity in HAND neuropathogenesis.
CB1 Receptor Antagonist, ATDP 32,456, as an Emergency Treatment Modality for Synthetic Cannabis Overdose in Mice
(2015-03) Alexander, Brian; Forster, Michael; Nejtek, Vicki
ATDP 32, 456 is a proven agent for blocking the effects of cannabinoids at the CB1 receptor. We sought to determine if the suppression of locomotor activity by Delta(9)-THC, JWH-018 and other synthetic cannabinoids could be alleviated with administration of ATDP 32, 456. The suppression of locomotor activity of mice was measured after administration of cannabinoids and after different rescue doses of ATDP 32, 456 given via the intraperitoneal route, 1-hour later. We noted a statistically significant reversal of locomotor activity suppression and inhibition of synthetic cannabinoid effects when mice received 3, 10 or 30 mg/kg ATDP 32, 456. These data support the use of ATDP 32, 456 as an acute treatment method for cannabinoid intoxication, though formulation compatible with intravenous administration would be required for a rapid response.
Human cerebral organoids generated using urinary epithelial cell-derived induced pluripotent stem cells
(2015-03) Lin, Victor; Zolekar, Ashwini; Mull, Amber; Wang, Yu-chieh Jack
Background: Human induced pluripotent stem cells (hiPSCs) provide a great promise for the success of novel disease model and regenerative medicine. Such remarkable cells may be derived using virtually any types of somatic cells through cell reprogramming and used for addressing questions relevant to the physiopathological development of human organs. The generation of hiPSCs using somatic cells that are obtained by noninvasive approaches would be ideal for the further development of hiPSC-based models to address childhood disease. Given the pluripotency in hiPSCs, we anticipate that hiPSCs generated from cells isolated in urine samples can be used for creating a three-dimensional organoid model system to recapitulate human brain development. Purpose: We desire the generation of transgene-free hiPSCs using urinary epithelial cells collected from the urine samples of health donors. We will further test cellular pluripotency and the capacity of forming three-dimensional cerebral organoids (minibrains) in these hiPSCs. Method: We use a polycistronic, self-replicating RNA system to express four transcription factors SOX2, KLF4, OCT4, and MYC in urinary epithelial cells that are isolated from the mid-to-late stream urine samples of healthy individuals. We also generate cerebral organoids from urinary epithelial cells-derived hiPSCs and other bona fide human pluripotent stem cell (hPSC) lines using an established differentiation protocol. Results: Urinary epithelial cells were successfully collected from four individuals, cultured, and reprogrammed into hiPSCs. Reprogrammed urinary epithelial cells express pluripotency markers and form cell types showing biomarkers for all three germ layers in undirected differentiation. The bona fide hPSCs have been used to optimize the procedure of building three-dimensional cerebral organoids. After matrigel embedding, the hPSC spheres that were precommitted to neuronal differentiation further developed complex structures that resemble discrete but interdependent cerebral regions, including forebrain, hippocampus, cortex, hindbrain, and a neural stem cell zone. These organoids stain positive for TUBB3 (a biomarker for neurons), GFAP (a biomarker for astrocytes), and SOX2 (a biomarker for neural stem cell populations). Conclusions: Our preliminary data suggest that hPSCs can reproducibly form minibrains in a defined culture condition. We will use the optimized procedure to test the capacity of urinary epithelial cells-derived hiPSCs in forming three-dimensional cerebral organoids (minibrains) and further establish a suitable model for studying brain developmental abnormalities associated with childhood disease.

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