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- Title
- ROLE OF TRANSIENT RECEPTOR POTENTIAL CANONICAL-6 (TRPC6) CHANNEL IN METASTASIS OF GLIOBLASTOMA MULTIFORME.
- Creator
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Venkataraman, Rajarajeshwari, Sugaya, Kiminobu, University of Central Florida
- Abstract / Description
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Glioblastoma multiforme (GBM) is one of the extremely fatal brain tumors. The main reason that makes it so lethal is its capability to invade and spread to other parts of CNS producing secondary tumors. Among other factors hypoxia, reduced oxygen availability, is linked to higher metastatic potential of cancers. Hypoxia causes numerous changes in genome and proteome of the cell. These changes help a normal cell to adapt to nutritional deficiency, but the same changes can increase the...
Show moreGlioblastoma multiforme (GBM) is one of the extremely fatal brain tumors. The main reason that makes it so lethal is its capability to invade and spread to other parts of CNS producing secondary tumors. Among other factors hypoxia, reduced oxygen availability, is linked to higher metastatic potential of cancers. Hypoxia causes numerous changes in genome and proteome of the cell. These changes help a normal cell to adapt to nutritional deficiency, but the same changes can increase the malignancy and metastasis in tumor cells. Extensive research by a number of curious scientists reveal that various pathways involving numerous proteins cross-talk and interact with each other and execute a response to hypoxia. We are trying to establish the link between two such pathways HIF1-alpha pathway and Notch pathway. Both, HIF1-alpha, which is a transcription factor that becomes active in hypoxic conditions and Notch, which is an evolutionarily conserved cell-fate determinant, are implicated in hypoxia-induced metastasis of cancer. In this given project, we confirm the cross talk between Notch and HIF1-alpha pathway and further continue our study to show that TrpC6 is the downstream mediator of this pathway, leading to metastasis of GBM. Expression analysis of hypoxia-induced U373 cells (Grade 3 glioblastoma cells), using Real-time PCR, western blot and immunocytochemistry, revealed elevated levels of Notch, Hif1 and TrpC6 indicating that these proteins might be important for the cellular response to hypoxia. Blocking Notch and/or HIF1-alpha, either by DAPT or HIF1-inhibitor, confirmed the communication between these two pathways. Role of TrpC6 in metastasis was demonstrated by knocking down this gene using siRNA against TrpC6. Inhibition of TrpC6 markedly decreased cell proliferation, migration, angiogenesis and tumorigenesis in these hypoxia-induced Glioblastoma cells. In summary, all these results reveal that TrpC6 is indeed an important member of the Notch-mediated metastasis of Glioblastoma under hypoxic conditions. This role of TrpC6 can therefore be utilized for pharmacological intervention to prevent hypoxia-induced metastasis in GBM.
Show less - Date Issued
- 2008
- Identifier
- CFE0002485, ucf:47691
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002485
- Title
- THE ENDOCYTIC PROTEIN NUMB REGULATES APP METABOLISM AND NOTCH SIGNALING: IMPLICATIONS FOR ALZHEIMER'S DISEASE.
- Creator
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Kyriazis, George, Chan, Sic, University of Central Florida
- Abstract / Description
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Increased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is...
Show moreIncreased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is an evolutionarily conserved endocytic adapter involved in the internalization of transmembrane receptors. Mammals produce four Numb isoforms that differ in two functional domains, a phosphotyrosine-binding domain (PTB) and a proline-rich region (PRR). Recent studies showed that the PTB domain of Numb interacts with the cytoplasmic tails of APP and Notch but the functional relevance of these interactions with respect to AD pathogenesis is not clear. In the current studies, we proposed to investigate the biological consequences of the interaction of the Numb proteins with APP and Notch in neural cells stably overexpressing each of the four human Numb proteins. In the first part of our studies, we found that expression of the Numb isoforms lacking the insert in the PTB (SPTB-Numb) caused the abnormal accumulation of cellular APP in the early endosomes, and increased the levels of C-terminal APP fragments and A-beta. By contrast, expression of the Numb isoforms with the insert in PTB (LPTB-Numb) leads to the depletion of cellular APP and coincides with significantly lower production of APP derivatives and A-beta. The contrasting effects of the Numb isoforms on APP metabolism were not attributed to differences in the expression of APP nor the activities of the various APP-processing secretases. In the second part of our studies, we found that expression of SPTB-Numb protein enhances neuronal vulnerability to serum deprivation-induced cell death by a mechanism involving the dysregulation of cellular calcium homeostasis. Neural cells expressing SPTB-Numb exhibited enhanced Notch activity, which markedly upregulated the expression of transient receptor potential canonical 6 (TRPC6) channels enhancing calcium entry in response to store depletion. We also found that serum deprivation increased TRPC6 expression, mediating the serum deprivation-induced death in neural cells. Interestingly, expression of LPTB-Numb protein suppressed serum deprivation-induced activation of Notch and the subsequent upregulation of TRPC6 and cell death. Finally, we showed that the Numb proteins differentially impact Notch activation by altering the endocytic trafficking and processing of Notch. Taken together, these studies demonstrate that aberrant expression of the Numb proteins may influence APP metabolism and Notch-mediated cellular responses to injury by altering their endocytic trafficking and processing.
Show less - Date Issued
- 2008
- Identifier
- CFE0002233, ucf:47917
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002233
- Title
- Plasticity of Central and Peripheral Nervous System: Effects of Oxygen-Glucose Deprivation (OGD), Chronic Intermittent Hypoxia (CIH) and hSOD1 Overexpression.
- Creator
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Chen, Jin, Cheng, Zixi, Naser, Saleh, Singla, Dinender, University of Central Florida
- Abstract / Description
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Transient receptor potential canonical 6 (TRPC6) channels are permeable to Na+ and Ca2+ and are widely expressed in the brain. In this study, we investigated the role of TRPC6 following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal...
Show moreTransient receptor potential canonical 6 (TRPC6) channels are permeable to Na+ and Ca2+ and are widely expressed in the brain. In this study, we investigated the role of TRPC6 following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal death. Using live-cell imaging to examine intracellular Ca2+ levels ([Ca2+]i), we found that OGD induced a significant higher increase in glutamate-evoked Ca2+ influx compared to untreated control and such an increase was reduced by TRPC6 deletion. Enhancement of TRPC6 expression using AdCMV-TRPC6-GFP infection in WT neurons increased [Ca2+]i in response to glutamate application compared to AdCMV-GFP control. Inhibition of N-methyl-d-aspartic acid receptor (NMDAR) with MK801 decreased TRPC6-dependent increase of [Ca2+]i, indicating that such a Ca2+ influx was NMDAR dependent. Furthermore, TRPC6-dependent Ca2+ influx was blunted by blockade of Na+ entry. Finally, OGD-enhanced Ca2+ influx was reduced, but not completely blocked, in the presence of voltage dependent Na+ channel blocker tetrodotoxin (TTX) and dl???amino?3?hydroxy?5?methyl?4?isoxazole propionic acid (AMPA) blocker CNQX. Altogether, we concluded that I/R-induced brain damage was, in part, due to upregulation of TRPC6 in cortical neurons. We postulate that overexpression of TRPC6 following I/R may induce neuronal death partially through TRPC6-dependent Na+ entry which activated NMDAR, thus leading to a damaging Ca2+ overload. These findings may provide a potential target for future intervention in stroke-induced brain damage. Obstructive sleep apnea (OSA) is a highly prevalent sleep disorder that is associated with many cardiovascular complications, such as autonomic dysfunctions, stroke and heart failure. Chronic intermittent hypoxia (CIH) is a prominent feature of OSA. In CIH exposed rodents (a model for OSA), CIH induces the similar cardiovascular complications as seen in OSA patients. In particular, OSA impairs baroreflex control of the heart rate (HR), which is used as an independent indicator for heart failure. Since the baroreflex control arc includes the aortic depressor nerve (ADN), vagal efferent and central components, we hypothesize that CIH induces dysfunctions of all three components. Since mice can be genetically manipulated, an understanding of the effects of CIH on multiple neural components in the baroreflex arc in wild type mice may lead to a future study of treatments. In this study, we have examined the effects of CIH on baroreceptor afferent, central and vagal efferent components of the baroreflex circuitry in normal wild type C57BL/6J mice. Mice (4-5 months) were exposed to room air (RA) or CIH for 35-50 days and were then anesthetized with isoflurane, ventilated and catheterized for measurement of mean arterial blood pressure (MAP) and HR. Baroreceptor function was characterized by measuring percent changes of integrated ADN activity (Int ADNA) relative to the baseline value in response to the vasodilator sodium nitroprusside and the vasoconstrictor phenylephrine-induced changes in MAP. Data were fitted to a sigmoid logistic function curve. HR responses to electrical stimulation of the left ADN and the right vagus nerve were assessed under anesthesia. Compared with RA controls, CIH significantly increased maximum baroreceptor gain or maximum slope, maximum Int ADNA, and Int ADNA range (maximum-minimum Int ADNA). In addition, CIH maintained the maximum amplitude of the bradycardic response to vagal efferent stimulation. In contrast, CIH significantly reduced the maximum amplitude of bradycardic response to left ADN stimulation. Thus, CIH decreased central mediation of the baroreflex, but augmented the baroreceptor afferent function and maintained vagal efferent control of HR in mice. Excessive reactive oxygen species (ROS) (such as the superoxide radical) is commonly associated with cardiac autonomic dysfunctions. Though superoxide dismutase 1 (SOD1) overexpression may protect against ROS damage to the autonomic nervous system, superoxide radical reduction may change normal physiological functions. Previously, we demonstrated that human SOD1 (hSOD1) overexpression did not change baroreflex bradycardia and tachycardia, but increased aortic depressor nerve activity (ADNA) in responses to arterial pressure changes in C57B6SJL-Tg (SOD1)2 Gur/J mice. Since the barorelfex arc includes afferent, central and efferent components, the objective of this study was to determine whether hSOD1 overexpression alters the central and vagal efferent mediation of the heart rate (HR) responses. Our data indicate that SOD1 overexpression decreased HR responses to vagal efferent nerve stimulations but did not change HR responses to aortic nerve stimulation. Along with the previous study, we suggest that SOD1 overexpression preserves the normal baroreflex function but may alter the functions of aortic depressor nerve, vagal efferent and central components differently. While SOD1 overexpression likely enhanced aortic depressor nerve function and central mediation of bradycardia, it decreased vagal efferent control of HR. Currently, we are using the hSOD1 overexpressing mouse model to determine whether hSOD1 overexpression can preserve normal afferent, efferent, and central components of the baroreflex arc in the CIH model of sleep apnea.
Show less - Date Issued
- 2017
- Identifier
- CFE0006576, ucf:51334
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006576