Browsing by Author "Nguyen, Derek"
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Item Alterations in Neural Stem Cell Fate Following Focal Ischemia(2014-02-04) Nguyen, Derek; Vemireddy, Vamsidhara; Mashimo, Tomoyuki; Battiste, James; Maher, Elizabeth; Bachoo, RobertINTRODUCTION: The purpose of this experiment is to determine the differentiation identity of the neural stem cells (NSC) in the subventricular zone (SVZ) of adult mouse brain after a middle cerebral artery occlusion (MCAO). Injury to the brain causes a large number of changes including inflammation and apoptosis, but the reaction of NSC's has been more difficult to characterize because of the transient nature of their response. Previously, adult neural stem cells (NSC) in the SVZ have been observed to differentiate predominantly into cells with neuronal characteristics. This theory is questioned via a tamoxifen-inducible cre-recombinase (Cre-ERT2) expression mouse model system. METHOD: The Cre-ERT2 expression mouse model system is driven by the Cystatin-C promoter to label NSC's in a time specific manner and track their cell fate after MCAO. After the ischemia, these brain sections were stained with different immunohistochemicals at three separate time points. One set was co-labeled with GFAP, an astrocyte marker, and BrdU, a proliferation marker. Another set was co-labeled with DCX, a neuronal marker, and BrdU. This was used to differentiate between latent NSCs and proliferating NSCs by comparing the ipsilateral side (ischemic) with the contralateral side (control) of the brain. RESULTS: Compared to the contralateral, the ipsilateral side had a significant increase in GFAP/BrdU positive cells between day 3 and day 7 time points. The cell quantity dropped between day 7 to day 14 time points. Compared to the contralateral, the ipsilateral side had a decrease in DCX/BrdU positive cells between day 3 and day 7 time points. The cell quantity significantly increased between day 7 to day 14 time points, and the quantity at day 14 was about twice to that of the day 3 time point. DISCUSSION: This data demonstrated that after the MCAO, the stem cells are not just undergoing neurogenesis, but are for certain period of time, also differentiating into astrocytes that are migrating towards the site of injury. This phenomenon is only witnessed in the NSCs towards the day 7 time point. Afterwards and leading up to day 14, the NSCs seem to be changing their cell fate programming from the astrocyte pathway back to the intended neuronal pathway. Thus, the staining results verify that after an ischemia, NSCs within the SVZ regions of the brain undergo a constant change of programmed cell fate, alternating between immature neurons and astrocytes implicating future aims for "programmed" neurogenesis in the development of therapeutic strategies for the treatment of brain damage and disease.Item Reciprocal Interactions of STIM1 with Orai1 and L-Type Ca2+ Channels in Cardiac Myocytes(2015-01-26) Nguyen, Derek; Luo, Xiang; Morales, Cyndi; Li, Dan; Gillette, Thoams; Hill, JosephBACKGROUND: Pathological cardiac hypertrophy can be triggered by abnormal Ca2+ levels. It has been shown that the mechanisms governing context-dependent changes in Ca2+ influx are linked to stromal interaction molecule 1 (STIM1). STIM1 is a sarcoplasmic reticulum Ca2+ sensor that regulates Ca2+ influx by directly activating store operated calcium channels such as Orai1 in response to stress such as intracellular Ca2+ depletion. STIM1 is also known to regulate L-type Ca2+ channels in cardiomyocytes, though the mechanism has not been elucidated. HYPOTHESIS: The Ca2+-sensing protein, STIM1, molecularly interacts with Orai1 and L-type Ca2+ channels in cardiomyocytes to facilitate Ca2+ influx as a master regulator of Ca2+ channels. METHODS: To address this, we monitored STIM1 interactions with either Orai1 or L-type Ca2+ channels in neonatal rat ventricular cardiomyocytes (NRVMs) and adult rat ventricular cardiomyocytes (ARVMs) using the Duolink in situ fluorescence assay. This technique utilizes proximity ligation assay technology to directly monitor interactions between proteins within 40nm. These channel interactions were observed under endogenous culture conditions as well as thapsigargin-mediated calcium store depleted conditions. Differences in STIM1 interactions between control (untreated) and thapsigargin-treated NRVM and ARVM cells were observed. RESULTS: In NRVM cells, STIM1 interactions with Orai1 increased by 44.79 ± 2.68% when treated with thapsigargin as compared to the control population (n=73-83). Conversely, STIM1 interactions with L-type Ca2+ channels decreased by 52.31 ± 3.45% compared to the control (n=64-69). In ARVM cells, STIM1 interactions with Orai1 decreased by 59.19 ± 2.39% when treated with thapsigargin as compared to the control population (n=11-16). STIM1 interactions with L-type Ca2+ channels on the other hand increased by 74.13 ± 0.21% compared to the control (n=11-19). CONCLUSIONS: We observed an opposite trend in STIM1 interactions with Orai1 and L-type Ca2+ channels in NRVM and ARVM cells. When treated with thapsigargin, their degree of interaction changed significantly. This would suggest that STIM1 undergoes dynamic changes in response to calcium conditions in order to regulate Ca2+ influx. At a molecular level, it would seem that STIM1 is a versatile Ca2+ channel regulator as it can alternately interact with both channels. Further studies will aid in the development of novel therapeutic strategies for the treatment of cardiac hypertrophy and disease.