Cellular and Molecular Science

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21707

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    Methamphetamine-induced activation of trace amine associated receptor (TAAR) 1 regulates astrocyte excitatory amino acid transporter (EAAT)-2 via differential CREB phosphorylation during HIV-associated neurocognitive disorders (HAND)
    (2016-03-23) Ghorpade, Anuja PhD; Cisneros, Irma
    Objective: Methamphetamine (METH) abuse commonly results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders. METH abuse exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for [greater than] 90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-kB, transcription factors activated by cAMP, calcium and IL-1b. Of these, cAMP and calcium are second messengers initiated via activation of TAAR1, that is upregulated by IL-1b. METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. Hypothesis: We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. Materials and Methods: To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference targeting cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Conclusions: Overall, this work revealed METH-induced differential CREB phosphorylation is critical for EAAT-2 regulation, and may serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.
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    IL-1Beta autocrine loop differentially regulates astrocyte inflammatory responses in HAND
    (2016-03-23) Ghorpade, Anuja PhD; Edara, Venkata Viswanadh
    Hypothesis: HIV-1 infection of the central nervous system (CNS) impairs brain function and leads to HIV associated neurocognitive disorders (HAND). Astrocytes are the most abundant cell type in CNS and provide structural and metabolic support under homeostasis and in diseases including HAND. It is well established that IL-1beta is regulated in an autocrine fashion. Given our prior work on astrocyte inflammatory responses in HAND, we sought to investigate the role of IL-1beta autocrine loop in differential outcomes. Materials and Methods: Successfully knocking down new synthesis of IL-1beta by RNAi, changes in levels of CXCL-8, TNF-alpha, AEG-1, EAAT-2, TAAR-1 and TIMP-1 were measured in response to IL-1beta stimulation. Astrocytes were infected with the help of doubly fluorescent labelled pseudotyped HIV-1, and latently infected ones were sorted. Results: As expected, RT2-PCR data confirmed that there was an increased mRNA expression of CXCL-8, TNF-alpha, TAAR-1 and TIMP-1 and no changes in EAAT-2 and AEG-1 levels. Conclusions and Future Directions: This suggests that IL-1beta autocrine loop likely plays a differential role in astrocyte inflammatory responses. Furthermore, we are particularly interested in differential response of healthy versus latently HIV-1-infected astrocytes that act as viral reservoir in CNS. Our long term goal is to delineate the specific role of IL-1beta autocrine loop in differential regulation of inflammatory responses in latently infected astrocytes. These studies are highly significant to address CNS reservoir issues in post ART HAND.
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    Potassium: a fifth “element” for the regulation of pluripotency and the cellular state in human pluripotent stem cells
    (2016-03-23) Koneru, Bhuvana; Shi, Yi; Zolekar, Ashwini; Dimitrijevich, Dan; Di Pasqua, Anthony; Wang, Jack; Lin, Victor
    Many inorganic elements are critically involved in the modulation of biochemical reactions and cell signaling pathways, suggesting that cells in unique states may display distinct elemental profiles and have specific requirements for different elements. Using X-ray fluorescence (XRF) spectrometry and inductively coupled plasma mass spectrometry (ICPMS) techniques, we measured the amounts of 56 major and trace inorganic elements in undifferentiated human pluripotent stem cells (hPSCs), their isogenic differentiated derivatives, and somatic cells used for cell reprogramming. While the amounts of most elements analyzed did not appear correlated with the pluripotent state of cells, the amount of potassium cation in undifferentiated hPSCs was significantly lower than that in multiple types of non-pluripotent cells. This phenomenon was reproducibly and consistently shown by both XRF spectrometry and ICPMS analyses in multiple hPSC lines and differentiated cells. Flow cytometry analysis using a cell-permeable fluorescence indicator for potassium, APG2-AM, also suggested that higher percentages of cells in pluripotent populations have a low level of intracellular potassium than those in non-pluripotent populations. To test whether the cellular pluripotency could be influenced by the manipulation of intracellular potassium, we used pharmacological tools to alter the permeability and intracellular concentration of potassium in hPSCs. The treatment with two potassium channel blockers, tetraethylammonium and 4-aminopyridine, increased intracellular potassium in human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), accompanied by the dose- and time-dependent downregulation of pluripotency markers POU5F1 and NANOG. In contrast, treatment with two types of potassium channel activators led to a decrease in intracellular potassium and the upregulation of POU5F1 and NANOG. Via 4-aminopyridine, we further exploited the link between cellular states and potassium thresholds, selectively eliminating hPSCs from differentiated derivatives within a dose window. Collectively, our data indicates that the amount of intracellular potassium is associated with the cellular states of hPSCs, and that the manipulation of intracellular potassium with pharmacological tools has functional impact on the regulation of pluripotency signaling in hPSCs. Potassium-altering agents may therefore be utilized in regenerative medicine for one of several purposes, including cellular purification and changing cellular identity. We demonstrate the first evidence that at the most basic level, a periodic element can be manipulated and have physiological, and potentially, therapeutic consequences.
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    Polymeric Nanoparticles for Gene Delivery to Human Astrocytes
    (2016-03-23) Labhasetwar, Vinod; Ghorpade, Anuja PhD; Joshi, Chaitanya
    Purpose: Currently available therapies for the treatment of neurodegenerative disorders (NDD) are inadequate. Challenges include low blood-brain barrier (BBB) permeability, brain structure complexity. Nanoparticles (NPs) and gene therapy are the two suggested approaches to overcome these problems. Small diameter of NPs (100-200 nm) may allow them to cross the BBB and gene therapy can target specific type of cells to alter gene regulation and cellular function. In this study, we combined both approaches and tested gene delivery to astrocytes, the principal type of glial cells in the brain, via two NPs formulations. Methods: A5P50, an Arginine-based polyethylenimine (PEI) polymer and poly-lactic-co-glycolic-acid (PLGA) NPs were tested for their gene delivery potential in primary human neural cells and cell lines. AFM imaging was carried out to determine A5P50 and PLGA NP dimensions. CMV- or GFAP-promoter-driven luciferase reporter plasmids (pGL3) served as test genes. Cytotoxicity was measured using MTT, LDH, and DNA fragmentation assays. Luciferase assay and Yoyo-dye labeling was used to evaluate the efficiency of gene delivery. Results: FDA-approved, biodegradable PLGA NPs were able to deliver pGL3 across the cell membrane in astrocytes. However, pGL3 expression was negligible or absent. In parallel, A5P50 successfully delivered and expressed the pGL3 in all tested cell types including astrocytes. But, it was not optimally biocompatible in human neurons at higher treatment concentrations. Then, both NPs were used in combination and a significant change in delivery and expression was seen in all types of cells including astrocytes. AFM imaging showed that the size of NPs remained similar when combined indicating absence of direct interaction. Live imaging with Yoyo-labeled pGL3 indicated that presence of A5P50 facilitated PLGA-mediated pGL3 delivery across the nuclear membrane by an unknown mechanism. Conclusions: A5P50-PLGA-combination system was successfully used for gene delivery to astrocytes as well as other cell types. Low A5P50 concentration in the combination eliminated biocompatibility issues in human neurons. Further in vivo testing is necessary to establish this system for future therapeutic use. Presented in vitro results are promising to progress in that direction.