Cellular and Molecular Science
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21731
Browse
Browsing Cellular and Molecular Science by Author "Ghorpade, Anuja"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item METH-induced, TAAR1-associated CREB signaling serves as a master regulator for astrocyte EAAT-2(2015-03) Cisneros, Irma E.; Ghorpade, AnujaMethamphetamine (METH) abuse accelerates the onset and severity of HIV-associated neurocognitive disorders (HAND) and astrocyte-mediated excitotoxicity. METH targets several receptors, particularly astrocyte trace amine associated receptor 1 (TAAR1), as we have previously reported. Molecular alterations of astrocyte TAAR1 correspond to changes in astrocyte excitatory amino acid transporter-2 (EAAT-2) levels and function; however, the signaling pathways downstream of METH-induced TAAR1 activation remain unclear. Astrocyte EAAT-2 is tightly regulated at the transcriptional and translational levels by cAMP and calcium, yet METH-mediated increases in these second messengers have not been shown to directly modulate astrocyte EAAT-2. Furthermore, HIV-1 relevant stimuli and IL-1b, increase TAAR1 and may exacerbate METH-mediated excitotoxicity via MAPK/ERK and NF-kB. We propose CREB activation serves as a master regulator of astrocyte EAAT-2. To investigate the temporal order of CREB activation we utilized genetically encoded calcium indicators, or GCaMPs, to visualize and quantify METH-induced calcium signaling. RNA interference targeting PKA and NF-kB subunit p65, in addition to PKA and MAPK/ERK specific inhibitors support their involvement in astrocyte EAAT-2 regulation. Furthermore, we investigated CREB phosphorylation at serine 133/142, the co-activator and co-repressor forms, respectively, following METH-induced activation. Overall, this work identifies critical signaling pathways and therapeutic targets for astrocyte EAAT-2 recovery.Item Post-Transcriptional Regulation of Astrocyte-Tissue Inhibitor Metalloproteinase-1 (TIMP-1) in HAND(2015-03) Thete, Mayuri V.; Ghorpade, AnujaPurpose (a): HIV-1 can lead to several central nervous system impairments together termed HIV-1-associated neurocognitive disorders (HAND). In acute versus chronic neuroinflammation, differential regulation of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) is relevant to HAND neuropathogenesis. However, the underlying mechanisms are still being uncovered. In our study, we investigated the post-transcriptional regulation of TIMP-1 3’UTR via miRNAs. Method (b): Microarray analysis was used to analyze miRNA changes in IL-1β-activated astrocytes. To investigate miRNA-mediated TIMP-1 3’UTR post-transcriptional regulation, TIMP-1 3’UTR and specific miRNA overexpression constructs were used. Primary human astrocytes were nucleofected with TIMP-1 3’UTR and Pmir 146b/Pmir 155, and treated with IL-1b 24 h post-transfection. Firefly luciferase activity and astrocyte TIMP-1 levels were analyzed in parallel experiments 24 and 72 hours after IL-1β treatment. Result (c): Microarray analysis showed an increase in 12 miRNAs and decrease in 4 miRNAs. Seven of those were further confirmed by RT-PCR. The most consistent increase was observed in miRNA 155 and miRNA 146b. Overexpression of miRNA 155 and miRNA 146b in IL-1β-activated astrocytes decreased both luciferase activity and endogenous TIMP-1 levels. Conclusion (d): In summary, our preliminary studies suggest that astrocyte-TIMP-1 may be regulated post-transcriptionally by miRNAs (146b and 155) during HAND.