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Quantification of TGFβ1 and Inflammatory Cytokines in Neurons Exposed to Astrocytes Transfected with Nef
Author(s) -
MartínezOrengo Neysha,
Velázquez Bethzaly,
Vargas Valerie,
Noel Richard
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.835.6
Subject(s) - transfection , transforming growth factor , astrocyte , immunology , microglia , medicine , neuroscience , microbiology and biotechnology , biology , inflammation , cell culture , genetics , central nervous system
Neurocognitive impairments affect 30‐50% of all HIV patients receiving antiretroviral treatment. Even when the therapy maintains viral loads under detectable levels, viral proteins such as Nef continue to be produced in distinct reservoirs. Our previous work with an animal model of Nef neurotoxicity suggests that TGFβ1 is linked to learning impairments and correlates with increased CCL2 expression. To further investigate the effect of Nef on TGFβ signaling and pro‐inflammatory cytokines we used a co‐culture system. Primary neurons were grown to confluence, at which time primary astrocytes transfected with either Nef or GFP were placed on culture inserts in each well containing primary neurons. After 24 and 48 hours, RNA was isolated and RT‐PCR was used to measure gene expression of TGFβ1, TGFβR1, TGFβR2, IL6, and TNF. TGFβ1 ligand and receptors gene expression appear to be higher in astrocytes after 24 hours while in neurons an increased expression is seen at 48 hours. On the other hand, TNF and IL6 were higher expressed in neurons when compared to astrocytes and control. These data indicates that expression of Nef in astrocytes alone is capable of activating the TGFβ1 gene while inducing inflammation in neurons, which may result in neuronal damage. Deciphering if and how Nef alters the TGFβ signaling cascade can help understand the molecular processes involved in HIV‐associated neurocognitive disorder. Further experiments will include analysis of protein levels as well as the effects of downregulation of TGFβ receptors. This research was supported by the MBRS‐RISE Program (R25GM082406), Dr. Noel's Lab (GM106970) and the PSM Molecular Biology Core Lab (HD007579).