Nef mediated learning impairment is associated with a TGF‐β inflammatory environment in astrocyte cultures in vitro

Abstract

The worldwide epidemic of HIV has been ameliorated with the development of antiretroviral therapies. Modern treatment has been efficient in impairing viral replication, resulting in an undetectable viral load in patients. Unfortunately, the process of viral protein production is still functional contributing to detrimental conditions such as HIV associated neurocognitive disorders (HAND). Recent studies have associated HAND with astrocyte mediated toxicity of neurons, stimulating further investigations regarding the mechanisms of these effects. Studies conducted in our lab have shown loss of neurons and deficiency in the spatial learning of rats infused with astrocytes expressing the viral protein Nef. The release of inflammatory cytokines and modulation of TGF‐b pathway could be a plausible explanation for the pathology herein observed. We hypothesize that astrocytes transfected with Nef increase the expression of TGF‐b and promote the secretion of inflammatory cytokines. Therefore, we aim to investigate the possible molecular explanation to the relation between learning impairment, inflammation and modulation of TGF‐b pathway in the presence of HIV‐Nef. In order to verify the production of these factors, astrocytes were transfected by electroporation with a Nef or GFP plasmid. During the following 48 and 72 hours samples were collected and assessed by western blot and immunofluorescence. Resulting from these studies we found an increased expression of iNOS in supernantants and increased expression of TGF‐b in lysates of astrocytes expressing Nef compared to GFP controls. These results were more noticeable at 48 hours. We also observed that cellular localization of TGF‐b varied depending on treatment. In astrocytes transfected with GFP, TGF‐b labeling was primarily nuclear whereas in astrocytes transfected with Nef there was a distribution of the labeling more towards the cytoplasm. These results suggest a stimulation of a pro‐inflammatory environment by HIV‐Nef that could lead to harmful effects to neurons. We are also conducting in vitro co‐cultures of neurons exposed to astrocytes expressing Nef to study the extent of the inflammation and toxicity in this environment. Because TGF‐b can be either beneficial or negative to cell processes and cell response can vary depending on the type of TGF‐b receptor, further studies include understanding more in depth activation of the pathway. Studies have shown that the production of TGF‐b ligand can correlate directly or inversely to the receptors. Thus we are interested in measuring SMADs proteins, expression of TGF‐b receptors, and include a TGF‐b receptor blocker to better understand the role of this pathway.Support or Funding InformationR25GM082406, GM106970, MD007579