In response to oxidative stress, the nuclear factor E2-related factor 2 (Nrf2) transcription factor translocates from the cytoplasm into the nucleus and transactivates expression of genes with antioxidant activity. Despite this cellular mechanism, oxidative damage is abundant in Alzheimer and Parkinson disease (AD and PD). To investigate mechanisms by which Nrf2 activity may be aberrant or insufficient in neurodegenerative conditions, we assessed Nrf2 localization in affected brain regions of AD, Lewy body variant of AD (LBVAD), and PD. By immunohistochemistry, Nrf2 is expressed in both the nucleus and the cytoplasm of neurons in normal hippocampi with predominant expression in the nucleus. In AD and LBVAD, Nrf2 was predominantly cytoplasmic in hippocampal neurons and was not a major component of beta amyloid plaques or neurofibrillary tangles. By immunoblotting, we observed a significant decrease in nuclear Nrf2 levels in AD cases. In contrast, Nrf2 was strongly nuclear in PD nigral neurons but cytoplasmic in substantia nigra of normal, AD, and LBVAD cases. These findings suggest that Nrf2-mediated transcription is not induced in neurons in AD despite the presence of oxidative stress. In PD, nuclear localization of Nrf2 is strongly induced, but this response may be insufficient to protect neurons from degeneration.
Transmembrane-signaling events are mediated and regulated by protein-protein interactions. The yeast two-hybrid screen has proven to be an effective approach for studying interaction between signaling molecules, such as ras and raf. This approach can be used to identify new binding partners for a protein of interest or define the interaction domains and relative affinity between two proteins known to interact. To determine interaction, one protein is produced as a fusion protein with a known DNA-binding domain and a second protein is produced as a fusion protein with an acidic activation in yeast. If there is interaction between the two proteins of interest, the DNA-binding domain is brought into the vicinity of the acidic-activation domain, which recreates a functional transcriptional activator, which drives transcription of reporter genes allowing for selection and/or quantification of interaction between the two proteins. Here we describe a two-hybrid yeast system that has been used to successfully characterize protein interactions among signaling molecules.
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