14 March 2004. Researchers who follow the ups and downs of protein expression in Alzheimer’s disease (AD) may well think they are being taken on a roller coaster ride. Early on, many proteins are upregulated, and at later stages, they become downregulated. In particular, members of signaling pathways implicated in AD—such as tumor necrosis factor (TNF) α and mitogen-activated protein kinase/c-jun N-terminal kinase (MAPK/JNK)—are known to vary in their expression levels during disease states, and are suspected of contributing to disease progression. Most recently, Keith Del Villar and Carol Miller, from the University of Southern California Keck School of Medicine in Los Angeles, reported in the March 8 PNAS that expression changes in two such players may pave the way for neuronal apoptosis in AD. Their names are a mouthful: Expression of the TNF receptor (TNFR1) binding protein DENN/MADD (aka Differentially Expressed in Normal versus Neoplastic/MAPK Activating Death Domain) is diminished in AD tissue, while TNFR1-Associated Death Domain (TRADD) is upregulated. The scientists propose that this reduction of DENN, which competes with TRADD for TNFR1 binding, might clear the way for TRADD to activate these key signaling pathways and trigger cell death.
The researchers first set out to characterize expression of DENN in AD brain hippocampal tissue. Both immunohistochemical and Western analyses revealed diminished DENN expression especially in neurons burdened with neurofibrillary tangles (NFTs), leading the authors to link AD pathology with DENN expression. Looking at a widely studied mouse model, they found that cortical extracts from Tg2576 mice also exhibited reduced DENN expression.
What led to this decline in DENN expression? To answer this question Del Villar and Miller exposed neuronal cultures to Aβ and after one day observed a persistent 70 percent decrease in DENN expression, along with the nuclear translocation of activated JNK (P-JNK). Furthermore, inhibiting this endogenous DENN expression with antisense DENN depletion (DENN-AS) not only reduced the expression of DENN, but also induced cell death, suggesting to the authors that DENN expression may be critical for cell survival.
In both the AD-affected human brain tissue and neuronal cultures studies, the researchers noted increases in TRADD, TNFR1, and P-JNK expression, all members of the TNFalpha signaling pathway, implicating them in the observed cell death. According to the authors, this increased TRADD expression may make it more available to bind TNFR1 and thus induce the TNFα signaling pathway, leading to cell death. To examine this further, Del Villar and Miller devised a competition assay, in which they co-expressed DENN, TRADD, and TNFR1 in N2A cells, and used immunoprecipitation to determine the interactions among these proteins. The immunoblots indicated that DENN and TRADD bind TNFR1 competitively, and the authors argue that DENN, which also colocalized with TRADD immunohistochemically, might be important in protecting cells against TRADD-induced apoptosis.
Thus, the researchers conclude, downregulation of DENN in an AD neuron may contribute to the characteristic decrease in the neuron’s viability, as DENN would bow out of the competition and allow TRADD to modulate TNFR1 signaling in response to stress, pushing the neuron toward death.—Erene Mina.
Del Villar K, Miller CA. Down-regulation of DENN/MADD, a TNF receptor binding protein, correlates with neuronal cell death in Alzheimer's disease brain and hippocampal neurons. Proc Natl Acad Sci U S A. 2004 Mar 8 [Epub ahead of print] Abstract