16 March 2010. A new study reveals an intriguing link between two top suspects in schizophrenia, neuregulin-1 (NRG1) and disrupted in schizophrenia-1 (DISC1). Published in PNAS online on March 8, the study reports two surprises: NRG1 signaling boosts levels of an isoform of DISC1 protein, and DISC1 itself is found inside multiple types of glial cells in addition to its better known location inside neurons. These findings suggest a web of complex interactions—both between molecules and between different cell types—in the disorder.
The result of a four-way collaboration among the laboratories of Akira Sawa and Philip Wong, both at Johns Hopkins University, Baltimore, Maryland; Eva Anton at the University of North Carolina, Chapel Hill; and Carsten Korth at Heinrich Heine University of Düsseldorf, Germany, the study outlines a chain of events leading from NRG1 to DISC1. In doing so, they also find a bonus link with BACE1 (β-site amyloid precursor protein cleaving enzyme), an Alzheimer disease-related protease that is of interest to schizophrenia researchers because it cleaves NRG1 (see SRF related news story).
Although variations in the genes for NRG1 and for DISC1 are well-known risk factor candidates for schizophrenia, the abnormal behaviors of transgenic mice led first author Saurav Seshadri and colleagues to explore a relationship between the two. Mice deficient in NRG1, DISC1, or BACE1 all have problems with working memory and prepulse inhibition, behavioral abnormalities also found in schizophrenia. These behavioral overlaps suggested that the molecules themselves may be somehow connected—something not too hard to imagine given that NRG1 is a nexus of molecular interaction. When cleaved by BACE1, NRG1 is freed to bind with its receptors, the ErbB receptors. These activated ErbB proteins trigger intracellular signaling cascades, which, among other things, promote cell growth, proliferation, and differentiation—all important elements for building a brain.
The researchers began by examining DISC1 protein expression in vitro. When NRG1 or its cousin NRG2 was applied to immature neurons taken from rat brains and maintained in culture, levels of a 130-kDa isoform of DISC1 went up; treatment with NRG3, by contrast, had no effect. The effect was most pronounced for NRG1, amounting to a 1.77-fold increase in DISC1 over that found in untreated cells. This stemmed mainly from increases in DISC1 in neurites, the immature processes branching out of neurons that ultimately wire them up to other neurons.
Next, the researchers probed which ErbB receptors mediated this effect, using RNAi to systematically knock down levels of ErbB2, ErbB3, or ErbB4 in vitro. Neuron cultures without ErbB2 or ErbB3 could not translate treatment with NRG1 into an increase in the DISC1 isoform, indicating that these receptors were required to boost its expression. NRG1 treatment did not raise the DISC1 isoform's expression when the PI3K/Akt intracellular signaling pathway was blocked with a drug, or when transcription was halted with actinomycin D. Together these findings suggest that NRG1 elevates this DISC1 isoform's expression by binding ErbB2 and ErbB3 receptors, which in turn activate the PI3K/Akt pathway downstream and, somewhere along the line, transcription occurs.
More evidence for this NRG1-DISC1 connection came from mice with low levels of NRG1. NRG1-deficient mice had substantial decreases in the 130-kDa DISC1 isoform: a mouse heterozygous for a null mutation in NRG1 had a 23.6 percent decrease in the isoform compared to wild-type controls, and homozygotes, which do not survive beyond embryonic day 11, also had marked reductions in the isoform relative to controls. When the researchers looked at BACE1 knockouts—mice originally designed to study Alzheimer disease—they confirmed that there was less cleaved NRG1 around to bind ErbB receptors. These animals had a 44.1 percent decrease of the DISC1 isoform in the cortex compared to controls, due mainly to a lack of the protein in neurites.
Because NRG1 signaling occurs both in neurons and glia, the researchers looked for DISC1 in glia. Using an antibody to human DISC1, not only did they find DISC1 protein in neurons of postmortem human brain tissue, they also found it in multiple types of glial cells, as measured by staining for DISC1 that colocalized with markers for astrocytes, oligodendrocytes, and microglia. The same applied for rat cells in culture, and these findings widen the scope of DISC1's possible sites of action in the brain.
Though the researchers were able to home in on the 130-kDa DISC1 isoform using two specific antibodies and RNAi, further studies will have to elucidate the exact nature of this isoform. The study also redirects attention to ErbB2 and ErbB3 receptors in schizophrenia, which have not been as tantalizing as ErbB4 receptors (see Chong et al., 2008). The authors note that ErbB2 lacks an extracellular binding site, and ErbB3 is missing the intracellular tyrosine kinase site necessary for activation. Together in a heterodimer, these molecules may successfully transduce NRG1 binding. Overall, the study demonstrates the potential dividends of looking beyond a pet molecule or cell type, particularly at other disease gene candidates.—Michele Solis.
Seshadri S, Kamiya A, Yokota Y, Prikulis I, Kano SI, Hayashi-Takagi A, Stanco A, Eom TY, Rao S, Ishizuka K, Wong P, Korth C, Anton ES, Sawa A. Disrupted-in-Schizophrenia-1 expression is regulated by beta-site amyloid precursor protein cleaving enzyme-1-neuregulin cascade. Proc Natl Acad Sci USA. 2010 March. Abstract