It has been suggested that AICD has an important biological role in the development of AD pathology through its ability to act as a cleavage product with transactivation potential (Cao and Sudhof, 2001
). Until now, few AICD-dependent candidate genes had been described, and a genome-wide approach to screening was lacking. In this study, we established an inducible neuroblastoma cell culture model with AICD, FE65, or AICD and FE65 under control of a tetracycline-responsive promoter. Analysis of transcriptional changes after expression of AICD and/or its cofactor FE65 by using Affymetrix U133A GeneChips revealed a list of 182 putative AICD target genes (see Supplemental Table).
Real-time quantitative PCR of selected genes demonstrated that some genes were dependent on induction of AICD alone; others showed elevated expression when FE65 was coexpressed, whereas a third subset was only differentially regulated after expression of both AICD and FE65.
Fe65-independent signaling might be explained by direct binding of AICD to TIP60 (Kinoshita et al., 2002
). However, the majority of target genes described in this work demonstrated a requirement for coexpression of FE65, suggesting an important role for this binding protein in AICD-dependent gene expression, an observation that has also been reported by other studies (Cao and Sudhof, 2001
; von Rotz et al., 2004
). Indeed, there is evidence that binding of FE65 to AICD is necessary for the nuclear translocation (Kimberly et al., 2001
; Muresan and Muresan, 2004
; von Rotz et al., 2004
). Two different AICD transcriptionally active complexes based on the binding of AICD-FE65 to TIP60 and to CP2/LSF/LBP1 have been suggested (Cao and Sudhof, 2001
; Kim et al., 2003
). TIP60 is part of a large nuclear complex with DNA binding, ATPase and DNA helicase activity (Ikura et al., 2000
). In Gal4 reporter gene experiments, the ternary complex AICD-FE65-TIP60 is a potent transactivator, dependent on the expression level of FE65 (Cao and Sudhof, 2001
). An alternative mechanism based on structural modifications of FE65 by AICD has been suggested that does not require AICD translocation from the cytoplasm to nucleus (Cao and Sudhof, 2004
). This hypothesis is not in contrast to our findings, because elevated cytosolic levels of AICD might be sufficient to cause a change in the conformation of FE65. This second ternary complex (AICD-FE65-CP2/LSF/LBP1) was suggested to modulate expression of glycogen synthase kinase-3β (Kim et al., 2003
). Therefore, different AICD transcriptional complexes might explain different regulation patterns.
Long-time induction of ectopically expressed genes may result in secondary regulation effects as primary induced genes itself could act as activator for other genes. Therefore, we used 72-h induction (dox washout) for our studies corresponding to the earliest time point of measurable induction of AICD and/or FE65. Under these conditions we did not observe differential regulation of previously described AICD target genes such as KAI1 (). We could not detect regulation of other putative target genes, including SERCA2B (Leissring et al., 2002
) or GSK3β (Kim et al., 2003
). However, our data are in agreement with other groups who could not detect AICD-dependent regulation of these genes in AICD/FE65 transgenic mice (Ryan and Pimplikar, 2005
) or in cell lines with pharmacological inhibition or deficiency of γ-secretase activity (Hebert et al., 2006
). Furthermore, it is probable that differential gene expression of AICD targets occurs in a cell type-specific manner and is dependent on expression levels of endogenous cofactors. Our work is the first study analyzing AICD-dependent gene expression in stable transfected SHEP-SF neuroblastoma cells. Importantly, primary cortical neurons transiently transfected with AICD50/FE65 also demonstrated increased expression of TAGLN, TPM1 supporting the hypothesis of a role for AICD in regulating the expression levels of these genes. Moreover, given the emerging evidence for a role for AICD signaling in the pathophysiology of AD, we were able to verify differential expression of several of the AICD target genes described in this work in the frontal cortex of Alzheimer disease versus control brains. α2-Actin, IGFBP3, and TAGLN were significantly higher expressed in AD brains compared with their age-matched control. Interestingly, IGFBP3 has previously been described to be associated with neurofibrillary tangles in AD brains (Rensink et al., 2002
). Furthermore, other groups have also reported differential expression of PRKC (Par-4) (El Guendy and Rangnekar, 2003
) and TPM1 (Galloway et al., 1990
) in AD brain samples compared with brains from unaffected patients. Moreover, neuritic plaques positive for neuregulin (Chaudhury et al., 2003
) and fibronectin 1 (Van Gool et al., 1994
) have been shown by immunohistochemistry. Given that several of our AICD target genes have previously demonstrated differential expression levels during AD progression, it is interesting to postulate that increased expression of AICD, and of its targets, might play an important role in AD. In contrast, we could not confirm differential regulation for KAI1 and other discussed target genes in AD brains versus controls.
Among the AICD-dependent regulated genes, we identified several candidates that have previously been described as regulators of actin dynamics, including α2-Actin, Transgelin, and Tropomyosin 1. Transgelin, an actin cross-linking protein, is involved in the organization of the actin cytoskeleton and is thought to link aging to actin stability (Goodman et al., 2003
; Nakano et al., 2005
). Other genes as Fibronectin1 and RAB3B were also described to be associated with cytoskeletal organization. Visualization of F-actin structures in neuroblastoma cells as well as in cortical neurons after AICD and FE65 expression revealed disorganization of actin fibers within the cell body and clumping of actin fibers at the periphery, suggesting increased actin dynamics. Given that this phenotype was absent in cells expressing FE65 without AICD, our results suggest that this signaling occurs independent of the family of Ena/Vasp proteins, which interact with the WW domains of FE65 proteins (Ermekova et al., 1997
). In contrast, our data suggest that AICD dependent transactivation is responsible for the reorganization of actin filaments, and hence may play an important role during development and in reorganization of cellular structures in response to injury. Moreover, APP itself is transcriptionally up-regulated in response to nerve transections, trauma, and cerebral ischemia in the nervous system (Banati et al., 1993
; Shi et al., 2000
; Ciallella et al., 2002
), all processes that result in cytoskeletal reorganization. In Drosophila
, APP expression is associated with increases in postdevelopmental axonal arborization and loss of neuronal connections, which was strictly linked to the intracellular domain of APP (Leyssen et al., 2005
). Moreover, Tg2576 transgenic mice from as early as 4 mo old are positive for rod-like inclusions in the posterior cortex which are composed of actin (Maloney et al., 2005
). A model in which APP and FE65 are involved in the complex regulation of actin-based growth cone motility is also supported by other data (Sabo et al., 2003
). Interestingly, blockage of axonal transport, which might be a result of disorganized cytoskeleton, is the earliest measured disturbance in the brains of transgenic mice expressing mutant human APP (Stokin et al., 2005
). Furthermore, recent studies demonstrate that meningeal fibroblasts from FE65−/−
mice reveal a disturbed F-actin pattern compared with wild-type mice (Guenette et al., 2006
). Together, our study suggests an important role of AICD in the regulation of cellular actin dynamics.