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Zyxin is an adaptor protein recently identified as a novel regulator of the homeodomain-interacting protein kinase 2 (HIPK2)-p53 signaling in response to DNA damage. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer ‘s disease (AD), because of a deregulation of HIPK2 activity, leading to an impaired and dysfunctional response to stressors. Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 cells and SH-SY5Y neuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Aβ) 1-40 and 1-42 induce zyxin deregulation, thus affecting the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A, which is in turn responsible for the induction of an altered conformational state of p53. We demonstrate for the first time that zyxin is a novel target of Aβ activities in AD. These results may help the studies on the pathogenesis of AD, through the fine dissection of events related to beta-amyloid activities.
The protein p53 responds to a variety of cellular stresses and is able to sense the intensity of the damage and modulate biological responses, ranging from transient growth arrest to permanent replicative senescence or apoptosis (Vousden and Prives 2005). One important mechanism that controls p53 function is its conformational stability (Joerger and Fersht 2007). An altered protein conformational state of p53, independent from point mutations, has been reported in tissues from patients with Alzheimer's disease (AD) (Uberti et al. 2006; Lanni et al. 2008; Zhou and Jia 2010). When investigating the mechanism of such alteration, we found that soluble nanomolar concentrations of beta-amyloid (Aβ) 1-40 peptide induced the expression of an unfolded p53 protein isoform and modulated p53 functions by interfering with the homeodomain-interacting protein kinase 2 (HIPK2) (Lanni et al. 2007, 2010), a fundamental protein in maintaining wild-type p53 function (Puca et al. 2008). In particular, soluble Aβ 1-40 inhibited HIPK2 activity, consequently inducting an altered conformational state of p53, and thus resulting in an inability to properly activate an apoptotic program when cells are exposed to a noxious stimulus (Lanni et al. 2010).
A novel regulator of the HIPK2-p53 signaling in response to DNA damage, named zyxin, has been recently identified (Crone et al. 2011). Zyxin is primarily localized at the focal adhesion plaque complex as an adaptor protein (Beckerle 1997; Wang and Gilmore 2003) and contains a proline-rich domain at the N-terminus and three LIM domains at the C-terminus that are cysteine-rich motifs involved in protein–protein interactions. Zyxin is involved in regulating cell adhesion, spreading, and motility but also in transducing signals into the nucleus, to regulate gene expression, cell proliferation, differentiation, and apoptosis (Sadot et al. 2001; Wang and Gilmore 2001). Consistently, zyxin has been demonstrated to shuttle between the cytosol and the nucleus, where it affects transcriptional activity (Degenhardt and Silverstein 2001). In cancer cells, zyxin has been involved in DNA damage-induced cell fate control through the modulation of the HIPK2-p53 signaling. In particular, depletion of endogenous zyxin resulted in proteasome-dependent HIPK2 degradation, thus compromising DNA damage-induced p53 Ser46 phosphorylation dependent from HIPK2 (Crone et al. 2011).
A deregulation of HIPK2 has also been observed in cells treated with Aβ 1-40 (Lanni et al. 2010); however, the molecular mechanisms underlying HIPK2 proteasomal degradation in conditions related to Aβ-exposure need to be investigated. Considering that an altered proportion between Aβ 1-40/Aβ 1-42 as well as an increased production of one of the two species has been indicated as dangerous for the pathogenesis of AD (Verdile et al. 2004; Pangalos et al. 2005; Barten and Albright 2008), our purpose was to first investigate whether a difference in the activation of this pathway exists between the species 1-40 and 1-42, by evaluating the capability of these species to affect the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A (MT2A). Furthermore, based on data from literature demonstrating that zyxin expression is important in maintaining HIPK2 protein stability (Crone et al. 2011), our second goal was to determine whether a modulation of zyxin is involved in AD pathogenesis, using two cellular models of altered Aβ production. In particular, since in AD an altered metabolism of the amyloid precursor protein (APP) occurred, in turn leading to an aberrant production of Aβ peptides (Verdile et al. 2004), our purpose was also to investigate the effect of Aβ peptides on zyxin mRNA and protein levels. The results showed here may help to better understand the pathogenesis of AD, through the fine dissection of events related to Aβ activities. The characterization of Aβ activity on zyxin-HIPK2 signaling pathway represents a relevant characteristic, since it concerns a research field yet unexplored in a neurodegenerative context.
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We describe here a link between zyxin-HIPK2-p53 signaling pathway and Alzheimer's disease. We previously demonstrated the existence of Aβ-dependent HIPK2 deregulation responsible for the induction of an unfolded state of p53 protein in fibroblasts from AD patients leading to an impaired and dysfunctional response to stressor (Uberti et al. 2002; Lanni et al. 2008). On the contrary, the non-amyloidogenic product of APP metabolism, sAPPalpha, was demonstrated to be not involved in the regulation of the pathway resulting in conformationally altered p53 (Uberti et al. 2007). Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 and SH-SY5H neuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein. Recent findings show that zyxin expression is important to maintain HIPK2 protein stability (Crone et al. 2011). Our data suggest that intracellular Aβ peptides may be responsible for zyxin deregulation. This is supported by the observation that Aβ peptides down-regulated zyxin protein levels, compromising HIPK2 stability and thus leading to HIPK2 disappearance from target promoters such as MT2A. In agreement, MT2A mRNA up-regulation was found in HEK-APP cells that over-express APP751. The induction of MT2A, depending on HIPK2 knockdown has been reported to be responsible for p53 misfolding and inhibition of p53 transcriptional activity (Puca et al. 2009); therefore, the present data suggest that zyxin deregulation induced by Aβ peptides might be involved in HIPK2 degradation and in p53 misfolding via MT2A up-regulation in HEK-APP cells. This signaling pathway is further affected in a similar way by both soluble Aβ 1-40 and 1-42 at sublethal concentrations.
To investigate the contribution of APP metabolic products in the modulation of zyxin expression, we used HEK cells over-expressing wild-type APP able to generate high levels of Aβ 1-40 and Aβ 1-42 both intracellularly and secreted in the medium (Uberti et al. 2007). We found that reducing APP amyloidogenic metabolism by treating HEK-APP cells with β- and γ-secretase inhibitors (Lanni et al. 2010) prevented the deregulation of zyxin. It is worth to note that the conditioned medium of HEK-APP cells was able to affect untransfected HEK-293 cells recapitulating the HEK-APP phenotype, in terms of zyxin deregulation.
The data presented in this study suggest that the modulator effects of Aβ peptides on zyxin deregulation are, at least in part, due to the intracellular peptides. Different observations support this conclusion. First, considering that internalization of Aβ can be prevented under experimental conditions that do not allow endocytosis (Knauer et al. 1992), the conditioned media of HEK-APP cells treated with the specific neutralizing antibody 6E10, that recognizes the first 17 aa of the Aβ sequence, were unable to influence zyxin protein levels in HEK-293 cells. Second, we found that the synthetic Aβ peptides added to the cells crossed the plasma membrane, as previously demonstrated (Uberti et al. 2007).
The deregulation of zyxin was further analyzed in SH-SY5Y neuroblastoma cells and their counterpart over-expressing wild-type APP differentiated with retinoic acid. When treating differentiated SH-SY5Y cells with soluble Aβ peptides, both Aβ 1-40 and 1-42 were found to deregulate HIPK2 and decrease zyxin protein levels. Furthermore, also differentiated SY-APP cells are characterized by a decrease in zyxin protein levels, besides showing an HIPK2 down-regulation, thus recapitulating the HEK-APP phenotype in terms of zyxin and HIPK2 deregulation.
Zyxin is preferentially expressed in developing brain and various adult tissues, including lungs, spleen, and testis (Fujita et al. 2009). Depending on its subcellular location, zyxin can have anti-apoptotic or pro-apoptotic function, since zyxin has been shown to promote cell death downstream of apoptotic stimuli such as UV-C irradiation (Hervy et al. 2010), but it has also been implicated in Akt-dependent cardiomyocyte survival pathways (Kato et al. 2005). Zyxin has been extensively studied within a tumoral context, whereas very limited information is present at this time in the literature concerning its putative role in neurodegeneration. In this context, zyxin has been recently identified as an interacting partner for a protein, SIRT1, involved in protection from neurotoxicity in cell-based models for AD/tauopathies, amyotrophic lateral sclerosis and Wallerian degeneration, showing that the interaction of these proteins could be implicated in cellular survival, especially in the brain and heart, during physiological senescence (Fujita et al. 2009). Here, we established for the first time a link between zyxin and Alzheimer's disease. This observation is intriguing, since recent data from the literature support the concept that one or more common molecular mechanisms may be involved in the development of both neurodegenerative diseases and many cancers (Jope et al. 2007; Li et al. 2007; Lu and Zhou 2007; Alves da Costa and Checler 2011; Lanni et al. 2012). Taking into account that cancer and AD share common signaling pathways directing cell fate toward either death or survival, the identification of the putative common mechanisms may be useful to direct neurodegeneration studies toward the same intracellular pathways that have been successfully studied and targeted in cancer.
In summary, we hypothesize that soluble Aβ 1-40 and 1-42 may be responsible for important modulatory effects at cellular level before triggering the amyloidogenic cascade. For the first time, we demonstrated that soluble Aβ 1-40 and 1-42 modulate zyxin protein levels, fundamental in maintaining HIPK2 stability and in turn p53 activity. When zyxin is down-regulated by Aβ peptides, HIPK2 activity is inhibited, with MT2A up-regulation, in turn responsible for the induction of an altered conformational state of p53. As a result of this conformational change, p53 lost its transcriptional activity and was unable to properly activate an apoptotic program when cells were exposed to a noxious stimulus (Fig. 6). The reason why both Aβ 1-40 and Aβ 1-42 have the same effects on this pathway is at the moment under investigation in our laboratory. It is well known that Aβ 1-42 has been reported to aggregate faster than Aβ 1-40 and thus it is considered the most neurotoxic species (Verdile et al. 2004). Moreover, physiologically the 40-amino acid long peptide is the most abundant form (Terai et al. 2001; Kamenetz et al. 2003; Walsh and Selkoe 2007), since the concentration of secreted Aβ 1-42 is about 10% that of Aβ 1-40 (Bitan et al. 2003). However, in pathological conditions, the ratio of their production may be altered, as observed in familial AD cases (Mayeux et al. 1999). On the basis of this observation, one of the issues we should investigate is the differential modulation of this pathway by different concentration ratios of these two species. The sequence of events driven by beta-amyloid here described might contribute to AD pathogenesis since it may result in the presence of dysfunctional cells. Consistently, Yang and co-workers demonstrated the existence of aberrant neurons in AD brain by showing that neurodegeneration is correlated with neurons reentering a lethal cell cycle (Yang et al. 2001; Copani et al. 2007, 2008), which suggests that dysfunctional p53 in non-dividing cells may play a role in aberrant cell cycle progression. Furthermore, the observation that in AD are involved proteins controlling the duplication and cell cycle control leads to the speculation that, in senescent neurons, derangements in proteins commonly dealing with cell cycle control and apoptosis could affect neuronal plasticity and functioning rather than cell duplication.
Figure 6. Working hypothesis for a putative role of zyxin in Alzheimer's disease pathogenesis. The Figure indicates zyxin deregulation mediated by soluble beta-amyloid (Aβ) peptides. When zyxin is down-regulated by sublethal concentrations of Aβ 1-40 or Aβ 1-42, homeodomain-interacting protein kinase 2 (HIPK2) expression and activity are inhibited through degradation via the proteasome system. HIPK2 deregulation results in the induction of metallothionein 2A (MT2A), that exerts its Zn2+ chelator function. As a consequence, p53 changes the wild-type conformation to a conformationally altered status, with subsequent abolishment of wild-type p53 transcriptional activity.
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