The aim of this study was to assess TG2 and its isoform expression levels in both OECs exposed to full native peptide of Aβ(1–42) and its toxic fragment Aβ(25–35). Epidemiological evidences report that the effects of Mediterranean Diet “MeDi” could be an alternative prophylaxis treatment for AD . In particular, it has been identified in Sicily an increased frequency of centenarians, a reduced occurrence of mental and cognitive diseases, when compared with other Italian or European regions [19, 20]. One of the factors that could contribute to this phenomenon is the large availability of some rare specific nutrients, largely present in some area of Sicily, as well as Indicaxanthin from Opuntia ficus-indica fruit. Therefore, for the first time, we tested the effect of Indicaxanthin pre-treatment on OECs exposed to Aβ. Since cytoskeleton plays an important role in the pathogenesis of neurodegenerative diseases, including AD , particular attention was focused on the effect of Indicaxanthin on some cytoskeletal proteins, such as Vimentin, GFAP, that have an important role in astrogliosis, a typical sign of AD . Furthermore, the expression levels of cyclin D1, which is induced in stem cell reprogramming and is co-expressed with Nestin, marker of neural stem cells , were assessed. In addition, the effect of Aβ(1–42), Aβ(25–35) and Aβ(35 − 25) in the absence and in the presence of Indicaxanthin was tested on cellular viability and on the activation of apoptotic pathway. Intracellular total ROS and O2− production was also evaluated. The experiments were performed on OECs because they represent a glial population of olfactory system that is also involved in AD . It is note that olfactory dysfunction, as well as hyposmia and olfactory memory loss, represent the early symptoms of AD [18, 35, 36]. Furthermore, it has been demonstrated that anterior olfactory nucleus (AON) projects to hippocampus  and that it is the earliest site involved in AD, associated with cell loss, the neurofibrillary tangles and senile plaques .
Previous our researches demonstrated that TG2 is overexpressed in OECs exposed to Aβ(1–42) and its toxic fragment Aβ(25–35) and that the treatment with some Growth Factors (GFs) was able to restore its levels to control values . In particular, TG2, a calcium-dependent protein with transamidanting activity, is involved in AD, inducing the formation of insoluble amyloid aggregates that can alter the properties of several proteins . TG2 activity is down-regulated in response to oxidative stress [29, 30] and this effect could be related to the increase of the intracellular Ca2+ levels due to Aβ toxicity . In fact, the accumulation of extracellular protein aggregates prevalently constituted by polymeric Aβ, caused by the aberrant transamidanting activity of TG2, are also related to a dysregulation of autophagy process . These conditions contribute to oxidative stress and neural cell death, in which TG2 plays a key role . It has been reported that hippocampal neurons are more responsive to Indicaxanthin . In particular, it has an important role in several metabolic functions both in vitro and in vivo, reducing inflammation and enhancing immune response [22, 23, 40].
In this study, for the first time, we highlight that the OEC exposure to Aβ(1–42), its fragments Aβ(25–35) and Aβ(35 − 25) induces a different expression pattern of TG2-L and TG2-S, demonstrating the opposite role played by TG2. Furthermore, we show the protective effect exerted by Indicaxanthin pre-treatment on total TG2 and its isoforms expression levels. In particular, we found that in Aβ(1–42) treated cells the two isoforms appeared at same expression levels, whereas in Aβ(25–35) exposed ones TG2-S was at higher levels than TG2-L, when compared with Aβ(1–42) exposed cells and with the controls. In OECs exposed to Aβ(35 − 25), a light modification between TG2-L and TG2-S expression levels was observed. The pre-treatment with Indicaxanthin was able to counteract the oxidative damage following the exposure of the cells to full native peptide of Aβ(1–42) and its toxic fragment Aβ(25–35), restoring the expression levels of total TG2 to control values. Furthermore, CLSM analysis performed on single cell showed that TG2 in OECs pre-treated with Indicaxanthin alone was localized in the cytosol. In contrast, when cells were pre-treated with Indicaxanthin and then exposed to Aβ(1–42), the protein appeared prevalently localized into the nuclear compartment. In the cells pre-treated with Indicaxanthin and then stressed with Aβ(25–35), TG2 was localized both in the cytosol and in the nucleus. Western blot analysis showed a significant increase in TG2-L in Indicaxanthin alone treated cells and in those then exposed cells to Aβ(1–42). This effect might be correlated to the role played by TG2 when it is localized into the nuclear compartment, in which it acts on the control of cell proliferation, regulating gene expression, cell survival and differentiation, exerting an anti-apoptotic function [10, 18, 41]. In OECs treated with Indicaxanthin alone and in those subsequently exposed to Aβ(25–35), an increase of TG2-S expression levels was observed. The effect appeared more evident in the cells pre-treated with Indicaxanthin. TG2-S, even if reduced respect to that found in Aβ(25–35) treated cells, exerts transamidanting activity and acts as apoptotic factor [6, 18]. Surprisingly, Indicaxanthin pre-treatment in Aβ(35 − 25) exposed cells, induced a significant increase of TG2-S expression levels, when compared with Aβ(35 − 25) alone and controls. This finding might be due to the strong protective effect of Indicaxanthin, since we hypothesize that Aβ(35 − 25) fragment, even if it was reported that is not toxic , was able to induce a low toxicity in OECs, as relieved by a very significant increase of TG2-S expression levels when compared with exposed cells to Aβ(35 − 25) alone. Thus, we suppose that this effect may be due to the protective role played by TG2, which stimulates its pro-apoptotic activity, in order to remove damaged cells and to induce cellular repair. We also found that Indicaxanthin counteracted the oxidative stress induced by Aβ, as relieved by the reduction of total ROS and O2− production, that appeared similar to those observed in the controls. Thus, Indicaxanthin pre-treatment, for its antioxidant properties, was able to reduce the Aβ-toxicity, oxidative stress-dependent and mitochondrial damage. In addition, Indicaxanthin, with its anti-inflammatory proprieties, decreased GFAP and Vimentin expression levels, that were enhanced in Aβ exposed OECs. These results highlighted that Indicaxanthin exerted a protective effect on reactive astrogliosis induced by Aβ responsive of cytoskeleton modifications. Furthermore, to clarify the protective role played by TG2 in the absence and in the presence of Indicaxanthin, the levels of Nestin, marker of neural stem self-renewal, co-expressed with cyclin D1, marker of cellular proliferation , were assessed. These results show an increase of positive cells for Nestin and cyclin D1 expression levels, demonstrating that Indicaxanthin pre-treatment, stimulating the activity played by nuclear TG2 on stem self-renewal OEC reprogramming, that stimulates cell proliferation repairing the damage induced by Aβ. We also observed that Indicaxanthin counteracted the TG2-aberrant cross-linking activity induced by Aβ-exposure on the cells, evaluating caspase-3 cleavage, that appeared reduced following to its treatment. This effect might be correlated to the function that TG2 exerts on the apoptotic pathway, as revealed by the increase of TG2-S expression levels observed in our experimental conditions, when cells were treated with Aβ(1–42) and Aβ(25–35) in the absence of Indicaxathin. In contrast, total TG2 did not show its opposite role on the basis of cellular localization and did not evidence the effect of Aβ both in the absence and in the presence of Indicaxanthin.
Taken together, our findings demonstrate that Aβ stress is responsible of TG2 up-regulation  and its structural modifications in two distinct conformational states with different functions . In fact, when the levels of Ca2+ are low and those of guanosin triphosphate (GTP) or guanosin diphosphate (GDP) are high, TG2-L acts as a GTPase, is involved in signaling pathway, is inactive and is present in “closed” conformation, promoting cell growth and survival (Fig. 11A). Aβ exposure of OECs, increasing intracellular Ca2+ and decreasing GTP or GDP levels, might cause a change of TG2-L from “closed” to “open” conformation, catalytically active. In addition, Aβ treatment induced an increase of the levels of TG2-S, an alternative splice variant of TG2 lacking of the portion of the carboxyl terminal essential for the maintenance of the protein in the “closed” conformation, that is responsible of apoptotic activation and cell death. The effect is more evident when the cells were exposed to the major toxic Aβ(25–35) fragment, that strongly enhanced intracellular Ca2+ levels (Fig. 11B). Indicaxanthin pre-treatment prevented total TG2 over-expression induced by the OEC exposure to full native peptide Aβ(1–42) and Aβ(25–35) fragment, probably binding to Ca2+ . The significant increase of TG2-L isoform expression levels induced by Aβ(1–42), accompanied by the decrease of TG2-S ones, is related to the role that the protein plays into the nucleus, in which it might stimulate OEC self-renewal and the reparative effect against Aβ toxicity (Fig. 11C). Furthermore, in Aβ(25–35) exposed OECs Indicaxanthin is able to significantly decrease TG2-S isoform expression levels enhancing at the same time those of TG2-L. The different expression pattern of TG2 isoforms in Aβ(25–35) exposed cells in the presence of Indicaxanthin might be due to the major toxicity of the fragment that induces a major enhancement of Ca2+. Thus, in this conditions, the protein was able to stimulate both apoptosis and self-renewal (Fig. 11D).