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  • bruton\'s tyrosine kinase Aside from induction of cold s

    2018-10-25

    Aside from induction of cold-shock proteins however, little is known of other fundamental cellular stress pathways in relation to cooling and their potential relevance to hypothermic preconditioning (Hofman et al., 2012; van der Harg et al., 2014). Hypothermia can induce protein unfolding and disrupt the cell secretory pathway (Saraste et al., 1986; Liu et al., 1994; Fujita, 1999), both of which would result in endoplasmic reticulum (ER) stress (Kim et al., 2008). However, mammalian cell lines have produced conflicting data regarding the ability of cooling to trigger ER stress and downstream events coordinated by the unfolded protein response (UPR) (Hofman et al., 2012; van der Harg et al., 2014). Although this may relate to the variable depths of hypothermia studied, it likely also reflects the resistance of immortal cell types to physiological stress (Abdel Malek et al., 2015; Cerezo et al., 2015). Furthermore, the ER-UPR cascade as a whole has never been explored at clinically-relevant hypothermic temperatures. Potentially, such a moderate level of cold-stress might bring about an adaptive proteostatic response in post-mitotic neurons (Mendes et al., 2009; Fouillet et al., 2012). Here we test this hypothesis by characterizing the cold-shock response to protective hypothermia in functional cortical neurons differentiated from human pluripotent stem bruton\'s tyrosine kinase (hCNs) (Bilican et al., 2014), using this model to explore the molecular basis of hypothermic preconditioning.
    Materials and Methods
    Results
    Discussion In acute injury, mildly enhancing the UPR can rescue neurons from programmed cell death and instigate adaptive ER preconditioning (Hetz and Mollereau, 2014). In hCNs, PERK activity was essential for hypothermic preconditioning against an oxidative challenge. Mild XBP1 splicing after 24h of cooling, together with a substantial increase in unspliced XBP1 mRNA (Figs. 2E, F, and S2B) indicate that Ire1α and ATF6 were active within the cooling period (Hetz and Mollereau, 2014). Moreover, the increase in BiP transcript after 24h is consistent with prior activation of ATF6 and splicing of XBP1 (Hetz and Mollereau, 2014). Enhanced injury with PERK inhibition at 37°C may reflect a constitutive proteostatic function of the UPR in long-term culture — potentially through buffering oxidative processes (Cullinan et al., 2003). It might also explain why hypothermic induction of some PERK branch-specific components was not observed; eIF2α phosphorylation does occur under deep hypothermic conditions (10°C) and contributes to the global suppression of protein translation in mammalian cell lines (Roobol et al., 2009; Hofman et al., 2012), but it may be undetectable biochemically in the context of mild ER stress (Rutkowski et al., 2006). Equally, since PERK-mediated translational repression is subject to homeostatic autoregulation by phosphatases (Lin et al., 2007), a resolving influence of cooling on eIF2α activation is supported by hypothermic induction of GADD34 in hCNs (Figs. 2H and 3C) (Ma and Hendershot, 2003). In this respect, rather than signifying the duration limit of protective cooling, the CHOP induction observed would be a pre-requisite for GADD34-mediated negative feedback on eIF2α (Halterman et al., 2010). Accordingly, others have highlighted the protective role of CHOP in neuronal systems (Chen et al., 2012; Engel et al., 2013). The fact that 24h cooling did not increase levels of the pro-apoptotic marker Bax (Fig. S2E) is in line with previous studies (Yenari et al., 2002) and further supports our conclusion that this duration and depth of hypothermia produced an adaptive UPR. Potentially, cold-shock proteins may complement this cascade by relieving translational repression of critical mRNAs (Peretti et al., 2015), and limiting CHOP-mediated apoptosis (Saito et al., 2010). During an adaptive stress response UPR branches undergo complex homeostatic self-regulation (Fig. 3C). Thus the cross-sectional UPR profile captured in our hCN system cannot convey the dynamic nature of these pathways. However, our analysis at 24h intimately links UPR activation to neuronal preconditioning, since this was the point at which H2O2 was applied, and it further indicates co-ordination of ER-hormesis with cold-shock protein induction. The lack of GRP94 induction after 24h cooling may reflect the half-life of its transcript or the selective nature of this chaperone, whose client list is smaller than that of BiP — in particular, GRP94 is not induced at high temperatures (Marzec et al., 2012). Furthermore, prolonged ER stress leads to sequential activation then deactivation of Ire1α, ATF6 and PERK pathways respectively — which might explain the bias of UPR components towards the PERK arm at 24h (Tabas and Ron, 2011). Nevertheless, our transcript analysis revealed distinct patterns of UPR responses resulting from two different stresses; whilst BiP, GRP94, XBP1s and CHOP dramatically increased with Tm, ATF4 and GADD34 induction were comparable between Tm and cooling. Therefore, in contrast to models described elsewhere (Rutkowski et al., 2006), the negative regulation of eIF2α appears to take precedence over unloading the ER in cooled human neurons. This relief of translational repression may confer tolerance to a prolonged hypothermic state (Peretti et al., 2015; Moreno et al., 2013).