A decrease in heme levels enables the phosphorylation
A decrease in heme levels enables the phosphorylation of eIF2α by HRI and thereby inhibition of eIF2α activity. Indeed, the relatively small change in cytosolic heme levels in SZ SANT-1 showed downstream functional consequences manifested by almost 50% elevation in the phosphorylation of eIF2α in SZ-derived LCLs as compared to control, complementing prior reports of reduction in global protein translation in SZ (English et al., 2015). Nevertheless, the exact mechanism responsible for eIF2a phosphorylation is yet to be determined. In mammals, four protein kinases are known to phosphorylate eIF2’s α subunit at Ser-51, one of which is HRI (Bauer et al., 2001). At least dozen other agents such as polyamines can also affect physphorylation of eIF2a (Landau et al., 2010). Unraveling the role of HRI in eIF2alpha phosphorylation in SZ cells, by direct measures of HRI levels and phosphorylation and its inhibition or silencing in CoI deficient cells as well as the mechanism linking HRI kinase and CoI activities is currently under investigation. In order to study whether there is a link between Co-I deficits and alterations in cytosolic heme metabolism we inhibited Co-I activity by rotenone. An initial increase (24 h) with a later decrease (48 h) in heme levels following Co-I inhibition was observed, suggesting that impaired Co-I activity affects heme metabolism, inducing an initial compensatory mechanism that eventually fails. The decrease in heme levels is in line with two previous studies indicating a positive correlation between decrease in Co-I activity and levels of heme (Gielisch and Meierhofer, 2015, Homedan et al., 2015). At the longest duration examined, 72 h, and after repeated rotenone treatments, heme levels have stabilized, and rotenone did not seem to elicit a further effect on heme levels. This could be the result of the induction of cell compensatory mechanism when heme level reaches a certain threshold or a direct effect of rotenone on heme metabolism, as rotenone is known to cause secondary impacts on cells such as apoptosis and ER stress, both activating a number of different pathways (Han et al., 2014, Newhouse et al., 2004). Alterations in heme levels due to Co-I inhibition were associated with a significant increase in HO-1 protein levels at all-time durations at the highest rotenone dosage. HO-1 mRNA levels also increased at highest rotenone dosage, but with a moderate increase at the longest duration examined, 72 h. This lag between HO-1 protein and mRNA could suggests that mRNA may not necessarily correlate with protein levels of this enzyme following long-term exposure to rotenone, in line with reports of an overall ∼40% correlation between mRNA and protein expression (Vogel and Marcotte, 2012). The mechanism by which Co-I affects cytosolic heme metabolism is still an open question. One possible mechanism is elevated oxidative stress state due to impairment in Co-I, which is a major site for superoxide production and if impaired, as observed in SZ cells and in repeated exposure to rotenone, can lead to reactive oxygen species (ROS) production and lipid peroxidation (Barrientos and Moraes, 1999). HO-1 is highly induced by elevate oxidative stress (Barone et al., 2012, Choi and Alam, 1996) which can then lead to increased degradation of heme. The reduction in heme due to Co-I inhibition by rotenone was functionally manifested by the opposite, yet with a time shift pattern of change in eIF2α phosphorylation similarly to our findings in SZ cells. The reduction in heme levels in SZ cells, probably due to increased HO-1 and its possible link to Co-I impairment, is supported by our findings in brains of Poly I:C offspring of mitochondrial impairments including that of Co-I, increased protein levels of HO-1 and alterations of eIF2α phosphorylation in a brain-area specific pattern. Mitochondrial impairments were most severe in the mPFC, where virtually all examined proteins, Co-I subunits NDUFV1, NDUFV2 and the fission/fusion proteins FIS1 and OPA1, were decreased. This is in line with previous reports of decreased NDUFV1, NDUFV2 and OPA1 in the prefrontal cortex postmortem specimens and in cells of SZ patients (Dror et al., 2002, Rosenfeld et al., 2011). Changes in mitochondrial proteins were also witnessed in the hippocampus, and to a lesser extent in the striatum of the Poly I:C offspring. These changes in mitochondria add to previously reported reduction in mPFC neurons of mitochondrial membrane potential (Δψm), to which Co-I is a major contributor, of ATP in mPFC neurons and splenocytes of Poly-I:C offspring (Giulivi et al., 2013, Robicsek et al., 2018) and in Poly-I:C transfected cell lines (Chen et al., 2016). HO-1 protein levels were elevated in the mPFC and DH of Poly I:C offspring, but not in the VH and the striatum, while phosphorylation of eIF2α was enhanced in both VH and DH. The increase in HO-1 and PeIF2α/eIF2α in the DH suggests a decrease in heme levels in this brain area, since HO-1 catabolizes heme and deficiency in heme is a known cause for eIF2α phosphorylation. In the VH, levels of HO-1 were not elevated but PeIF2α/eIF2α was still increased. In the mPFC a decrease in PeIF2α/eIF2α was witnessed in the of Poly I:C treated offspring despite elevated levels of HO-1. A plausible explanation is that additional factors that are unrelated to heme/HRI pathway such as oxidative stress or other kinases affect phosphorylation of eIF2α in these brain areas (Holcik and Sonenberg, 2005). Different susceptibility to an insult between brain area are a common phenomenon and examples include differences in VH and DH responsiveness to oxidative stress specific (Steullet et al., 2010), eIF2α phosphorylation in the frontal cortex but not in the cerebellum in AD rat model (Ma et al., 2013) and brain-area dependent pattern of impairments in Co-I in SZ patients (Ben-Shachar and Karry, 2007). Interestingly, the only brain area that did not exhibit altered heme metabolism is the striatum, the brain area in which we did not find altered levels of Co-I subunits.