Original Research ArticleChronic PERK induction promotes Alzheimer-like neuropathology in Down syndrome: Insights for therapeutic intervention
Graphical abstract
Introduction
Down syndrome (DS) or trisomy 21 is one of the most common genetic causes of intellectual disability. Improved medical care in DS has led to a significantly improved lifespan and quality of life (Bittles et al., 2007; Glasson et al., 2002), but also to increased risk of developing Alzheimer’s disease (AD). DS and AD neuropathology have common pathological hallmarks that include deposition of senile plaques and neurofibrillary tangles (Head et al., 2016). Virtually all people with DS have sufficient neuropathology for a diagnosis of AD by 40 years (Lott and Head, 2005; Mann and Esiri, 1989; Wisniewski et al., 1978, 1985). Increased AD risk is likely due to the genetic imbalance present in DS: triplication and altered expression of genes located on chromosome 21 (Wiseman et al., 2015). Triplication of the amyloid precursor protein (APP) gene is considered the major precursor in which both DS-AD and frank AD converge, but other genes associated with tau hyperphosphorylation, mitochondrial defects, altered glucose metabolism, insulin resistance, protein dys-homeostasis and antioxidant response (AOR) contribute to AD onset and progression (Di Domenico et al., 2017, 2015; Di Domenico et al., 2018; Lemere et al., 1996; Lott and Head, 2019; Perluigi et al., 2014; Wiseman et al., 2015).
Oxidative stress (OS) impacts the development and the progression of DS cognitive impairment. Increased OS during fetal stages negatively affects brain development; our group and others have demonstrated that OS is an early event in the onset of the DS phenotype (Butterfield et al., 2014; Cenini et al., 2012; Di Domenico et al., 2014, 2019; Garlet et al., 2013; Pagano and Castello, 2012; Perluigi et al., 2011; Zis et al., 2012). In later stages of the disease, OS contributes to the neurodegenerative DS phenomena (Perluigi and Butterfield, 2012). Further investigation of the molecular mechanisms driving the accumulation of oxidized/unfolded/misfolded proteins and toxic aggregates identified aberrant regulation of pathways involved in protein degradation, namely the ubiquitin proteasome system (UPS) and autophagy (Di Domenico et al., 2013; Perluigi et al., 2014; Tramutola et al., 2017, 2018).
We recently established that the unfolded protein response (UPR) is chronically and aberrantly active in a murine model of DS (Lanzillotta et al., 2018). The UPR restores proteostasis during ER stress conditions such as those present in neurodegenerative diseases characterized by the toxic aggregates (Butterfield and Halliwell, 2019; Hetz and Saxena, 2017; Moreno et al., 2012; Radford et al., 2015). The adaptive nature of the UPR promotes the folding and degradative capacity of the ER (Halliday et al., 2017); moreover, it reduces translation to limit input of nascent proteins into an overburdened ER. The UPR is controlled by BiP, which regulates three transmembrane endoplasmic reticulum proteins: protein kinase RNA (PKR)- like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) (Bell et al., 2016). Of the three arms, the PERK pathway is the most commonly linked to neurodegenerative disease (Hetz and Saxena, 2017; Scheper and Hoozemans, 2013, 2015; Smith and Mallucci, 2016). Under non-stressed conditions, the PERK monomer traverses the ER membrane associated in its luminal N-terminus with the ER chaperone binding immunoglobulin protein (BiP) (also known as GRP78), while its cytoplasmic C-terminus contains the kinase domain (Halliday et al., 2017). Upon induction of ER stress, PERK dissociates from BiP, which allows dimerization and autophosphorylation into p-PERK. Activated PERK in turn phosphorylates the alpha subunit of eukaryotic initiation factor eIF2, which attenuates the initiation of translation (Harding et al., 1999). Our data, in the Ts65Dn mouse model of DS, demonstrated that PERK is early and selectively activated, and this may contribute to the observed failure of proteostasis and to the progression of neurodegeneration (Lanzillotta et al., 2018). Furthermore, the aberrant induction of the integrated stress response (ISR), as result of the increased PKR/eIF2α axis, was recently observed in DS mice (Zhu et al., 2019). Specific markers of UPR activation, such as p-PERK, p-eIF2α, p-IRE1 and BiP, are increased in AD brain tissue (Chang et al., 2002; Hoozemans et al., 2009, 2005; Stutzbach et al., 2013; Unterberger et al., 2006) and studies on mouse models of AD, PD, and prion disease demonstrated that p-PERK modulation rescues memory deficits (Ma et al., 2013; Mercado et al., 2018; Moreno et al., 2013; Radford et al., 2015; Trinh et al., 2014; Yang et al., 2016). In addition to eIF2α, p-PERK also regulates the nuclear factor (erythroid- derived 2)-like 2 (Nrf2), a transcription factor involved in the regulation of a variety of antioxidant genes that are regulated by an antioxidant response element (ARE). Nrf2 activity declines during aging (Rahman et al., 2013; Suh et al., 2004) and AD pathology, which correlates with increased OS and progression of neuropathology (Ramsey et al., 2007; von Otter et al., 2010v; Wyss-Coray, 2016). Furthermore, AD mice show reduced Nrf2/ARE pathway induction with aging, while its activation in AD mice alleviates cognitive deficits (Halliday et al., 2017; Tian et al., 2019). Nrf2 competes with Bach1, a repressor of transcription, for ARE binding. In unstressed cells, Nrf2 is maintained in an inactive state by Keap1, while Bach1 is linked to ARE (Itoh et al., 1999; Zhang et al., 2018). Under ER stress, PERK phosphorylates Nrf2 promoting nuclear translocation and transcriptional activation of antioxidant genes (Cullinan et al., 2003). In doing so, Nrf2 removes Bach1 from AREs. Despite that the basis for changes in Bach1 regulation during advanced age or AD have not been fully elucidated, reduced transcription of Nrf2-regulated genes could be driven by increased Bach1 expression (Rojo et al., 2018; Zhang et al., 2018; Zhou et al., 2018). However, the exact role of Nrf2/Bach1 balance during the progression of neurodegenerative disease is still unclear. Intriguingly, Bach1 is encoded by HSA21, and previous data by our laboratory demonstrated increased Bach1 protein levels in the brain of all DS cases and DS mice coupled with reduced induction of brain antioxidant response (Di Domenico et al., 2015). These results suggest that under physiological acute ER stress, PERK-mediated regulation of Nrf2/ARE is uncoupled under DS-induced, chronic ER stress.
The scope of this work was to establish, in human specimens, the consequences of abnormally sustained PERK activity and the induction of PERK-related downstream signals involved in the regulation of protein translation and antioxidant response. Further, by pharmacologically targeting PERK, in the brain of DS mice, we sought to investigate the benefit of its inhibition on the regulation of protein translation and antioxidant response. Our data suggest that the PERK pathway is central to the toxic consequences of DS.
Section snippets
Human subjects
Human DS brain samples were obtained from the University of California-Irvine-ADRC Brain Tissue Repository, the Eunice Kennedy Shriver NICHD Brain and Tissue Bank for Developmental Disorders, and the University of Kentucky Alzheimer Disease Center (ADC). Table 1 shows the characteristics and demographic data of the included subjects in the study. DS cases were divided into 2 groups, with or without sufficient neuropathology for the diagnosis of AD. The age of DS cases is under 40 years (DSy),
Aberrant induction of PERK/eIF2α pathway in DS human cortex
Given our previous results demonstrating the early and specific alteration of the UPR in DS mice (Lanzillotta et al., 2018), we tested the hypothesis that ER stress-related processes mediate AD-like development in human brain. We investigated the UPR status using postmortem tissue of DSy and DS-AD, compared with their age-matched control. Using post-mortem tissue, we found increased BiP levels in DSy compared to HSy (+90 %, p = 0.01) and in DS-AD individuals compared with HSo (+148 %, p = 0.03)
Discussion
Previous studies from our laboratory proposed that chronic PERK activation is an early and toxic mechanism, which precedes tau and Aβ deposition (Lanzillotta et al., 2018); this feature is concomitant to increased OS, thus suggesting a putative role for trisomic-related dysregulation of gene expression in the observed chronic UPR induction. Beyond these findings, there is little knowledge about ER stress in DS and how DS genetics may alter the UPR. In the present work, we describe the early
Conclusions
The results of the present study support the involvement of chronic PERK activation in DS brain pathology. Furthermore, since this alteration also occurs in peripheral cells, it conceivably may characterize trisomic-related genomic imbalance. On one side, over-induced PERK increases eIF2α deregulation promoting translation repression and may favors synaptic failure in DS (Contestabile et al., 2017; Meier et al., 2016; Roncace et al., 2017; Trinh et al., 2014). On the other side, we demonstrate
Author contributions
FDD, CL, JFA and IZ contributed to the study conception and design. EH, VF, MC, FL, DV and EV have been involved in human sample collection and preparation. CL, IZ, AT, EB, CB, JFA and MP contributed to data collection and analysis. FDD, JFA, CL and DAB wrote the first draft and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This work was funded by Sapienza University of Rome - Progetti d’AteneoRG1181642744DF59, RM11715C773949E3 and RG116154C9214D1A, by Istituto Pasteur Italia – Fondazione Cenci Bolognetti Under 45 U-4.IT, and by Ministry of HealthGR-2018-12366381to F.D.D.; J.F.A support pertinent to this work came from Alzheimer’s AssociationNIRG-14-322441, Department of DefenseAZ140097, NIH/NIMHD L32 MD009205-01, NIH/NINDS 1R01 NS091329-01. D.A.B and E.H were supported in part by NIHAG055596.
Declaration of Competing Interest
C.L., I.L., A.T., V.F., M.C., D.V., D.A.B., E.H., M.P., J.F.A. and F.D.D state no conflict of interest.
Acknowledgments
We thank Jeffrey M. Axten (GlaxoSmithKline, Collegeville, PA USA) for supplying GSK2606414.
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