Research Article: PKR involvement in Alzheimer’s disease

Date Published: October 5, 2017

Publisher: BioMed Central

Author(s): Jacques Hugon, François Mouton-Liger, Julien Dumurgier, Claire Paquet.


Brain lesions in Alzheimer’s disease (AD) are characterized by Aβ accumulation, neurofibrillary tangles, and synaptic and neuronal vanishing. According to the amyloid cascade hypothesis, Aβ1-42 oligomers could trigger a neurotoxic cascade with kinase activation that leads to tau phosphorylation and neurodegeneration. Detrimental pathways that are associated with kinase activation could also be linked to the triggering of direct neuronal death, the production of free radicals, and neuroinflammation.

Among these kinases, PKR (eukaryotic initiation factor 2α kinase 2) is a pro-apoptotic enzyme that inhibits translation and that has been implicated in several molecular pathways that lead to AD brain lesions and disturbed memory formation. PKR accumulates in degenerating neurons and is activated by Aβ1-42 neurotoxicity. It might modulate Aβ synthesis through BACE 1 induction. PKR is increased in cerebrospinal fluid from patients with AD and mild cognitive impairment and can induce the activation of pro-inflammatory pathways leading to TNFα and IL1-β production. In addition, experimentally, PKR seems to down-regulate the molecular processes of memory consolidation. This review highlights the major findings linking PKR and abnormal brain metabolism associated with AD lesions.

Studying the detrimental role of PKR signaling in AD could pave the way for a neuroprotective strategy in which PKR inhibition could reduce neuronal demise and alleviate cognitive decline as well as the cumbersome burden of AD for patients.

Partial Text

With aging populations, Alzheimer’s disease (AD) has become a major public health problem in developed countries [1]. The pathology of AD involves senile plaques made of accumulated Aβ peptide, neurofibrillary tangles with abnormally phosphorylated tau protein, and synaptic and neuronal losses [2]. The cause of AD is not known, but the Aβ peptide could be toxic according to the “amyloid cascade hypothesis” [3]. Aβ is formed after the cleavage of amyloid precursor protein (APP) by β secretase (BACE1) and γ secretase. The amyloid cascade hypothesis proposes that the accumulation of Aβ or its oligomeric forms could be responsible for deleterious consequences, including neuronal and synaptic demise, as well as dementia. The cause of Aβ accumulation in sporadic forms of AD is not fully understood but it might be linked to increased Aβ production due to enhanced BACE1 and γ secretase activities or to reduced Aβ degradation [4]. Many pathogenic mechanisms have been assessed in AD, and the role of kinases has often been linked to tau phosphorylation [5, 6]. However, few studies have explored the links between kinase activation and other brain lesions [7]. This review analyzes recent reports implicating the kinase PKR in the pathogenesis of the neuronal degeneration observed in AD (Table 1).Table 1Published reports on PKR and Alzheimer’s diseaseReportSampleResultsChang et al. 2002 [45]BrainpPKR neuronal accumulationPeel et al. 2003 [39]BrainpPKR neuronal accumulationOnuki et al. 2004 [32]BrainpPKR neuronal accumulationPaccalin et al. 2006 [71]PBLpPKR increased levelPage et al. 2006 [40]BrainpPKR increased concentrationBullido et al. 2008 [74]DNAPKR gene associationDamjanac et al. 2009 [72]PBLPKR-dependent increases in P53, Redd1Couturier et al. 2010 [35]PBLPKR control of inflammationBose et al. 2011 [47]BrainCo-localization of pPKR and ptauPaquet et al. 2012 [69]BrainIncreased levels of PKR activator PACTMouton-Liger et al. 2012 [76]CSFIncreased levels of PKR and pPKRBadia et al. 2013 [73]PBLIncreased levels of PKR RNA in ApoE4 patientsDumurgier et al. 2013 [77]CSFCSF pPKR predicts cognitive declinePaquet et al. 2015 [79]BrainAβ vaccine reduces pPKR loadTaga et al. 2017 [70]BrainCorrelations cognitive scores and pPKR loadNon-exhaustive list of published studies that assessed the levels of PKR signals in human AD samples, including brain, peripheral blood lymphocytes (PBL), and cerebrospinal fluid (CSF)

Various stresses can be induced, and eukaryotic cells have an adaptive response called the integrated stress response (ISR) that can restore cell homeostasis [10]. The main molecular event in this response is the phosphorylation of eIF2α, which can lead to a global reduction in translation and the induction of selected genes, including the transcription factors ATF4 and BACE 1. Four eIF2 kinases catalyze this phosphorylation, PKR, PERK, GCN2, and HRI, which are all induced by specific or common stresses. The ISR is primarily a pro-survival pathway, but prolonged stress can lead to cell death. ATF4 has been implicated in cell survival or apoptosis and memory formation. Conflicting results have been proposed by different groups, showing either a constraint of memory [11] or that ATF4 is a key physiological regulator of memory [12]. In addition to PKR, PERK has also been implicated in neurodegeneration through the induction of the unfolded protein response (UPR) [13]. The UPR is a protective reaction triggered by the occurrence of ER stress that decreases the unfolded protein load and assures a normal protein‐folding process [14]. Prolonged UPR might be detrimental to cell survival. PERK is also a new therapeutic target in AD [15]. ISR is depicted schematically in Fig. 1. Converging cellular stresses, such as ER stress, can concurrently activate PKR and PERK. The question that should be addressed is what involvement PKR has in the genesis of lesions, including neuronal apoptosis and autophagy, neuroinflammation, and Aβ formation and toxicity. This review focuses on the participation of this eIF2α kinase in the development of abnormal signaling pathways associated with neurodegeneration and memory disturbances.Fig. 1The possible signaling stress pathways contributing to the integrated stress response and PKR activation in neurodegenerative diseases, as well as the molecular consequences of PKR activation in AD, Parkinson’s disease (PD) and Huntington’s disease (HD)

Many reports have shown that PKR is a pro-apoptotic kinase in various cells, including in neurons. For example, in cultures of retinal ganglion neurons, tunicamycin exposure induced ER stress, PKR activation, and widespread neuronal apoptosis. The pretreatment of cell cultures with the PKR inhibitor C16 (also designated PKRi) or with PKR siRNA attenuated neuronal or retinal ganglion cell death induced by tunicamycin [16, 17]. The authors concluded that inhibiting PKR activation is neuroprotective. In 2007, data from mixed cortical cultures revealed that the toxic protein GP-120 of the human immunodeficiency virus 1 increased PKR phosphorylation and caspase 3 activation. The pharmacological pretreatment of cultures with two PKR inhibitors reduced neuronal apoptosis [18]. The exposure of human neuroblastoma cells SH-SY5Y to interferon-β induced the activation of PKR and caspase 3 cleavage, and this effect was inhibited by the PKR inhibitor C16 [19]. In 2014, a report demonstrated that acute striatal injection of the excitotoxic compound quinolinic acid locally produced PKR activation neuroinflammation and neuronal apoptosis in vivo. An intraperitoneal injection of the PKR inhibitor C16 reduced PKR activation IL-1β levels and neuronal apoptosis [20]. PKR is also involved in the induction of autophagy, and this process could be linked to the protein STAT 3, which directly interacts with PKR [21]. In a recent study, Bordi et al. [22] have demonstrated that abnormal autophagy could contribute to the pathogenesis of AD lesions. Increased triggering of autophagy associated with reduced lysosomal clearance of substrates could lead to autophagic pathology and neuritic dystrophy detected in AD. Further research will be needed to determine whether PKR can participate in this autophagic induction of neurodegeneration.

Previous reports have demonstrated that PKR is an active player in innate immunity and could participate in several inflammatory pathways [23]. Published data have shown that PKR is involved via an interaction with NLRP3 in HMGB1 release and IL-1β production [24, 25], although these findings are still being debated [26]. In addition, PKR can trigger the NF Kappaβ pathway necessary for TNFα expression via direct protein interactions with I Kappaβ [27]. Finally, PKR can interact with the MAPK pathways and can trigger the activation of JNK and P38 kinases, which are also implicated in neuronal death and inflammation [28]. In conclusion, PKR is activated during three detrimental cellular events, that is, apoptosis, autophagy, and inflammation, which are prominent features of AD brain lesions. Future studies will be necessary to determine the exact starting time of brain PKR activation in the long evolution of preclinical AD brain lesions.

In vitro studies have revealed that PKR is activated by Aβ peptide toxicity. In 2002, a report showed that, in a human neuroblastoma cell line and in primary neuronal cultures, Aβ exposure induced PKR activation, eIF2α phosphorylation, and apoptosis. The use of dominant-negative PKR cell lines or PKR knockout neurons and calcium blockers reduced the levels of neuronal apoptosis, which suggested that PKR could be involved in calcium-mediated Aβ neurotoxicity [29]. Further studies have revealed that caspase 3 could modulate PKR activation and apoptosis [30, 31]. Using a randomized ribozyme library, the authors found that PKR was involved and activated in ER stress induced by tunicamycin in human neuroblastoma cells [32]. Surprisingly, another report did not detect UPR activation in cultured neurons exposed to Aβ, whereas PKR was clearly activated [31]. Tunicamycin exposure has also been explored in human neuroblastoma cells, and the results have shown that the PKR inhibitor C16 or the overexpression of a dominant-negative PKR attenuates neural cell apoptosis [17, 33]. Recently, it was shown in primary neuronal cultures from wild-type and PKR knockout mice that Aβ toxicity was blocked by genetic deletion of PKR and the JNK inhibitor XG 102, suggesting that dual kinase inhibition might be efficient for enhanced neuroprotection [34]. In addition, it was demonstrated that the PKR inhibitor C16 reduces the release of the inflammatory cytokines TNFα and IL-1β in mixed co-cultures of neurons and microglial cells [35]. PKR can control the levels of BACE 1 protein in human neuroblastoma cells exposed to oxidative stress, which suggests that PKR could modulate Aβ production [36]. In addition, the increased activity of BACE1 could also lead to synthesis of the β-cleaved carboxy-terminal fragment of APP (βCTF), which can recruit APPL to rab5 endosomes and can abnormally increase endocytosis and impair axonal transport [37]. Overall, PKR inhibition reduces Aβ-induced apoptosis neuroinflammation and BACE1 levels in cell cultures.

It was demonstrated in early reports on AD human brains [39, 45] that phosphorylated PKR could co-localize with phosphorylated tau in affected neurons. The question raised by these findings was: could PKR directly or indirectly participate in tau phosphorylation? Two studies have addressed this subject. The first report demonstrated that, in rat neuronal cultures, the phosphatase inhibitor okadaic acid can induce tau and PKR phosphorylation, can trigger the induction of transcription factor 4 (ATF4), and can lead to apoptosis [46]. Another study demonstrated that tunicamycin or Aβ treatment can induce PKR in human neuroblastoma cells and can trigger GSK3β activation, as well as tau phosphorylation. The pretreatment of cell cultures with the PKR inhibitor PRI peptide reduced GSK3β and PKR activation, as well as tau phosphorylation, which suggests that PKR can indirectly control GSK3β activation [47]. These results could partially explain the histological co-localization of neuronal PKR and tau detected in AD brains and could implicate PKR in signaling pathways that lead to tau phosphorylation.

AD is very often marked by initial memory disturbances, and patients are often followed over the course of the disease with memory tests such as the free and cued selective reminding test [48]. Several experimental reports have shown that the activation of PKR signaling could be associated with decreased memory performance. Previous studies have shown that local protein synthesis at synapses is required for long-lasting strength induced by, for example, BDNF [49]. Inhibitors of protein synthesis have been used to experimentally induce amnesia.

Recent studies have supported the possibility that AD could be a form of type III diabetes in which insulin resistance could play a major role [59]. Clinical trials are underway to test whether the administration of intranasal insulin could modulate cognitive decline in AD [60]. An experimental report has shown that PKR can phosphorylate insulin receptor substrate 1 (IRS1), which is a cellular event linked to insulin resistance in peripheral organs [61]. In addition, high glucose can disturb insulin signaling through the activation of PKR in muscle cells [62]. Increased apoptosis and production of ROS can be reduced by pharmacological inhibition of PKR. Another study has shown that PKR can control insulin sensitivity under physiological conditions in normal experimental animals, as well as in obese mice. The authors showed improvements in insulin sensitivity and glucose tolerance in PKR knockout mice [63]. The activation of PKR can also decrease the proliferation of pancreatic β cells once this kinase is triggered by lipotoxicity of pro-inflammatory cytokines. Cell proliferation is stopped at the G1 phase [64]. To determine whether PKR activation has a similar detrimental function in human brains will require appropriate research in AD patients. It is possible to assume that the various neuronal stresses associated with increased levels of PKR activation detected in AD brains could interfere with neuronal insulin signaling, as observed in the cells of peripheral organs during metabolic stress.

As reported previously in this review, several studies have used PKR genetic blockade or PKR pharmacological inhibition to modulate the molecular process of memory formation or AD brain lesions in experimental models and transgenic AD mice. Unfortunately, no pharmacological PKR inhibitors have reached clinical phase 1 or subsequent phases of clinical trials. The consequences of the active anti-amyloid therapy AN 1792 on brain PKR and tau loads have recently been studied [79]. In non-immunized patients, the magnitude of axonal degradation (neuritic curvature ratio) and spongiosis was correlated with the levels of phosphorylated PKR load assessed by immunohistochemical methods. In immunized patients, the reduction of PKR load was associated with Aβ1-42 removal and the decrease of microglial markers. These results underlined the links between Aβ1-42 accumulation, PKR activation, and neuroinflammation.

The initial cause of AD is unknown, and the trigger for inducing the accumulation of Aβ oligomers in sporadic AD is also unknown. Whether activation of PKR in AD follows the accumulation of Aβ or is located upstream of this amyloid pathway and leads to BACE 1 induction has not yet been determined. PKR can be activated by so many stresses that lead to ISR that an association with subtle brain inflammation (viral or infectious), ER or oxidative stress, and metabolic abnormalities could increase BACE 1 translation and Aβ synthesis. These events, which may be associated, for example, with an infection, trauma, or an unknown aging process, could occur decades before the first clinical signs and may be reinforced by Aβ oligomer production. As mentioned before, brain PKR activation is independent of Aβ accumulation in PD and HD and could be linked to α-synuclein and abnormal metabolism of huntingtin. It is plausible that once the neurotoxic cascade is switched on, several abnormal molecular pathways could contribute to this vicious circle and lead to AD through tau phosphorylation, synaptic degradation, and initial memory disturbances. A comparable cascade involving other proteins could theoretically be proposed for other neurodegenerative disorders, such as PD and HD. In the future, early pharmacological inhibition of kinases associated with a reduction of Aβ oligomer synthesis might support efficient multi-target therapy. The discovery of new PKR inhibitors seems to be an appropriate goal for a new therapeutic approach, especially if the sum of the early initial brain cellular events can contribute to the activation of PKR and other potential toxic kinases. In addition, new biological methods detecting subtle PKR anomalies in the blood and/or in the CSF in pre-symptomatic or prodromal AD patients could facilitate the validation of target engagement. Early detection and treatment of AD brain lesions, including PKR deregulation, might provide a sensitive way to put in place secondary prevention to reduce the relentless burden on patients and their caregivers.




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