Pathogenesis and promising therapeutics of Alzheimer disease through eIF2α pathway and correspondent kinases
Abstract
Eukaryotic initiation factor 2 (eIF2α) pathway is overactivated in Alzheimer disease and is probably associated with synaptic and memory deficiencies. EIF2α protein is principally in charge of the regulation of protein synthesis in eukaryotic cells. Four kinases responsible for eIF2α phosphorylation at ser-51 are: General control non-derepressible-2 kinase (GCN2), double- stranded RNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI) are the four kinases. They lead to reduced levels of general translation and paradoxical increase of stress-responsive mRNAs expression including the B-secretase (BACE1) and the transcriptional modulator activating transcription factor 4 (ATF4), which in turn accelerates the beta-amyloidogenesis, tau phosphorylation, proapoptotic pathway induction and autoph- agy elements formation leading to the main pathological hallmarks of AD. Findings suggest that genetic or pharmacological inhibition of correspondent kinases can restore memory and prevent neurodegeneration. This implies that inhibition of eIF2α phosphorylation through respondent kinases is indeed a feasible prospect of clinical application. This review discusses recent therapeutic approaches targeting eIF2α pathway and provides an overview of the links between correspondent kinases overactivation with neurodegeneration in AD.
Keywords : Eukaryotic initiation factor 2 (eIF2α) . Alzheimer disease (AD) . HRI . PERK . PKR . GCN2
Background
As the most common neurodegenerative disorder, Alzheimer disease (AD) pathology is conventionally attributed to aber- rant accumulation of misfolded proteins in the endoplasmic reticulum (ER) (Hoozemans et al. 2009). Accumulation of amyloid precursor protein (APP), its cleavage into amyloid β by B-site APP cleaving enzyme 1 (BACE1), and aggrega- tion of amyloid β in extracellular plaques and intracellular neurofibrillary tangles, are key histologic findings of AD, while an increasing interest is being put onto the significance of tau protein accumulation (Ballatore et al. 2007).
Highlights • Chronic phosphorylation of eIF2α pathway due to correspondent kinases overactivation is harmful to neuronal plasticity and memory formation.
• Genetic or pharmacological inhibition of eIF2α pathway activity can restore memory and prevent neurodegeneration.
• Gastrodin is capable of Suppressing BACE1 expression under oxidative stress condition via inhibition of the PKR/eIF2α signaling pathway in AD.
• BACE1 translation is regulated by NO through HRI.
• Fine-tuning the eIF2α inhibition genetically or by using the repurposed drugs like Trazodone or dibenzoylmethane (DBM) can be a promising therapeutic target for AD.
Accumulation of these intracellular aggregates in the ER evokes the unfolded protein response (UPR) which leads to neuronal death (Hetz et al. 2013).Eukaryotic initiation factor 2 (eIF2) protein is principally in charge of regulation of protein synthesis in eukaryotic cells and is inhibited through phosphorylation. In mammalians, four different protein kinases can phosphorylate α subunit of eIF2 at Ser-51 in different environmental stress conditions. Phosphorylation of eIF2 changes it from a substrate to an inhibitor of eIF2B and can lead to either apoptosis or cell damage reversing. This lowers general translation, which pro- vides cells sufficient time to repair the stress damage during cellular stress conditions, and upregulates gene-specific trans- lation to remediate damage (Aarti et al. 2010). Moreover, eIF2α translational regulatory mechanism plays an important role in prevention of the synthesis of unwanted proteins that could interfere with the cellular stress. (Onyango and Khan 2006; Onyango and Research 2006; Wek et al. 2006).
eIF2α pathway and synaptic plasticity
Synapses are composed of proteins that play direct roles in synaptic transmission and synaptic function regulation. De novo protein synthesis is essential for synaptic plasticity while synaptic proteins have a short lifetime and should continuous- ly be replaced with newly synthesized ones (Jiang et al. 2010). Synaptic plasticity is also essential for the formation of both short-term and long-term memory. Long-term potentiation (LTP) of signals requires both morphological and functional adaptions in neural endings associated with various intracel- lular signaling pathways and gene up or down regulations (Korb and Finkbeiner 2011). The persistent changes in syn- aptic strength, specifically long-term potentiation, and depres- sion depending on new protein synthesis (Rosenberg et al. 2014). These changes are controlled by engaging the signaling pathways that regulate mRNA translation. One highly con- served mechanism of translational control in eukaryotic cells involves phosphorylation of eukaryotic initiation factor 2. Phosphorylation of eIF2α halts protein elongation in cells, an intermediate mechanism that leads to suppression of new memory formation in mice, while inducing neuronal death in the hippocampus (Ma et al. 2013). This evidence justifies investigating the regulation of long-term potentiation and syn- aptic plasticity in AD through eIF2α. Finally, EIF2α phos- phorylation induces proapoptotic pathways through formation of autophagy elements, both mechanisms proved to be path- ogenic in AD (Wei et al. 2015). Importantly, phosphorylation of eIF2 during UPR assures minimal protein synthesis as a protective response (Clemens 2001).
An increase in eIF2α phosphorylation has been identified in post mortem specimen of patients with sporadic AD pa- tients, as well as transgenic mice with AD (Kim et al. 2007). Chang et al. first indicated that tau accumulates in AD brain are immunoreactive for eIF2α, and that elF2α upregulation is concomitant with an increase in kinases, like PKR, that regu- late eIF2α activation under physiological conditions (Chang et al. 2002). EIF2α phosphorylation at Ser 51 disrupts binding of the activated β subunit of protein and proper binding of the entire complex to GTP and tRNA initiator, further inhibiting protein synthesis and in turn, upregulating the BACE1, and another transcriptional modulator activating transcription fac- tor 4 (ATF4) (Sathya et al. 1970). ATF4 is a repressor of long- term synaptic plasticity and memory formation through ham- pering the cAMP-response element binding protein (CREB), which potentiates long-term memory formation and synaptic plasticity (Ma et al. 2013; Ohno 2014). This is a common pathway between ATF4 and amyloid β, which itself alters synaptic plasticity through interfering with CREB induction of synaptic adaptations seen with LTP (Dehghani et al. 2018). Finally, EIF2α phosphorylation induces proapoptotic path- ways through formation of autophagy elements, both mecha- nisms proved to be pathogenic in AD (Wei et al. 2015). Importantly, phosphorylation of eIF2 during UPR assures minimal protein synthesis as a protective response (Clemens 2001).
In general, elF2 is important in mediating the signalling cas- cade that follows the ER stress in AD. Indeed, increased eIF2α phosphorylation also appears to be a prodromal sign for preclin- ical sporadic AD. Mild cognitive impairments (MCI) are noted in mice that overexpress apolipoprotein E4 (ApoE4), which is the greatest genetic risk factor for AD, and this has been discovered to be associated with a concomitant rise in eIF2 phosphorylation (Segev et al. 2013). Additionally, the glycogen synthase kinase-3 (GSK3) as the main phosphorylator of tau that mediates its ab- errant aggregation as neurofibrillary tangles can prevent eIF2α complex formation, accentuating the ER stress and ER induced neuronal death (Morel et al. 2009).
Four eIF2α kinases are known to be phosphorylating eIF2α at the Ser51 position, hence inactivating the complex: the general control non-derepressible-2 kinase (GCN2), the double-stranded RNA-activated protein kinase (PKR), the PKR-like endoplasmic reticulum kinase (PERK), and the heme-regulated inhibitor kinase (HRI) (Ma et al. 2013). Following, we thoroughly discuss the mechanisms and poten- tials provided through modulation of the above-mentioned kinases in preventing AD progression.
GCN2 kinase
Protein kinase GCN2 is a sensor of amino acid starvation and regulates translation by phosphorylating the eIF2α. GCN2 can be triggered by nutrient deprivation including glucose deprivation, viral infections, oxidative stress, and UV irradia- tion. GCN2 applies its function through phosphorylating eIF2α at ser-51, leading to reduced global protein synthesis and increased upregulation of stress-responsive mRNAs in- cluding ATF4 Fig. 1 (Baird and Wek 2012; Chaveroux et al. 2010; Hinnebusch 2005).
Other than amino acid hemostasis, GCN2 also affect syn- aptic plasticity and plays an important role in long-term mem- ory formation. Costa-Mattioli and colleagues aimed to eluci- date impairment in long-term potentiation and long-term memory in GCN2 kinase knock out mice (Costa-Mattioli et al. 2007, 2005). At base line, GCN2 phosphorylated eIF2α which then evoked ATF4 mediated suppression of translation and expression dependent synaptic plasticity, which happens with learning. Moreover, a paradoxical in- crease in the translation of selective mRNAs that happens with ATF4 activation precipitates metabolic stress and abnormal Aβ accumulation observed in AD.
ATF4 is an activating transcription factor and its translation is increased during the micro-environmental stresses includ- ing oxidative stress, amino acid starvation and ER stress. Lewerenz and Maher reported that in the cortex of AD brains, the protein level of ATF4 is increased in comparison to age- matched controls (Lewerenz and Maher 2009). Baleriola et al. demonstrated that synthesized ATF4 is a mediator for the spread of AD pathology and its translation levels are increased in axons in the brain of AD patients (Baleriola et al. 2014). ATF4 is an initiator of memory deficits and pathological hall- marks of AD brains, suggesting that several proteins in the C/ EBP family including the ATF4 act as a constrainer of long term synaptic plasticity and memory formation (Chen et al. 2003). The binding of ATF4 to the regulatory region of hu- man presenilin-1 (PS1) gene is essential for the activity of PS1 which is an important cofactor for stimulating the γ-secretase, which in turn accelerates the beta-amyloidogenesis leading to the formation of amyloid plaques (Mitsuda et al. 2007). ATF4 acts as a promoter of GSK-3 and PP1 expression in AD pa- tients and thereafter phosphorylates tau (Yoshizawa et al. 2009). Also, increases in ATF4 plays an important role in neuronal death including autophagic cell death and apoptosis. ATF4 combining with C/EBP-homologous protein (CHOP) can trigger apoptosis genes expression such as Bim, TRB3 and PUMA and thereafter promotes neuronal apoptosis (Avery et al. 2010; Galehdar et al. 2010; Verfaillie et al.2012). ATF4 also increases the expression of autophagic genes like Map1lc3b, leading to autophagosome formation (Rouschop et al. 2010). However, whether ATF4 is involved in the necrosis procedure in AD or not is not clear yet and further investigation is needed.
Deleting PERK or GCN2 in the brains of APP / PS1 mice reduces eIF2α-P concentrations, saving synaptic plasticity and cognition. Moreover, removal of GCN2 kinases prevented deterioration of spatial memory and enhanced signs of long-term memory deficits by reducing eIF2α phosphory- lation in transgenic APP/presenilin 1 (PS1) mice. Behavioral assessments indicated significantly increased escape latencies in APP/PS1 mice compared to the controls, and inhibition of GCN2 kinases could ameliorate this result (Ma et al. 2013). Similarly, Costa-Mattioli et al. showed that GCN2 −/− mice responded to LTP in hippocampal neurons characterized by enhanced performance in spatial memory in Morris water maze in short term training but deteriorated in LTP for longer training hours (Costa-Mattioli et al. 2007).
Surprisingly, the deletion of GCN2 kinase aggravated rath- er than suppressed the elevations of BACE1 and ATF4 in the hippocampus of 5XFAD mice partially due to the activation of the PERK-eIF2α pathway (Devi and Ohno 2013). Unlike previous studies, Devi and Ohno mentioned that GCN2−/− and GCN2± deficiencies deteriorate rather than suppress eIF2α phosphorylation in 5XFAD mice, thus in the contextual fear conditioning task, are not capable of inhibiting memory deficiencies. The most striking result of this study was to show that the deletion of GCN2 overactivates the PERK-dependent eIF2α phosphorylation pathway and thus exacerbates the el- evations of BACE1 and ATF4 in 5XFAD mice. This results in the dysfunction of CREB and β-amyloidogenesis. In addition, the overactivation of PERK in reaction to GCN2 deletion is particular to 5XFAD mice as GCN2−/− mice demonstrate decreased eIF2α phosphorylation relative to wild-type con- trols, as phosphorylated PERK concentrations in wild-type control mice were equal to GCN2−/−. In conclusion, these findings suggest that signalling mechanisms of eIF2α phos- phorylation are versatile in various stressful conditions and stages of AD and further studies are required to enhance our understanding of the precise mechanisms underlying interac- tions between these pathways.
Fig. 1 GCN2/eIF2α pathway in AD. (GCN2: General control nonderepressible 2, eIF2a: Eukaryotic Translation Initiation Factor 2A, BACE1: Beta-site amyloid precursor protein cleav- ing enzyme 1, AB: Amyloid β, ATF4: Activating transcription factor 4, PS1: Presenilin-1, GSK- 3: Glycogen synthase kinase 3, PP1: Protein phosphatase 1, CHOP: C/EBP homologous pro- tein). Amino-acid starvation, nu- trient deprivation, viral infections, oxidative stress, and UV irradia- tion can trigger GCN2 kinase.GCN2 applies its function through phosphorylating eIF2α leading to reduced global protein synthesis and increased upregula- tion of stress-responsive mRNAs including ATF4 and BACE1.Overexpression of stress- responsive mRNAs accelerates AB production, tau phosphoryla- tion, and induces neuronal apoptosis.
PKR kinase
PKR, a 551 amino acid interferon-induced serine-threonine protein kinase, is found to be one of the key elements to evoke a cellular response under stress conditions leading to transla- tion inhibition of several molecular pathways leading to AD. It can also be activated by Aβ1–42 neurotoxicity. PKR phos- phorylates eIF2α in response to viral infections or upon bind- ing to double-stranded RNAs, leading to the downregulation of viral mRNAs expression and causing apoptosis (Onuki et al. 2004). Interestingly, the amount of PKR in human cere- brospinal fluid is an early indication of cognitive decline and activated levels of PKR are increased in AD/MCI brains as well as AD/MCI cerebrospinal fluid, concluding that early MCI should be the most suitable stage for performing PKR inhibition (Dumurgier et al. 2013).
While eIF2α is the main target for PKR, PKR exerts at least a portion of the cascade of metabolic stress through other apoptosis intermediates, including p53 and NF-kβ and other caspase fam- ily members. P53 itself inhibits the mTOR pathway and permis- sively activates a wide range of p53-induced tumor-suppressive genes (Feng et al. 2007). On the other side, PKR activates the GSK3, which phosphorylates both eIF2α and P53, giving a cross-link between the two pathways. Importantly mTOR inhi- bition results in mitigation of the late LTP response observed by synaptic stimulation in pretreated brain slices with rapamycin (Morel et al. 2009). Later in 2010, Bose et al. showed that neu- ronal PKR co-locates with GSK-3b and tau protein in AD brains and is capable of modulating GSK-3b activation, tau phosphor- ylation and apoptosis in neuroblastoma cells (SH-SY5Y cell cultures) exposed to Tunicamycin or AB peptides, all of which are attenuated by PKR inhibitors or PKR siRNA (Bose et al. 2011 Baltzis et al. 2007).
Moreover, vital regulatory roles were demonstrated for PKR in protein synthesis and cell growth (Lee et al. 1997). Data have revealed an initiating role for PKR in the dysfunction of controlling translation, associated with neuro- nal death, and later in controlling the cell cycle and the inflam- matory reaction in peripheral mononuclear cells of AD pa- tients. Results have indicated a significant increase in phos- phorylated PKR and eIF2α levels in lymphocytes of AD pa- tients (Morel et al. 2009). A co-localization of the phosphor- ylated tau protein with phosphorylated PKR and tau protein was also detected in AD brains (Chang et al. 2002). In a virus- based research in 2008, EIF2αK2 (alpha kinase 2; PKR cod- ing) stated the shutdown of translation and increased concen- trations of activated PKR in AD patients ‘brains, although it is not obviously established whether this is an early disease oc- currence or a late neurodegeneration result (Bullido et al. 2008). PKR is a pro-apoptotic kinase that phosphorylates eIF2α and modulates the activation of c-jun N-terminal kinase (JNK) in different cell stresses Fig. 2. The JNK pathway is accountable for elevated expression of Beta-site APP cleaving enzyme 1 (BACE1) after cellular stress (Mouton-Liger et al. 2012). Mouton-Liger et al. 2012 explored the relationship between PKR, eIF2α, and BACE1 in hydrogen peroxide- induced oxidative stress in human neuroblastoma (SH- SY5Y) cell cultures and in AD brains of APP/PS1 knock-in mice. Results of immunoblotting showed that concentrations of activated PKR (p-PKR), activated eIF2α (p-eIF2α) and BACE1 in AD cortices correlate with phosphorylated concen- trations of eIF2α. BACE1 protein concentrations in SH- SY5Y neurons are elevated in reaction to OS and Specific PKR-eIF2α inhibitions in this model attenuate BACE1 pro- tein concentration. The insoluble 40–42 amino acid Aβ pep- tides are produced by BACE1, which is essential for AD pathogenesis.
PKR inhibition is a way for early treatment of MCI in AD patients. Researchers found that injecting PKR inhibitor (PKRi) to different AD mouse models can suppress ATF4 overexpression levels and restore the memory decline in early stages of AD, suggesting PKR inhibition as a way for early treatment of MCI in AD patients (Segev et al. 2015) (Hwang et al. 2017). In 2016, a group reported that Gastrodin is capa- ble of Suppressing BACE1 expression in hippocampi of Tg2576 mice under oxidative stress condition via inhibition of the PKR/eIF2α signaling pathway in AD. According to their findings, Gastrodin can improve the learning and mem- ory of Tg2576 mice and ameliorated OS in the hippocampi of mice, suggesting that Gastrodin can be a useful candidate to treat AD (Zhang et al. 2016a).
Moreover, studies suggest that genetic inhibition of PKR leads to neuroprotection. Results have indicated that LPS- induced neuroinflammation and beta-amyloidogenesis could be altered by genetic PKR downregulation (Carret-Rebillat et al. 2015). Demonstrated for the first time that the deletion of the PKR gene in a transgenic model of AD can reduce several func- tional, biochemical, and pathological brain alterations observed in the 5xFAD model (Tible et al. 2019). The researchers found that neuroinflammation and AB production are significantly ele- vated in the hippocampus of LPS-injected wild type mice and not in LPS-treated PKR knock-out mice. Besides, the elevation of BACE1 and activated STAT3 levels, BACE1 Putative Transcription Regulator, was not found in the brain of PKR knock-out mice as compared to wild type mice. These data sug- gest that the PKR pathway contributes to AD pathogenic mech- anisms and that inhibition of PKR has the potential to be used as a disease-modifying and symptomatic treatment for AD.
Fig. 2 PERK/eIF2α pathway in AD. (eIF2a: Eukaryotic Translation Initiation Factor 2A, BACE1: Beta-site amyloid pre- cursor protein cleaving enzyme 1, AB: Amyloid β, ATF4: Activating transcription factor 4, GSK-3: Glycogen synthase ki- nase 3, ISRIB: Integrated stress response inhibitor, DBM: Dibenzoylmethane). As an im- portant phosphorylator of eIF2α, PERK can mediate memory defi- cits and neurodegeneration. ALA and GSK2606414 can directly suppress the PERK pathway resulting in neuroprotection.Also, Trazodone, DBM, and ISRIB proved to inhibit UPR in- duced eIF2α signalling and re- store protein synthesis rates.
Prolonged overactivation of PERK, leading to hyperphosphorylation of its downstream target eIF2α, was demonstrated to lead to a sustained down-regulation of protein synthesis, proceeding to memory impairment and neuronal loss. As an upstream amyloid-β genesis and tauopathy regu- lator, PERK significantly involves in neuroprotection and mnemonic function through blocking PERK-eIF2α pathway. Genetic deletion of this kinase has prevented excessive phos- phorylation of eIF2α and deficits in protein synthesis, improv- ing synaptic plasticity and spatial memory in mice model of AD, proposing PERK as a potential therapeutic target for the treatment of individuals with AD (Ma et al. 2013). Its haploinsufficiency was reported to block overactivation of the PERK-eIF2α pathway, leading to significant down regu- lations in phosphorylation of PERK and eIF2α, in 5XFAD mice (Devi and Ohno 2014) Fig. 3. Additionally, to the role of PKR in B-amyloidogenesis, it also plays an important role in tau-mediated neurodegeneration. PERK-eIF2α overactivation also contributes to the pathological phosphorylation of tau in rTg4510 mice, and levels of phosphorylated tau are decreased by PERK inhibitor treatment. Oral Treatment with the PERK inhibitor, GSK2606414, in mutant tau-expressing mice re- stores protein synthesis rates to normal, protecting against further neuronal loss and reducing brain atrophy (Radford et al. 2015).
Genetic or pharmacological inhibition of eIF2α-P activity based on prior research can restore memory and prevent neu- rodegeneration (Trinh et al. 2012; Ohno 2014). Unfortunately, the preclinically used experimental compounds are unsuitable for human use owing to related toxicity or poor pharmacoki- netic characteristics. Investigating a clinically appropriate compound with anti-eIF2α-P activity, Halliday et al. per- formed phenotypic screenings on an NINDS small molecule library of 1040 drugs. First, by using the tunicamycin (glyco- sylation inhibitor) the ability of 1040 drugs to prevent ER stress-induced developmental delay in Caenorhabditis elegans was tested. Tunicamycin exposure makes it impossi- ble for most animals to reach the last larval developmental stage. Using this in vivo, 20 compounds were discovered in Caenorhabditis elegans that could overcome ER stress caused developmental delay. Later, Halliday et al. selectivity assessed cell lines from UPR-reporters, limiting the drugs to five com- pounds that lowered the expression of CHOP under ER stress. Finally, they were able to select two neuroprotective compo- nents (trazodone hydrochloride and DBM) as candidates for future clinical trials. Both components proved to inhibit UPR induced eIF2α signalling and restore protein synthesis rates in vitro and in vivo (Halliday et al. 2017). Both drugs are highly neuroprotective and capable of restoring memory def- icits, and reducing hippocampal atrophy without systemic tox- icity. Besides, Trazodone reduced tau phosphorylation bur- den. The pharmacological analysis revealed a remarkable blood-brain barrier (BBB) penetration and stability for both compounds. Regarding the fact that Trazodone and DBM are being currently used in patients, make these drugs a promising candidate of novel disease-modifying agents for many dis- eases with no current cure. Previous efforts to target the UPR were restricted by pancreatic toxicity using the PERK inhibitor GSK2606414. In mice treated with Trazodone or DBM, however, no such toxicity was apparent, implying that eIF2α P inhibition is indeed a feasible prospect of clinical application.
Fig. 3 PKR pathway and neurodegeneration. (eIF2a: Eukaryotic Translation Initiation Factor 2A, BACE1: Beta-site amyloid precursor protein cleaving enzyme 1, AB: Amyloid beta, ATF4: Activating tran- scription factor 4, GSK-3: Glycogen synthase kinase 3). Celluar stresses, pro-inflammation cytokines, and viral infections can trigger PKR kinase.PKR, when become activated, can trigger different neurodegenerative pathways including the JNK pathway and eIF2α pathway leading to memory deficits and neurodegeneration (Peel 2004; Peel and Bredsen 2003).
Researchers have also attempted to break the limitation in translation conferred by the integrated stress response, by PERK and eIF2α by ISRIB (Sidrauski et al. 2013). ISRIB as a novel small inhibitor of ATF4 expression has a safe tox- icological profile in addition to its ability to cross the BBB, unlike the GSK2606414. ISRIB does so by hampering the interaction of phospho-eIF2α with eIF2β which prevents the formation of the ternary complex from eIF2α, eIF2β, and GTP Without negative pancreatic impacts. It was shown in advance that mutation in eIF2β could mitigate its affinity for phosphorylated eIF2α and confer resistance to P-eIF2α (Pavitt et al. 1997). ISRIB is likely to do the same by preventing GTP from loading into the complex, having the advantage of not interacting with the phosphorylation site of other subunits of eIF2α, including GCN2, that might change its affinity for the GCN kinase, against the initial effect. ISRIB was also proposed to cause posttranslational regulations in eIF2β, making it inherently more active. They had previously introduced GSK2606414 to overcome translational attenua- tion and inhibit the UPR, by direct PERK inhibition (Moreno et al. 2013). GSK2606414 was extremely neuropro- tective to prevent clinical disease in prion-infected mice but to the detriment of tissue toxicity, Where UPR activation is crit- ical. Saliently, in comparison with the complete protein syn- thesis recovery seen with GSK2606414, ISRIB treatment can only partially restore global translation rates. Findings indicate that ISRIB offers adequate protein synthesis rates for neuronal survival while enabling certain residual protective UPR func- tions in secretory tissue. Without pancreatic toxicity, this ma- terial had additional neuroprotective characteristics and was shown to be effective in subsequent clinical studies (Moreno et al. 2013).
Recently researchers reported that Long-term alpha- linolenic acid (ALA) treatment can ameliorate age-related cognitive impairment and can decrease AD-like pathology during natural aging (Gao et al. 2016). ALA plays neuropro- tection via suppressing the PERK/eIF2α branch of UPR sig- nalling resulting in inhibition of AB production. Chronic ALA supplement can remarkably suppress BACE1, downregulate ATF4, enhance CREB function, and suppress tau phosphory- lation by (GSK-3β) pathway inhibition. Together these find- ings suggest that fine-tuning the level of PERK inhibition using the repurposed drugs can be a promising therapeutic target for developing drugs against prion disease or other neu- rodegenerative diseases where the UPR is implicated.
Fig. 4 PKR, PER, HRI and GCN2 kinases combined effects in AD. (eIF2a: Eukaryotic Translation Initiation Factor 2A, BACE1: Beta-site amyloid pre- cursor protein cleaving enzyme 1, AB: Amyloid beta, ATF4: Activating transcription factor 4, ISRIB: Integrated stress response inhibitor, DBM: Dibenzoylmethane, ALA: Alpha- linolenic acid, PKRi: protein ki- nase R inhibitor).
HRI kinase
HRI limits protein synthesis in heme-deficient erythroid cells, therefore, reduces the oxidative stress and promotes maturation and activation of macrophages. A general trans- lation attenuation in response to ER stress is due to dephos- phorylation of eIF2α mediated by a special protein phos- phatase (PP1). Therefore, the activation of eIF2α kinase, down-regulates general protein synthesis (Cao and Kaufman 2012). In 2015 ILL-Raga et al. indicated that HRI and eIF2α work under the influence of nitric oxide (NO) and glutamate signals in brain’s physiological cir- cumstances, and also that BACE1 has a physiological role in the function of the hippocampus (ILL-Raga et al. 2015). They found that BACE1 is expressed on glutamate activa- tion by causing eIF2α phosphorylation with NO being the downstream effector, Like the Western blot and luciferase assay obtained. This is due to NO activation of HRI as tested by Western blot and immunofluorescence with an HRI inhibitor and HRI siRNA. BACE1 translation was early detected at synaptic spines contributing to synapto- genesis and the hippocampal memory consolidation as evaluated with HRI or NO synthase inhibitors treated mice. Concluding that in glutamatergic hippocampal synapses,
BACE1 translation is regulated by NO through HRI. However, the possible pathways that directly link this ki- nase to AD development, remains to be further investigated.
EIF2α phosphorylation in AD
Four different kinases can phosphorylate eIF2α. The main known difference between these four kinases is their activa- tion in response to different stimuli. Phosphorylated eIF2α-p binds to eIF2B, causes a decrease in global protein synthesis, and increases the translation of stress responsive-mRNAs like ATF4 and BACE1. Various stress conditions like nutrient deprivation, viral infections, and cellular stresses including oxidative stress and ER stress can trigger the GCN2 kinase leading to eIF2α phosphorylation. PKR phosphorylates eIF2α in response to viral infections, upon binding to double-stranded RNAs, or cellular stresses leading to the downregulation of viral mRNAs expression and causing apo- ptosis. PERK mainly becomes activated upon the accumula- tion of misfolded proteins in time of cellular stress, especially ER stress. It is not acceptable that each eIF2α kinase responds to only one type of stressor. The current studies support the idea that in some conditions multiple eIF2α kinases will be activated. For example during oxidative stress, multiple eIF2α kinases, especially PERK, and GCN2 are recruited either si- multaneously or sequentially to reset cellular homeostasis, moreover, during the ER stress, all four kinases can be acti- vated. (Hamanaka et al. 2005). Studies showed that the dele- tion of either PERK or GCN2 can fine-tune eIF2α hyperphosphorylation. Researchers reported that although re- moval of PERK by itself resulted in decreased basal phosphorylation of eIF2α, removal of GCN2 alone did not affect basal eIF2α phosphorylation. Moreover, the deletion of Eif2ak2, which encodes the eIF2α kinase PKR, also did not alter the basal phosphorylation of eIF2α. Supporting the idea that multiple kinases should function to cooperatively regulate eIF2α phosphorylation (Fig. 4).
Conclusion
This review tends to illustrate the complexity of eIF2α path- way signaling in MCI/AD brains at various developmental stages and cellular compartments. As discussed, eIF2α pro- tein phosphorylation at Ser 51 disrupts protein synthesis, while upregulating the translation of a subset of mRNAs, in- cluding the B-secretase, BACE1, and the transcriptional mod- ulator activating transcription factor 4 (ATF4) (Sathya et al. 1970). These changes lead to increased B-amyloidogenesis through BACE1 upregulation. In addition, the upregulation of ATF4 leads to the promotion of GSK-3 and PP1 expression in AD patients and thereafter phosphorylates tau. ATF4 also increases the expression of autophagic genes and induces proapoptotic pathways. Four kinases including GCN2, PKR, PERK, and HRI directly phosphorylate eIF2α and disrupt general protein synthesis. Chronic phosphorylation of this pathway due to correspondent kinases overactivation is harm- ful to neuronal plasticity and memory formation. Recent stud- ies have come up with novel therapeutic agents that can effec- tively target the eIF2α pathway and fine-tune its inhibition. Recently, selective inhibitors of PERK and PKR pathways have been widely utilized to investigate possible disease- modifying therapeutic interventions regarding AD. Gastrodin is reported to be a potential candidate for AD treat- ment. Through BACE1 expression suppression in hippocampi of Tg2576 mice under oxidative stress condition via inhibition of the PKR/eIF2α signaling pathway, Gastrodin can improve learning and memory (Zhang et al. 2016b). GSK2606414 molecule can also directly inhibits the PERK/eIF2α pathway and provides strong neuroprotection but to the detriment of pancreatic toxicity (Radford et al. 2015). By contrast, ISRIB as a novel therapeutic agent can inhibit ATF4 expression with- out tissue toxicity. Although ISRIB can not fully recover pro- tein synthesis as seen with GSK2606414, it can provide a decent protein synthesis rate capable of neuroprotection. Moreover, this molecule has a great potential in easily cross- ing the BBB, making it an impressive therapeutic molecule in AD (Pavitt et al. 1997). In recent years, researchers have pre- sented novel neuroprotective compounds; including Trazodone, DBM, alpha-linolenic acid and (Halliday et al. 2017). These compounds are highly neuroprotective and can restore memory deficits. Besides, their potential in crossing BBB makes them great therapeutic potentials regarding AD. All together, the current literature suggests that modulating the eIF2α inhibition genetically or through utilization of repurposed drugs, such as Trazodone or DBM, can be a prom- ising therapeutic target regarding AD. Table 1 illustrates the SBI-0640756 promising therapeutics regarding eIF2a pathway in AD.