Julien Couturier

Interaction of double-stranded RNA-dependent protein kinase (PKR) with the death receptor signaling pathway in amyloid beta (Abeta)-treated cells and in APPSLPS1 knock-in mice.

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-β peptide (Aβ) neurotoxicity and in APPSLPS1 KI transgenic mice. In SH-SY5Y cells, results showed that Aβ42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APPSLPS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT451-PKR and pS194-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.

PKR, the double stranded RNA-dependent protein kinase as a critical target in Alzheimer's disease

•  Introduction •  Control of translation •  Dysfunctions of protein synthesis mediated by PKR‐ and mTOR‐dependent signalling pathways •  Crosslink between the up‐regulation of PKR/eIF2α and the down‐regulation of mTOR/RS6K in AD •  PKR: a potential biomarker of AD diagnosis •  Conclusion and perspectives

Evidence of molecular links between PKR and mTOR signalling pathways in Abeta neurotoxicity: role of p53, Redd1 and TSC2.

The control of translation is disturbed in Alzheimers disease (AD). This study analysed the crosslink between the up regulation of double-stranded RNA-dependent-protein kinase (PKR) and the down regulation of mammalian target of rapamycin (mTOR) signalling pathways via p53, the protein Regulated in the Development and DNA damage response 1 (Redd1) and the tuberous sclerosis complex (TSC2) factors in two beta-amyloid peptide (Abeta) neurotoxicity models. In SH-SY5Y cells, Abeta42 induced an increase of P(T451)-PKR and of the ratio p66/(p66+p53) in nuclei and a physical interaction between these proteins. Redd1 gene levels increased and P(T1462)-TSC2 decreased. These disturbances were earlier in rat primary neurons with nuclear co-localization of Redd1 and PKR. The PKR gene silencing in SH-SY5Y cells prevented these alterations. p53, Redd1 and TSC2 could represent the molecular links between PKR and mTOR in Abeta neurotoxicity. PKR could be a critical target in a therapeutic program of AD.

Pharmacological inhibition of PKR in APPswePS1dE9 mice transiently prevents inflammation at 12 months of age but increases Aβ42 levels in the late stages of the Alzheimer's disease.

The double-stranded RNA-dependent protein kinase (PKR) is switched on by a wide range of stimuli, including the amyloid peptide. Then, PKR transmits signals to the translational machinery, apoptosis and inflammatory signaling pathways by interacting with some adapters. In virus-infected cells, PKR engages the nucleus factor κB (NF-κB) pathway. In many models of Alzheimers disease (AD) and patients with AD, PKR was activated. Furthermore, there is strong evidence implicating the inflammatory process in the AD brain. However, the PKR involvement in inflammatory responses in AD is not elucidated. Based on our previous in vitro results, the aim of this study was to evaluate the effects of a pharmacological inhibition of PKR in inflammation in APPswePS1dE9 transgenic mice. Our results showed that PKR inhibition prevented the NF-κB activation and production of tumor necrosis factor alpha (TNFα) and interleukin (IL)-1β at 12 months of age without decrease of Aβ42 levels and memory deficits. Surprisingly, PKR inhibition failed to prevent IL-1β- mediated inflammation and induced a great increase in β-amyloid peptide (Aβ42) levels at 18 months of age. In this model, our findings highlight the lack of relationship between inflammation and Aβ42 levels. Moreover, the age-dependent inflammatory response must be carefully taken into account in the establishment of an anti-inflammatory therapy in AD.

Inhibition of double-stranded RNA-dependent protein kinase strongly decreases cytokine production and release in peripheral blood mononuclear cells from patients with Alzheimer's disease.

Alzheimers disease (AD), a neurodegenerative disorder, is the most common form of dementia in the elderly individuals. Among the pathogenic mechanisms in AD, chronic systemic inflammation is described and characterized by massive production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMCs), which may contribute to an altered immune response and exacerbation of neurodegeneration. Studies have also reported increased double-stranded RNA-dependent protein kinase (PKR) activation in the PBMCs of patients with AD. Interestingly, PKR could be involved in NF-κB activation, leading to production of a wide range of cytokines. We proposed to decrease proinflammatory cytokines production and release by treating the PBMCs in 25 patients with AD with a specific inhibitor of PKR. Our results showed that PKR inhibition greatly decreased tumor necrosis factor , interleukin (IL)-1α, IL-1β, and IL-6 production and release but did not affect the chemokine RANTES. Moreover, inhibition of the proinflammatory factors was correlated with prevention of caspase-3 activation. These results indicated that specific inhibition of PKR at the peripheral level might decrease the inflammatory response in AD.

C24 Inflammation et maladie d’Alzheimer

La maladie d’Alzheimer (MA) est la principale cause de demence d’origine degenerative. Les travaux physiopathologiques concernent essentiellement les plaques amyloides et la degenerescence neurofibrillaire (DNF). Il apparait evident que la composante inflammatoire participe egalement au mecanisme de la mort neuronale. Ainsi, le peptide amyloide Aβ peut stimuler la voie classique du complement qui joue un role important dans le recrutement des astrocytes et de la microglie. Ces cellules produisent plusieurs mediateurs de l’inflammation dont les cytokines : IL-1β, IL-6 et TNFalpha;, fortement exprimees a proximite des plaques amyloides et des lesions de DNF et impliquees dans la synthese d’Aβ. Les neurones produisent egalement plusieurs cytokines susceptibles de participer au processus inflammatoire. Des travaux ont montre des correlations entre les taux plasmatiques de certaines cytokines et le declin cognitif a des stades legers a moderes et suggerent une interaction entre les cellules gliales et les lymphocytes T. Bien d’autres parametres de l’inflammation sont impliques au cours de la MA comme les chemokines, les facteurs de croissance, ou le stress oxydant. Le role de l’inflammation au cours de la MA n’est cependant pas totalement elucide. En effet, il a aussi ete demontre que la microglie activee facilitait la clairance d’Aβ. Ainsi, la reaction inflammatoire est a la fois deletere et protectrice. Sur le plan therapeutique, des etudes epidemiologiques ont montre que l’utilisation au long cours de traitements anti-inflammatoires dans les maladies rhumatismales, semblait diminuer le risque de MA. Neanmoins les resultats sont contradictoires en fonction de l’AINS utilise suggerant que l’inhibition de la COX-2 n’etait pas la seule explication. Les essais concernant l’interet des molecules anti-TNF meritent confirmation. L’agregation d’Aβ dans le cerveau entraine une activation des cellules gliales et la production de mediateurs de l’inflammation, mais le doute persiste quand a la signification exacte de cette neuroinflammation.

P2b-15 Inhibition de la kinase PKR dans la prévention de l’inflammation au cours de la maladie d’Alzheimer

Introduction Des etudes soulignent la composante inflammatoire dans la physiopathologie de la maladie d’Alzheimer (MA). Le spectre des mediateurs inflammatoires dont TNFα, IL-1β et IL-6 implique de nombreuses voies de signalisation, conduisant a la mort neuronale. Il est bien connu que la MA se caracterise aussi par une activation de la voie PKR (double-stranded RNAdependent Protein Kinase). Des travaux ont egalement montre que PKR peut controler l’expression de differents mediateurs inflammatoires via l’activation du facteur NF-κB. Cependant, le role de PKR dans la reaction inflammatoire au cours de la MA est inconnu. Cette etude a pour but de mettre en evidence l’implication de PKR dans l’inflammation au cours de la MA en suivant les facteurs moleculaires de la voie NF-κB. Materiel et methodes Une premiere etude a ete realisee sur des co-cultures primaires comprenant neurones (45%), astrocytes (40%) et microglie (15%) dans des proportions physiologiques, traitees par le peptide amyloide Aβ42 (20μM) avec ou sans pre-traitement par un inhibiteur specifique de PKR (C16 a 210 nM). Les cytokines (TNFα, IL-1β et IL-6) ont ete dosees par ELISA dans des lysats cellulaires. La seconde etude realisee sur des lymphocytes de patients atteints de la MA remis en culture et traites par le C16 a 1μM, consiste en un dosage des cytokines (TNFα, IL-1α et β, IL-6 et RANTES) produites et liberees, grâce a la technique xMAP®. L’expression de PS32-36IκBα, PS536NF-κB et de la caspase-3 clivee a ete analysee par Western Blot. Resultats Aβ42 augmente significativement le taux des cytokines a 72h, ainsi que l’expression de PS32-36IκBα, PS536NF-κB et induit une activation de la caspase-3. Ces modifications sont prevenues par un pre-traitement au C16 a 210 nM. Ces resultats ont ete confirmes sur des lymphocytes de patients atteints de la MA. En effet, le C16 a 1 μM previent totalement la production des cytokines, exceptee celle de RANTES. L’activation de la voie NF-κB est egalement prevenue. Conclusion Ces resultats montrent l’implication de PKR dans l’inflammation au cours de la MA et confirment que cette kinase constitue une cible tres interessante dans les strategies therapeutiques futures de la MA.