Poly (I∶C) induces BACE1 translation in a mechanism depending on PKR-catalysed eIF2-alpha phosphorylation.

<p>BACE1 5′UTR fragment was inserted into the HindIII site of a luciferase reporter construct (5′UTR-luc). The plasmids bear the strong cytomegalovirus promoter (CMV), which allows sufficient reporter gene expression for luciferase determinations. HeLa cells were transfected with 5′UTR-luc or empty plasmids (named as CMV) for luciferase reporter studies (A). 5′UTR-luc repressed translation in comparison with the empty 5′UTR-free luciferase construct (B). Inhibition of eIF2-alpha phosphatase PP1c by Sal003 de-repressed the reporter signal elicited by 5′UTR-luc (C). Stimulation (3 h) with poly (I∶C) also de-repressed the reporter signal yielded by 5′UTR-luc. This effect was abolished when cells were preincubated with a specific and potent PKR inhibitor that acts via the PKR ATP-binding site. Data are mean ± SEM of 3 independent experiments performed in triplicate. *p<0.05 by Student's <i>t</i> test (D). Incubation with the specific PKR inhibitor -in the presence/absence of poly (I∶C)- did not result in a decrease in cell viability as shown by an MTT assay. Data are mean ± SEM of 3 independent experiments performed in triplicate (Inset in panel D).</p

Presence of activated PKR in human brain tissue.

<p>The presence of activated PKR -autophosphorylated at Thr446- was studied in human brain sections (temporal lobe) from one non-demented control and one AD patient by immunohistochemistry (A). Colocalization of BACE1 and p-PKR(T446) in neurons from an AD patient. All brain sections analysed were positive for HSV1 infection as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021456#pone.0021456-Wozniak3" target="_blank">[39]</a> (B).</p

HSV1 infection activates PKR.

<p>SH-SY5Y cells were infected with HSV1 (1 pfu/cell, 24 h) or were not infected (controls). Immunocytochemistry analysis was carried with the following Abs: anti-p-PKR, anti-p-eIF2-alpha and anti-BACE1 (A). Protein extracts of SH-SY5Y cells infected with HSV1 (3 pfu/cell, 24 h) and uninfected cells (controls) were analysed by Western blotting using the following Abs: anti-BACE1, anti-p-PKR, anti-PKR, anti-p-eIF2-alpha, anti- eIF2-alpha and anti-tubulin. Bands were quantified by densitometric analysis. Results are expressed as the mean ± SEM of 3–4 independent experiments. * p<0.05, ** p<0.01, *** p<0.0005 by Student's <i>t</i> test (B). Sections from mouse dorsal ganglion root (DRG) were obtained from HSV1-infected mice. Contralateral uninfected ganglia were used as controls. Immunohistochemistry analysis was carried out to detect p-PKR (C).</p

Biochemical pathway linking HSV1 infection and AD.

<p>Despite the viral ability to circumvent host defensive mechanisms, including PKR activation, HSV1 activates PKR <i>in vitro</i> and <i>in vivo</i>. Activated PKR increases eIF2-alpha phosphorylation levels, leading to BACE1 translation de-repression, BACE1 protein up-regulation and Aß production (left track; continuous line). In the right track (dotted line) we present the pharmacological and biological tools that we used to study this pathway: poly (I∶C), to mimic the effect of viral dsRNA; a specific imidazolo-oxindole compound that acts as a potent PKR inhibitor; and a 5′UTR-luc reporter construct used for the evaluation of the translational effect exerted by the PKR-eIF2-alpha pathway over BACE1 5′UTR. Also, we have utilized the PP1c inhibitor salubrinal, which prevents the dephosphorylation of eIF2-alpha.</p