Neuroinflammation and Aβ accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder, marked by senile plaques composed of amyloid-β (Aβ) peptide, neurofibrillary tangles, neuronal loss and neuroinflammation. Previous works have suggested that systemic inflammation could contribute to neuroinflammation and enhanced Aβ cerebral concentrations. The molecular pathways leading to these events are not fully understood. PKR is a pro-apoptotic kinase that can trigger inflammation and accumulates in the brain and cerebrospinal fluid of AD patients. The goal of the present study was to assess if LPS-induced neuroinflammation and Aβ production could be altered by genetic PKR down regulation. The results show that, in the hippocampus of LPS-injected wild type mice, neuroinflammation, cytokine release and Aβ production are significantly increased and not in LPS-treated PKR knock-out mice. In addition BACE1 and activated STAT3 levels, a putative transcriptional regulator of BACE1, were not found increased in the brain of PKR knock-out mice as observed in wild type mice. Using PET imaging, the decrease of hippocampal metabolism induced by systemic LPS was not observed in LPS-treated PKR knock-out mice. Altogether, these findings demonstrate that PKR plays a major role in brain changes induced by LPS and could be a valid target to modulate neuroinflammation and Aβ production.

Figure 1. Activation of PKR in WT mice cortex and hippocampus after LPS systemic challenge.

Double-labeling in immunofluorescence of DAPI (blue) and pPKR_Thr446 (red) in hippocampus (A) and frontal cortex (B) sagittal sections of WT mice treated with saline (sal) or LPS. Percentage of pPKR_Thr446 positive cells (C) and relative IOD (integrated optical density) of pPKR_Thr446 immunoreactivity (D) are increased in LPS-treated mice. WT [n = 4], *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. Microglial activation in WT and PKR^-/- mice hippocampus.

Double-labeling in immunofluorescence of DAPI (blue) and IBA1 (red) in hippocampus sagittal sections of WT and PKR^-/- mice treated with saline or LPS (A). Relative IOD (integrated optical density) of IBA1 immunoreactivity in activated microglial cells. LPS induces microglial activation in WT mice. PKR inhibition prevents microglial activation after LPS treatment (B). mRNA level of TNF-α evaluated by qRT-PCR and normalized to GAPDH mRNA levels (C). Brain Il-6 levels in controls or after LPS injection in wild type mice and PKR knock-out mice (D). WT [n = 4], PKR^-/- [n = 4], *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Inhibition of PKR decreases BACE1 levels and Aβ production in mice hippocampus after LPS systemic challenge.

Immunoblot analysis (A) and protein levels of mature BACE1 (B) and APP (C) and quantification of Aβ_1–42 by ELISA (D) in WT and PKR^-/- mice treated with saline or LPS. LPS induces BACE1 maturation and Aβ production without altering APP levels in WT mice. PKR inhibition prevents BACE1 maturation and Aβ production after LPS treatment. Transcriptional activity on BACE1 in hippocampus assessed with qRT-PCR and normalized to GAPDH mRNA levels (E). Immunoblots (A) have been cropped in this figure. Full length version of BACE1 and APP blots are available in the supplementary Figure 5. WT [n = 4], PKR^-/- [n = 4], **p < 0.01.

Figure 4. Activation of transcription factor STAT3 after LPS systemic challenge is down-regulated by PKR but not translation initiation factor eIF2α.

Immunoblot analysis (A) and corresponding quantification of peIF2α_Ser51/eIF2α (B) and pSTAT3_Tyr705/STAT3 (C) ratios in hippocampus of WT and PKR^-/- mice treated with saline or LPS. LPS induces STAT3 but not eIF2α activation in WT mice. PKR inhibition prevents STAT3 activation. Cropped immunoblots are presented in this figure. Full length immunoblots of eIF2α (phosphorylated and total forms) and STAT3 (phosphorylated and total forms) are available in the supplementary Figure 6. WT [n = 4], *p < 0.05, **p < 0.01.

Figure 5

(A) Sagittal view of a co-registered PET-CT whole-body mouse. (B) Coronal view of a co-registered murine cerebral MRI-PET. Regions of interest have been drawn on the hippocampus, bilaterally. (C) Hippocampal, Ammon's horn, in vivo metabolism through [¹⁸F]FDG-PET.

Figure 6. Schematic representation of PKR-dependent Aβ production in brain after systemic inflammation.

Peripheral inflammation communicates with the brain through the blood-brain barrier to induce microglial activation and pro-inflammatory cytokines production, involving PKR pathway. Cytokines, including TNFα, activate PKR in neurons leading to Aβ production. Thereof could be controlled by the activation of transcription factor STAT3.