Interleukin 1β triggers synaptic and memory deficits in Herpes simplex virus type-1-infected mice by downregulating the expression of synaptic plasticity-related genes via the epigenetic MeCP2/HDAC4 complex

Abstract

Extensive research provides evidence that neuroinflammation underlies numerous brain disorders. However, the molecular mechanisms by which inflammatory mediators determine synaptic and cognitive dysfunction occurring in neurodegenerative diseases (e.g., Alzheimer's disease) are far from being fully understood. Here we investigated the role of interleukin 1β (IL-1β), and the molecular cascade downstream the activation of its receptor, to the synaptic dysfunction occurring in the mouse model of multiple Herpes simplex virus type-1 (HSV-1) reactivations within the brain. These mice are characterized by neuroinflammation and memory deficits associated with a progressive accumulation of neurodegenerative hallmarks (e.g., amyloid-β protein and tau hyperphosphorylation). Here we show that mice undergone two HSV-1 reactivations in the brain exhibited increased levels of IL-1β along with significant alterations of: (1) cognitive performances; (2) hippocampal long-term potentiation; (3) expression synaptic-related genes and pre- and post-synaptic proteins; (4) dendritic spine density and morphology. These effects correlated with activation of the epigenetic repressor MeCP2 that, in association with HDAC4, affected the expression of synaptic plasticity-related genes. Specifically, in response to HSV-1 infection, HDAC4 accumulated in the nucleus and promoted MeCP2 SUMOylation that is a post-translational modification critically affecting the repressive activity of MeCP2. The blockade of IL-1 receptors by the specific antagonist Anakinra prevented the MeCP2 increase and the consequent downregulation of gene expression along with rescuing structural and functional indices of neurodegeneration. Collectively, our findings provide novel mechanistic evidence on the role played by HSV-1-activated IL-1β signaling pathways in synaptic deficits leading to cognitive impairment.

Keywords: Anakinra; Memory; Neurodegeneration; Neuroinflammation; SUMOylation; Synaptic function.

Fig. 1

Inhibition of IL-1R restores LTP and hippocampal-dependent memory in HSV-1-infected mice undergoing 2TS. A Bar graph showing IL-1β levels in hippocampal homogenates from mock- and HSV-1-infected mice sacrificed 24 h (n = 5 for each group) and 1 week (n = 3 and 4, respectively) after 2TS; B, C Bar graphs showing the mean values of PI for the novel object in NOR test performed: B 1 week after 2TS (n = 16 mock, n = 15 HSV-1, n = 10 mock + A and n = 12 HSV-1 + A); C 3 weeks after 2TS in mock- and HSV-1-infected mice treated and untreated with Anakinra (n = 13 mice/group); D mean time course of fEPSP amplitude before and after high-frequency stimulation (indicated by arrow) in hippocampal slices from: mock- (n = 15 slices from 7 mice) and HSV-1-infected mice (n = 15 slices from 6 mice) and mice treated with Anakinra [mock + A (n = 11 slices from 8 mice) and HSV-1 + A (n = 15 slices from 7 mice)] sacrificed 1 week afteṙ 2TS; E Bar graphs showing mean LTP in the last 5 min of recording in controls and infected mice. *p < 0.05, **p < 0.01 and ***p < 0.005 vs. mock; ^§p < 0.05 vs. mock + A; ^#p < 0.05 and ^##p < 0.005 vs. HSV-1 assessed by Mann–Whitney Rank Sum Test (A) and by two-way ANOVA followed by Student–Newman–Keuls post-hoc (B–E). n.s. not significant difference

Fig. 2

Anakinra rescues synaptic deficits induced by IL-1β in HSV-1-infected mice. A Representative WB images of pre- and post-synaptic proteins in hippocampal lysates from mock- and HSV-1-infected mice treated or untreated with Anakinra; B, C Bar graphs showing the densitometric analysis of SYN1 (B) and SYP (C) in mock- (n = 6 and n = 7, respectively) and HSV-1-infected mice (n = 6 and n = 5), and in Anakinra-treated mock- (n = 6 and n = 5) and infected mice (n = 6 and n = 5); D–F Densitometric analysis of postsynaptic proteins: D GluA1 (n = 6 mock, n = 7 HSV-1, n = 9 mock + A and n = 8 HSV-1 + A); E NR2B (n = 7 for both mock- and HSV-1-infected mice, n = 5 mock + A, n = 6 HSV-1 + A); and F PSD95 (n = 6 mock, n = 7 HSV-1, n = 6 mock + A and n = 9 HSV-1 + A); G Relative expression of mRNA encoding for SYN1 (n = 5 mock, n = 6 HSV-1 infected mice, n = 5 Anakinra-treated controls and n = 3 Anakinra-treated HSV-1-infected mice). *p < 0.05, **p < 0.01 and vs. mock, ^#p < 0.05 vs. HSV-1-infected mice assessed by Kruskal–Wallis One Way Analysis of Variance on Ranks. n.s. not significant difference

Fig. 3̇

Dendritic spine density and morphology are restored in Anakinra treated HSV-1-infected mice. A Representative images of Golgi staining of apical and basal dendrites of CA1 pyramidal neurons of mock- and HSV-1-infected animals with and without Anakinra treatment (n = 4 mice/group, at least 10 neurons/animal); B Bar graph showing mean values of spine density in all neurons examined in A; C, D Bar graphs showing the percentage of mushroom and thin spine types of apical (C) and basal (D) dendrites in Anakinra treated and untreated mice. **p < 0.01 and ***p < 0.005 vs. mock, ^#p < 0.05 and ^##p < 0.005 vs. HSV-1, assessed by two-way ANOVA and Newman-Keuls post-hoc. n.s. not significant difference. Scale bar 10 µm

Fig. 4

IL-1β upregulates the epigenetic repressor MeCP2 in HSV-1-infected mice. A, B Representative images (A) and quantification (B) of WB analysis for MeCP2 protein levels in hippocampal homogenates from mock- (n = 4) and HSV-1-infected mice (n = 4) sacrificed 24 h after 2TS; D, E Representative WB images (D) and quantification (E) of MeCP2 protein levels in untreated mock- (n = 6) and HSV-1-infected mice (n = 6) and in animals treated with Anakinra (n = 4 for mock; n = 6 for HSV-1-infected mice analyzed 1 week after 2TS; C, F Relative expression of mRNA encoding for MeCP2 (n = 7 for both) analyzed 24 h (C) and 1 week (F) after 2TS (n = 8 for untreated control mice and n = 7 for untreated infected ones; n = 4 and n = 3 for Anakinra-treated controls and HSV-1-infected mice). Tubulin was used as a loading control. *p < 0.05, **p < 0.01 vs. mock, ^#p < 0.05 vs. HSV-1, assessed by Mann–Whitney Rank Sum Test (for B, C) and Kruskal–Wallis One Way Analysis of Variance on Ranks (for E, F). n.s. not significant difference

Fig. 5

In HSV-1-infected mice nuclear HDAC4 interacts with MeCP2 and promotes its SUMOylation. A, B Cellular fractioning showing HDAC4 distribution in the nuclear and cytoplasmic compartments in hippocampal extracts from mock- and HSV-1-infected mice and their densitometric analyses (n = 4 mice/group). Dotted line indicates the cytoplasmic and nuclear levels of HDAC4 in the controls; C representative Co-IP experiment showing HDAC4 interaction with MeCP2 in hippocampal tissues form mock- and HSV-1-infected mice (n = 4 mice/group) and its quantification (D); HDAC2 was used as control of interaction; E, F Co-IP showing SUMO-1-MeCP2 levels in mock- and HSV-1-infected mice (n = 5 mice/group) and its quantification; G, H ChIP assay showing HDAC4 recruitment on c-fos and syn1 promoters performed on HSV-1- and mock-infected animals (n = 4 mice/group). *p < 0.05 vs. mock, assessed by Mann–Whitney Rank Sum Test