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. 2018 Feb 13;115(7):1611-1616.
doi: 10.1073/pnas.1711977115. Epub 2018 Jan 29.

Hippocampal expression of a virus-derived protein impairs memory in mice

Alexandre Bétourné  1 , Marion Szelechowski  1   2 , Anne Thouard  1 , Erika Abrial  3   4 , Arnaud Jean  2 , Falek Zaidi  1   2 , Charlotte Foret  1 , Emilie M Bonnaud  1 , Caroline M Charlier  1 , Elsa Suberbielle  1 , Cécile E Malnou  1 , Sylvie Granon  5 , Claire Rampon  2 , Daniel Gonzalez-Dunia  6
Affiliations

Hippocampal expression of a virus-derived protein impairs memory in mice

Alexandre Bétourné et al. Proc Natl Acad Sci U S A. .

Abstract

The analysis of the biology of neurotropic viruses, notably of their interference with cellular signaling, provides a useful tool to get further insight into the role of specific pathways in the control of behavioral functions. Here, we exploited the natural property of a viral protein identified as a major effector of behavioral disorders during infection. We used the phosphoprotein (P) of Borna disease virus, which acts as a decoy substrate for protein kinase C (PKC) when expressed in neurons and disrupts synaptic plasticity. By a lentiviral-based strategy, we directed the singled-out expression of P in the dentate gyrus of the hippocampus and we examined its impact on mouse behavior. Mice expressing the P protein displayed increased anxiety and impaired long-term memory in contextual and spatial memory tasks. Interestingly, these effects were dependent on P protein phosphorylation by PKC, as expression of a mutant form of P devoid of its PKC phosphorylation sites had no effect on these behaviors. We also revealed features of behavioral impairment induced by P protein expression but that were independent of its phosphorylation by PKC. Altogether, our findings provide insight into the behavioral correlates of viral infection, as well as into the impact of virus-mediated alterations of the PKC pathway on behavioral functions.

Keywords: dentate gyrus; hippocampus; memory; protein kinase C; virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Lentiviral expression of the P protein interferes with PKC-dependent phosphorylation in mouse hippocampal neurons. (A) Schematic representation of the P protein, displaying the protein kinase C (PKC) and casein kinase II (CK2) phosphorylation sites. NES, nuclear export sequence; NLS, nuclear localization sequence. Map of the lentiviral vectors (LVs) expressing GFP, wild-type P (PWT), or mutant P (PAASS). Also shown are: positions of the cytomegalovirus enhancer/chicken β-actin (CAG) promoter and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). (B) Western blot analysis of phospho-SNAP25 levels upon PKC stimulation. Protein extracts were prepared from neurons transduced with LVs expressing GFP (as a control), PWT, or PAASS, after stimulation or not with PMA. Nontransduced (NT) neurons were also processed in parallel. Levels for β-actin and total SNAP25 were used to normalize phosphorylation levels. Data shown are those of a representative experiment out of four that gave similar results. (C) Stereotaxic procedure for in vivo LV delivery and experimental timeline. (D) Expression of GFP, PWT, or PAASS in the DG of mice, 4 mo after surgery. Representative pictures of brain sections expressing GFP, PWT, or PAASS in the dorsal and ventral DG. (Scale bar, 100 μm.) Insets show enlarged view to visualize P expression in the dendrites.
Fig. 2.
Fig. 2.
Impact of hippocampal P expression on locomotor activity and basal anxiety. (A) Distance traveled and (B) mean velocity during exploration in the open field. (C) Analysis of anxiety-like behavior in the elevated plus maze. PWT decreased the percent of time spent in the open arms (one-way ANOVA, P < 0.01). (D) PWT also decreased the number of visits in the open arms of the elevated plus maze (one-way ANOVA, P < 0.05). Data are expressed as means ± SEM (GFP n = 8; PWT n = 11; PAASS n = 10). **P < 0.01, #P < 0.05 by post hoc Fisher’s least significant difference test for, respectively, PWT vs. GFP and PWT vs. PAASS.
Fig. 3.
Fig. 3.
Effects of P expression on contextual memory. (A) Fear expression in GFP, PWT, and PAASS mice during conditioning. Repeated ANOVA ran on 30-s blocks during the whole session (P < 0.05), and independent one-way ANOVA ran on block 4 (*P < 0.05). All groups displayed a similar increase in their freezing response after delivery of the electric shock (P > 0.05, independent one-way ANOVA ran on block 6). The lightning bolt icon indicates the time of shock delivery; the speaker indicates the tone delivery block. (B) Contextual memory assessed 24 h after conditioning and expressed as normalized data (SI Materials and Methods), showing the selective impairment of contextual memory due to PWT expression in the DG. One-way ANOVA, P < 0.01; #P < 0.05 for PWT vs. GFP and **P < 0.01 for PWT vs. PAASS by post hoc Fisher’s least significant difference test. (C) Analysis of residual freezing to the modified context before tone emission, 48 h after training. (D) Cued memory, assessed in a modified context, 48 h after training. Data are expressed as means ± SEM (GFP n = 8; PWT n = 10; PAASS n = 9).
Fig. 4.
Fig. 4.
Effects of P expression on long-term spatial memory in the object location task. (A) Schematic representation of the object location task and experimental timeline. (B) Cumulated time spent exploring the objects during training sessions (seconds). (C) Analysis of the preference index (as detailed in SI Materials and Methods). The horizontal dotted line represents equal exploration of both objects (50%). Comparison with 50%: *P < 0.05, **P < 0.01 for index vs. chance level, Wilcoxon signed-rank test. Data are expressed as means ± SEM (GFP n = 8; PWT n = 11; PAASS n = 10).
Fig. 5.
Fig. 5.
Effects of P expression on long-term spatial memory in the Morris water maze. (A) Schematic representation of the setup and experimental timeline. (B) Mean distance traveled to find the hidden platform during training (one-way ANOVA with repeated measures *P < 0.001). (C and D) Effects of P expression on spatial memory during the 24 h posttraining probe test. (C) Time (seconds) spent in each quadrant of the pool, showing that all mice spent more time in the target quadrant compared with the three others (***P < 0.001 by one-way ANOVA intragroup analyses). Comparison of time spent in the target quadrant between PWT mice and GFP and PAASS animals. One-way ANOVA, #P < 0.05. (D) Spatial search precision: analysis of the number of annuli crossings between groups of mice. *P < 0.05 for PWT and ***P < 0.001 for GFP and PAASS, by one-way ANOVA intragroup analyses. (P > 0.65 and P > 0.07 for, respectively, target vs. east and target vs. west, by post hoc Fisher’s least significant difference test). Data are expressed as means ± SEM (GFP n = 20; PWT n = 25; PAASS n = 23).
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