Multiple Herpes Simplex Virus-1 (HSV-1) Reactivations Induce Protein Oxidative Damage in Mouse Brain: Novel Mechanisms for Alzheimer's Disease Progression

Abstract

Compelling evidence supports the role of oxidative stress in Alzheimer's disease (AD) pathophysiology. Interestingly, Herpes simplex virus-1 (HSV-1), a neurotropic virus that establishes a lifelong latent infection in the trigeminal ganglion followed by periodic reactivations, has been reportedly linked both to AD and to oxidative stress conditions. Herein, we analyzed, through biochemical and redox proteomic approaches, the mouse model of recurrent HSV-1 infection we previously set up, to investigate whether multiple virus reactivations induced oxidative stress in the mouse brain and affected protein function and related intracellular pathways. Following multiple HSV-1 reactivations, we found in mouse brains increased levels of oxidative stress hallmarks, including 4-hydroxynonenal (HNE), and 13 HNE-modified proteins whose levels were found significantly altered in the cortex of HSV-1-infected mice compared to controls. We focused on two proteins previously linked to AD pathogenesis, i.e., glucose-regulated protein 78 (GRP78) and collapsin response-mediated protein 2 (CRMP2), which are involved in the unfolded protein response (UPR) and in microtubule stabilization, respectively. We found that recurrent HSV-1 infection disables GRP78 function and activates the UPR, whereas it prevents CRMP2 function in mouse brains. Overall, these data suggest that repeated HSV-1 reactivation into the brain may contribute to neurodegeneration also through oxidative damage.

Keywords: Alzheimer’s disease; HSV-1; Herpes simplex virus-1; oxidative stress; redox proteomics.

Figure 1

Experimental design. We infected two-month-old BALB/c mice with Herpes simplex virus-1 (HSV-1, HSV1-M) or a mock solution (CTRL-M). Six weeks after the infection, mice underwent several cycles of thermal stress (TS) that were spaced 6–8 weeks from each other. After the 7th TS, the mice were sacrificed, and brain tissues were collected for biochemical analyses (redox proteomics, Western blot, WB, and immunoprecipitation, IP) or perfused with paraformaldehyde (PFA) for confocal immunofluorescence analysis (IF).

Figure 2

Total and protein-specific levels of 4-hydroxynonenal (HNE) in the brain of mice representing an in vivo model of recurrent HSV-1 reactivation. (A) Representative WB of HNE–protein levels in cortical lysates from HSV1-M or CTRL-M sacrificed after 7 cycles of TSs at 13 months of age. Densitometric analysis of oxidative modifications observed in HSV1-M (n = 6) are shown in the graph as fold increase compared to CTRL-M (n = 6). Each HNE densitometric value was normalized with total load within the same lane. Error bars represent SEM, (* p < 0.05 assessed by Student’s t-test). (B) Representative 2D-Map-blot proteomic profiles of HNE-oxidized proteins in cortical lysates from HSV1-M and CTRL-M sacrificed after 7 cycle of TS at 13 months of age. (C) Representative total protein spots detected in an HSV1-M sample by SYPRO Ruby staining in a 2D gel. Numbered spots indicate all the HNE-modified proteins with significant altered levels in HSV1-M with respect to CTRL-M. (D) Heat map and fold oxidation table of protein-specific HNE levels (see also Table 1).

Figure 3

STRING analysis of proteomics data. (A) Network statistics reporting data concerning the number of nodes and edges, the average node degree, the average local clustering coefficient, the expected number of edges, and the protein-protein interaction (PPI) enrichment p-value. (B) Network interaction data table that reports all the significant interactions (min 0.4) between proteins of HSV1-M vs. those of CTRL-M; (C) Network interaction image showing nodes and edges between the proteins identified. The thickness of a line indicates the strength of the interaction between the proteins it connects. The colors of the spheres indicate the biological processes in which the proteins participate. (D) Biological process table reporting the main pathways in which each protein of the networks is involved. For each biological process identified, the corresponding Gene Ontology (GO) pathway, the number and identity of proteins, and the False Discovery Rate (FDR) are reported. The colors of the spheres indicate the biological processes in which the proteins participate.

Figure 4

Multiple HSV-1 reactivations affect GRP78 oxidation but not its expression levels. (A): image and bar graph of the immunoprecipitation analysis of GRP78 HNE modification in HSV1-M compared to CTRL-M cortex samples (mean + SEM; * p < 0.05); (B): Representative western blot showing GRP78 expression in cortical homogenates from CTRL-M and HSV1-M. Tubulin expression level was used as sample loading control. Densitometric analyses of immunoreactive signals, normalized to tubulin levels, are shown in the graphs: values represent the normalized fold changes in protein levels from n = 6 HSV-M with respect to n = 6 CTRL-M (mean ± SEM). (C): Confocal immunofluorescence analysis of coronal brain slices from HSV1-M and CTRL-M (for each group, n = 3). Panels show representative neurons of the cortex that were immunostained for GRP78 (green) and NeuN (red). Cell nuclei were stained with DAPI (blue). Bar graphs show mean GRP78 fluorescence intensity quantified in the frontal cortex and expressed as fold change with respect to CTRL-M (mean ± SEM).

Figure 5

Multiple HSV-1 reactivation activates UPR. (A) Representative western blots showing total levels and phosphorylation of PERK and IRE1 and total and cleaved ATF6 levels in cortical homogenates from CTRL-M and HSV1-M. (B) Densitometric analysis of immunoreactive signals of phosphorylation/expression ratio normalized to total load is shown in the graphs: values represent the fold increase of HSV1-M with respect to CTRL-M, set to 1 (mean ± SEM); * p < 0.05; ** p < 0.02.

Figure 6

Multiple HSV-1 reactivations do not affect CRMP2 expression but inhibit its phosphorylation in T514. (A) Blots and bar graph of the immunoprecipitation analysis of CRMP2– HNE modification in HSV1-M compared to CTRL-M cortex samples; (B) Representative western blot showing the levels of CRMP2 phosphorylation at threonine 514 (p-CRMP2) and total CRMP2 expression in cortical homogenates from CTRL-M and HSV1-M. Tubulin expression level was used as a sample loading control. Densitometric analysis of immunoreactive signals normalized to CRMP2 (for p-CRMP2) or tubulin (for CRMP2) are shown in the graphs. Values represent the normalized fold changes in protein levels from n = 6 HSV-M with respect to n = 6 CTRL-M (mean ± SEM; * p < 0.05). (C): Confocal immunofluorescence analysis of coronal brain slices from HSV1-M and CTRL-M (n = 3). Panels show representative neurons of the cortex that were immunostained for CRMP2 (green) and NeuN (red). Cell nuclei were stained with DAPI (blue). Bar graphs show mean CRMP2 fluorescence intensity quantified in the frontal cortex and expressed as fold change with respect to CTRL-M (mean ± SEM).