Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Sep;132(3):433-51.
doi: 10.1007/s00401-016-1597-2. Epub 2016 Jul 25.

Herpes Simplex Encephalitis Is Linked With Selective Mitochondrial Damage; A Post-Mortem and in Vitro Study

Affiliations
Free PMC article

Herpes Simplex Encephalitis Is Linked With Selective Mitochondrial Damage; A Post-Mortem and in Vitro Study

Małgorzata Wnęk et al. Acta Neuropathol. .
Free PMC article

Abstract

Herpes simplex virus type-1 (HSV-1) encephalitis (HSE) is the most commonly diagnosed cause of viral encephalitis in western countries. Despite antiviral treatment, HSE remains a devastating disease with high morbidity and mortality. Improved understanding of pathogenesis may lead to more effective therapies. Mitochondrial damage has been reported during HSV infection in vitro. However, whether it occurs in the human brain and whether this contributes to the pathogenesis has not been fully explored. Minocycline, an antibiotic, has been reported to protect mitochondria and limit brain damage. Minocycline has not been studied in HSV infection. In the first genome-wide transcriptomic study of post-mortem human HSE brain tissue, we demonstrated a highly preferential reduction in mitochondrial genome (MtDNA) encoded transcripts in HSE cases (n = 3) compared to controls (n = 5). Brain tissue exhibited a significant inverse correlation for immunostaining between cytochrome c oxidase subunit 1 (CO1), a MtDNA encoded enzyme subunit, and HSV-1; with lower abundance for mitochondrial protein in regions where HSV-1 was abundant. Preferential loss of mitochondrial function, among MtDNA encoded components, was confirmed using an in vitro primary human astrocyte HSV-1 infection model. Dysfunction of cytochrome c oxidase (CO), a mitochondrial enzyme composed predominantly of MtDNA encoded subunits, preceded that of succinate dehydrogenase (composed entirely of nuclear encoded subunits). Minocycline treated astrocytes exhibited higher CO1 transcript abundance, sustained CO activity and cell viability compared to non-treated astrocytes. Based on observations from HSE patient tissue, this study highlights mitochondrial damage as a critical and early event during HSV-1 infection. We demonstrate minocycline preserves mitochondrial function and cell viability during HSV-1 infection. Minocycline, and mitochondrial protection, offers a novel adjunctive therapeutic approach for limiting brain cell damage and potentially improving outcome among HSE patients.

Keywords: Gene-expression; Herpes simplex virus encephalitis; Human; Minocycline; Mitochondria.

Figures

Fig. 1
Fig. 1
Histological features of HSE: a HSE patient, histological features of pyknotic and karyolytic pyramidal neurons (arrows). b, inset neuronal necrosis and parenchymal rarefaction in association with astrocytes containing intranuclear eosinophilic inclusion bodies. c Early degenerative and inflammatory changes of the grey matter associated with two astrocytes containing intranuclear inclusion bodies (arrows). d Discrete area of grey matter rarefaction with marked inflammatory cell infiltrate. e Control RTA patient showing unaltered neurons. f Inset hippocampus, dentate gyrus, immunoperoxidase HSV-1 antigen stain [brown (DAB)] is largely restricted to nuclei. Inset image astrocyte with enlarged dense HSV-1 staining of the nucleus. Scale bars 50 μm (a, b, df), 20 µm (c)
Fig. 2
Fig. 2
Heat-map of relative transcript abundance in the brain of HSE cases and controls. Samples are manually organised on the x axis; AC represent HSE patients, 1–5 represent control patients. Transcripts are hierarchically clustered by similarity of transcript abundance on the y axis (n = 2882). Transcripts with high abundance (relative to median value for the set) are exhibited as magenta tiles; those with low abundance—green; and those at median—black. Although there was variation in transcript abundance between the HSE cases, with cases B and C exhibiting a greater proportion of down-regulated transcripts compared to case A, there remained a cluster of 287 transcripts [green tiles in the top left of the heat-map (asterisk)] that exhibited significantly (FDR <5 %) lower abundance in HSE cases (n = 3) compared to controls (n = 5). This set of transcripts was significantly (p < 0.0001) over-represented with transcripts corresponding to mitochondrial DNA encoded genes. Gene-expression was visualised as a heat-map using Treeview 1.60 (http://www.eisenlab.org)
Fig. 3
Fig. 3
Relative abundance for mitochondrial and apoptotic transcripts in HSE. Y axis represents mean log (base 2) fold change in abundance for individual transcripts among HSE cases relative to controls. Replicate transcripts have been collapsed to their arithmetic mean. Negative values represent lower abundance in HSE tissue. a Mitochondrial DNA encoded transcripts exhibit lower abundance compared to nuclear DNA encoded mitochondrial transcripts (right compared to left side of X axis). Among transcripts corresponding to the cytochrome c oxidase enzyme complex, mitochondrial encoded transcripts (red hatched bars) exhibit lower abundance than nuclear encoded transcripts (red solid bars). For succinate dehydrogenase, nuclear encoded transcripts (green solid bars) exhibit relatively high abundance compared to mitochondrial encoded transcripts. b Transcripts encoding for apoptotic mediators did not show any consistent or significant changes in abundance in HSE tissue compared to controls
Fig. 4
Fig. 4
Pattern of distribution of CO1 and HSV-1 antigen expression in brain tissue from HSE patients. Consecutive brain tissue sections were immunoperoxidase stained for either CO1 or HSV-1 antigen. All regions show an inverse pattern of staining for CO1 and HSV-1. a Hippocampus, CA2 region, diffuse granular CO1 staining in the neuropil, with a relative lack of CO1 staining in the dentate gyrus. b Hippocampus, CA2 region (consecutive section); absence of HSV-1 staining in the neuropil, with strong HSV-1 staining restricted to the dentate gyrus. c Frontal cortex, moderate granular CO1 staining in neurons. d Frontal cortex (consecutive section); strong HSV-1 antigen staining in neurons. e Amygdala, strong CO1 reaction among large neurons. f Amygdala (consecutive section); limited HSV-1 antigen staining among large neurons. Scale bars 250 μm (a, b), 100 μm (cf). Note; although strong HSV-1 antigen staining was observed in neurons, no intranuclear inclusions were observed in these cells
Fig. 5
Fig. 5
Quantification of CO1 and HSV-1 antigen expression in brain tissue from HSE patients. Consecutive brain tissue sections were immunoperoxidase stained for either CO1 or HSV-1 antigen. a Cingulate gyrus, moderate CO1 expression. b Cingulate gyrus, neurons with strong HSV-1 reaction. Scale bars 100 μm. c Quantification of immunostaining in consecutive sections of the cingulate cortex for two HSE cases (A, C); cingulate and insula for the third HSE case (B). The proportion (%) of the area of tissue staining positively for either CO1 or HSV-1antigen was measured in sets of ‘paired’ images of the same field of view [n = 4 images for case 1 (squares) and n = 5 for case 2 (circles)]. There is a significant negative correlation between HSV-1and CO1 antigen staining for two cases (A, C) with the third case (B) showing the same negative trend (Spearman rank correlation coefficient; r = −0.66, p = 0.044; r = −0.9, p = 0.005; r = −0.8, not significant; respectively, for HSE patients A, C, B). d Measurement of CO1 transcript abundance in tissue from amygdala, frontal and cingulate regions among HSE cases and controls. Abundance for CO1 is significantly lower in cases compared to controls (p = 0.032); 2-way repeated measures ANOVA over 3 brain regions (two tissue sections per region) for cases (black circles) against controls (grey squares). CO1 abundance measured as 1/C T (PCR fractional cycle number for threshold fluorescence). Mean and 95 % CI presented [cases (n = 3) and controls (n = 5)]
Fig. 6
Fig. 6
HSV-1 and host transcript abundance during in vitro HSV-1 infection. Viral DNA and host RNA from the infected human astrocytes were examined at indicated time-points via qPCR and qRT-PCR, respectively. a Increase in HSV-1 DNA (solid circles) abundance (C T) during the course of infection, reaching a maximum at 48 h pi. b Decrease in transcript abundance (C T) for CO1 (circles) and DAD1 (squares) among infected (solid symbols) relative to non-infected cells (open symbols), reaching a nadir at 72 h pi. Early decrease in CO1 (solid circles) over the first 36 h, with relative abundance [δC T (CO1-DAD1)] significantly lower at 36 h pi (p = 0.036). At the same time-point, no change in DAD1 abundance between infected and non-infected cells. c TNF (circles) exhibits a significant increase in relative abundance [δC T (TNF-DAD1)] at 72 h pi (p = 0.029) among infected (solid circles) relative to non-infected astrocytes (open circles). CASP3 (squares) exhibits no significant change in relative transcript abundance [δC T (CASP3-DAD1)] during infection. Data presented as mean ± 95 % confidence interval (CI) for each experimental group (minimum of 3 replicate experiments per group). Differences in C T at 36 and 72 h pi assessed using the Mann–Whitney U test
Fig. 7
Fig. 7
CO1 and HSV-1 antigen expression during in vitro HSV-1 infection. Dual immunofluorescence: infected human astrocytes were labelled CO1 (green) and HSV-1 (red) antigens with DAPI nuclear counter-stain (blue). a At 6 h pi, a granular reaction for CO1 is detected in majority of cells, with HSV-1 antigen expression observed in occasional cells. b At 72 h pi, HSV-1 antigen is expressed in majority of cells, with no apparent CO1-positive cells. The nuclei appear slightly larger compared to early infected and non-infected cells. This may represent nuclei swelling following infection [see TEM images (Fig. 10)]. c Non-infected astrocytes are positive for CO1, with no HSV-1 expression at 72 h pi. Scale bars 100 μm. d Plot of proportion of astrocytes expressing HSV-1 and/or CO1 antigen among non-infected (ni) and HSV-1 infected cultures at 6, 24, 48 and 72 h pi. Y axis—proportion. X axis—hours pi. Grey bars CO1 expressing cells. Hatched bars HSV-1 expressing cells. There is progressive reduction in CO1 and steady rise in HSV-1 antigen expression over time pi. Proportions are corrected for number of positive DAPI (nuclear) staining cells
Fig. 8
Fig. 8
Mitochondrial enzyme function during in vitro HSV-1 infection. ad Sequential enzyme histochemistry of cytochrome c oxidase CO (brown) and succinate dehydrogenase SDH (blue) mitochondrial enzyme activity demonstrate an early loss of CO function during infection. a At 6 h pi, uniform brown stain indicates that CO is functional. b At 24 h pi, early impairment of CO activity, with sustained SDH activity, is evidenced by patches of blue, CO-negative, SDH-positive cells. c, d At 48 and 72 h pi, both CO dysfunction and cell death is evidenced by further blue patches and loss of continuity of the cell monolayer. eh In non-infected astrocytes, CO remained active throughout the time-course [6 h (e), 24 h (f), 48 h (g) and 72 h pi (h)]. Scale bars 200 μm (ad), 300 μm (eh)
Fig. 9
Fig. 9
Quantification of cytochrome c oxidase enzyme function during in vitro HSV-1 infection. The proportion of cells adherent to the flask surface (within the monolayer) and exhibiting intact cytochrome c oxidase (CO) function was quantified. ad The area (%) of the monolayer staining positively for DAB (false red) was measured at 6, 24, 48 and 72 h pi. e Area of DAB staining declined from 87.52 to 50.54, 25.53 and 19.04 % over time pi. f Area of DAB staining remained above 80 % throughout time-course among non-infected cells, data are presented as mean ± 95 % CI for each time-point. Replicate cultures were examined per time-point (n = 3)
Fig. 10
Fig. 10
Ultra-structural cellular changes following in vitro HSV-1 infection. a In non-infected cells, mitochondria are scattered throughout the cytoplasm (arrows) with a centrally located nucleus exhibiting granular chromatin. b ‘Early’ morphological changes: HSV-1 infected cell exhibit central clustering in the cytoplasm (arrow) of ‘condensed’ mitochondria (top inset). Viral particles are detected in the nucleus, cytoplasm and budding on the surface of the cell (bottom inset). c ‘Intermediate’ morphological changes: mitochondria are swollen (arrow/inset) with intra-cristal enlargement and whirling of the mitochondrial membrane (inset). The nucleus exhibits loss of chromatin granularity. d ‘Late’ morphological changes: mitochondria are markedly enlarged (inset); the cell is dramatically swollen, the nucleus exhibits a severe loss of chromatin granularity and its membrane appears fragmented. Scale bars 2 μm
Fig. 11
Fig. 11
Comparison of host transcript and HSV-1 DNA abundance in minocycline treated and non-treated astrocytes during in vitro HSV-1 infection. Viral DNA and host RNA from infected human astrocytes were examined at indicated time-points via qPCR and qRT-PCR, respectively. Relative transcript abundance. a CO1 (circles) and DAD1 (squares), b BCL2, c BAX, d IL-6, e TNF in HSV-1 infected non-treated (solid symbols) compared to HSV-1 infected minocycline treated (open symbols) astrocyte cultures; f HSV-1 DNA in non-treated (solid circles) compared to minocycline (60 µM) treated (open circles); minocycline + aciclovir (20 µM) co-treatment (open squares) and aciclovir (20 µM) alone (solid squares) among HSV-1 infected astrocytes. Y axis—relative transcript abundance δC T (difference in C T in infected minus non-infected; a or δδC T (difference for target gene—DAD1 in infected minus non-infected cultures; bf X axis—hours pi. Data are presented as mean ± 95 % CI for each experimental group (minimum of 4 experiments per group). a CO1 exhibits sustained abundance during infection in minocycline treated compared to non-drug exposed cells. There is a significant difference in relative transcript abundance [δδC T (CO1-DAD1)] between exposures (p = 0.029) at 48 h pi. DAD1 exhibits a smaller decrease in transcript abundance (δC T) in minocycline compared to non-drug exposed cells (not-significant). b BCL2 and c BAX exhibit significant increases in relative transcript abundance among minocycline exposed compared to non-drug exposed cultures between 2 and 24 h pi (p = 0.004, p = 0.012, respectively). d IL-6 exhibits significant decrease in transcript abundance among minocycline treated compared to non-drug exposed astrocyte cultures between 4–72 h pi (p < 0.0001). e TNF exhibits similar trend to IL-6. f HSV-1 DNA shows no significant change in relative transcript abundance [δδC T (HSV-1 minus DAD1) in infected minus non-infected cultures] in either minocycline treated compared to non-treated or minocycline and acyclovir co-treated compared to aciclovir alone. Significant differences in δδC T were assessed using 2-way repeated measures ANOVA (column factor—drug exposure; row factor—time pi) using time-points: 2, 4, 6, 24 h (b, c) and 4, 6,24, 32, 48, 72 h (e). The Mann–Whitney U test was used to compare corrected CO1 abundance at 48 h pi (a)
Fig. 12
Fig. 12
Comparison of cytochrome c oxidase function in minocycline treated and non-treated astrocytes during in vitro HSV-1 infection. Sequential enzyme histochemistry of cytochrome c oxidase CO (brown) and succinate dehydrogenase SDH (blue) activity demonstrates protective properties of minocycline. a, b At 24 and 48 h pi, vast majority of cells stain brown indicating intact CO function in minocycline treated cells. c, d At 24 and 48 h pi, obvious patches of cells stain blue, indicating impaired CO, but sustained SDH activity among non-treated cells. There is also a greater loss of the cell monolayer (reduced area stained for CO or SDH activity) among non-treated cells Area (proportions) for monolayer staining is documented in Table S7 (Online resource 5). Scale bars 200 μm

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

References

    1. Agwuh KN, MacGowan A. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother. 2006;58:256–265. doi: 10.1093/jac/dkl224. - DOI - PubMed
    1. Allaman I, Belanger M, Magistretti PJ. Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci. 2011;34:76–87. doi: 10.1016/j.tins.2010.12.001. - DOI - PubMed
    1. Aravalli RN, Hu S, Rowen TN, Gekker G, Lokensgard JR. Differential apoptotic signaling in primary glial cells infected with herpes simplex virus 1. J Neurovirol. 2006;12:501–510. doi: 10.1080/13550280601064921. - DOI - PubMed
    1. Athmanathan S, Vydehi B, Sundaram C, Vemuganti G, Murthy J. Neuronal apoptosis in herpes simplex virus-1 encephalitis (HSE) Indian J Med Microbiol. 2001;19:127. - PubMed
    1. Booss J, Kim JH. Biopsy histopathology in herpes simplex encephalitis and in encephalitis of undefined etiology. Yale J Biol Med. 1984;57:751. - PMC - PubMed

Publication types

Substances

Feedback