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Journal of Alzheimer’s Disease 39 (2014) 849–859 DOI 10.3233/JAD-131706 IOS Press

Inflammatory and Neurodegeneration Markers during Asymptomatic HSV-1 Reactivation Carolina Martina , Blanca Aguilaa , Paulina Arayab , Karin Viob , Sharin Valdiviaa , Angara Zambranoc , Margarita I. Conchac and Carola Ottha,d,∗ a Instituto

de Microbiolog´ıa Cl´ınica, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile

b Instituto de Anatom´ıa, Histolog´ıa y Patolog´ıa, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile c Instituto

de Bioqu´ımica y Microbiolog´ıa, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile de Investigaci´on Sur-Austral en Enfermedades del Sistema Nervioso (CISNE), Universidad Austral de Chile, Valdivia, Chile

d Centro

Accepted 17 October

Abstract. Background: Currently, it is unclear whether asymptomatic recurrent reactivations of herpes simplex virus type 1 (HSV-1) occur in the central nervous systems of infected people, and if these events could lead to a progressive deterioration of neuronal function. In this context, HSV-1 constitutes an important candidate to be included among the risk factors for the development of neuropathies associated with chronic neuroinflammation. Objective: The aim of this study was to assess in vivo inflammatory and neurodegenerative markers in the brain during productive and latent HSV-1 infection using a mouse model of herpes simplex encephalitis. Methods: Neuroinflammation and neurodegeneration markers were evaluated in mice trigeminal ganglia and cerebral cortex during HSV-1 infection, by immunohistochemistry, western blot, and RT-PCR. Results: Neuronal ICP4 viral antigen expression indicative of a reactivation episode during asymptomatic latency of HSV-1 infection in mice was accompanied by upregulation of neuroinflammatory (toll-like receptor-4, interferon ␣/␤, and p-IRF3) and early neurodegenerative markers (phospho-tau and TauC3). Conclusions: HSV-1 reactivation from latency induced neuroinflammatory and neurodegenerative markers in the brain of asymptomatic mice suggesting that recurrent reactivations could be associated with cumulative neuronal dysfunctions. Keywords: Herpes simplex encephalitis, herpes simplex virus type 1, neurodegeneration, neuroinflammation, TauC3, toll-like receptor 4, trigeminal ganglion

INTRODUCTION The pathogenic mechanisms of herpes simplex virus type 1 (HSV-1) at the brain level are not well known [1–4]. An interesting finding is that the affected limbic structures in herpes simplex encephalitis (HSE) are the ∗ Correspondence to: Carola Otth, PhD, Instituto de Microbiolog´ıa Cl´ınica, Facultad de Medicina, P.O. Box 567, Universidad Austral de Chile, Valdivia, Chile. Tel.: +56 63 2221923; Fax: +56 63 2293300; E-mail: [email protected].

same ones affected in Alzheimer’s disease (AD) leading to the hypothesis proposed in the early eighties that HSV-1 could be involved in the pathogenesis of AD [5]. Ball [5] suggested in 1982 that HSV-1 reactivation occurs in the trigeminal ganglia and the virus then travels to the brain. An important support for this idea was the finding that demonstrated the presence of the HSV-1 DNA in brains of immunosuppressed patients previously infected by the virus [6], which suggested that HSV-1 can establish latency in central nervous

ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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system (CNS) neurons and possibly undergo reactivations. Since then a growing body of evidence has accumulated suggesting a link between HSV-1 infection and AD. Among these are relevant studies showing the presence of HSV-1 in the brains of patients carrying the type 4 allele of the apolipoprotein E gene (APOE), a well-known susceptibility factor for AD [7, 8] and of anti-HSV IgG antibodies in cerebrospinal fluid (CSF) of adult and AD patients, but not in children younger than 7 years old, suggesting that the virus can reach the brain during adulthood perhaps favored by a decline in the immune system [9]. In agreement with this view, a recent study reported an increased risk of AD in a large population-based prospective study in elderly subjects with a positive detection of antiHSV (HSV-1 and HSV-2) IgM antibodies [10]. During HSV-1 infection, the IgM response tends to decline within one to two months, therefore the presence of IgM antibodies in the blood is indicative of a recent HSV-1 reactivation [10]. Although these findings are not enough to prove a relationship between HSV-1 and AD, they suggest the existence of recurrent reactivations of HSV-1 in CNS of adult people, pointing out a possible risk of chronic neuronal damage capable of causing neurological dysfunction. Additionally, HSV1 has been identified as a cause of deficits in memory and executive functioning following replication within the CNS in diverse neurological pathologies, such as chronic psychiatric diseases like schizophrenia and bipolar disorders, meningitis, mild, severe and diffuse encephalitis, and epilepsy [11–18]. The possibility that HSV-1 reactivates in CNS neurons asymptomatically, but causes chronic progressive damage at the cellular level and alters neuronal functionality, has not been completely investigated [3, 18, 19]. Brain inflammation due to infection, aging, and other deleterious processes is associated with activation of the local innate immune system. This could be a crucial, if not a causal mechanism leading to the neuronal damage seen in various CNS diseases. In fact, increasing evidence indicates that Toll-like receptors (TLRs) play a major role in several inflammatory CNS pathologies [20]. In this context, the neurotropic persistent pathogen HSV-1 constitutes an important candidate to be included among the risk factors for the development of neuropathies associated with chronic neuroinflammation. Previously, we found that in vitro HSV-1 infection triggered a clear induction in TLR2 and TLR4 expression in astrocytes and showed increased levels of interferon regulatory factors IRF3 and IRF7 transcripts, as well as phospho-IRF3 protein

indicating the activation of TLR-dependent pathways [21]. Furthermore, HSV-1 infection of neuronal and astrocytes cultures showed increased early neurodegenerative markers such as caspase-3 cleaved- (TauC3) and phosphorylated tau protein (pTau) [22, 23]. These findings were confirmed and further detailed by other groups identifying the protein kinases and the phosphorylation sites induced by the virus [24] and also showed that phospho-tau accumulation is dependent on HSV1 DNA replication [25]. Therefore, the aim of this study was to assess in vivo inflammatory and neurodegenerative markers in the brain during productive and latent HSV-1 infection using a mouse model of HSE. MATERIAL AND METHODS Animals The animal studies were approved by the Animal Care and Experimentation Committee of the Universidad Austral de Chile; and CONICYT guidelines 2008 for the performance of animal studies and Bio-safety of pathogen studies were followed during all procedures in agreement with the laboratory animals limited organization recommendations (http://www.lal.org.uk). Three-month-old female BALB/C mice (average weight 24–25 g) were maintained under a 12 h light–dark cycle with free access to food and water, in the breeding unit at the Immunology Institute, Universidad Austral de Chile [26]. Mice HSV-1 experimental infection Three experimental units were used consisting of six groups of three mice: i) three groups were HSV-1 infected and sacrificed at 7, 15, and 60 days postinoculation (dpi); and ii) three groups were mock infected and sacrificed at 7, 15, and 60 dpi. For HSV1 infection procedures, nine mice were subjected to intranasal inoculation of 20 ␮L of HSV-1 suspension (equivalent to 105 plaque forming units), as previously described [26–28]. The HSV-1 strain F used, which is a neurovirulent wild-type strain of HSV, was propagated and titrated after several passages, as described previously [22]. As negative controls, nine animals were mock-inoculated with sterile buffered-phosphate solution. Previous to intranasal inoculation, mice were anesthetized with intravenous sodium pentabarbitone (50 mg/kg of total weight) by trained personnel using Tuberculin syringe 29G, supervising loss of tactile stimuli, decrease of heartbeat and respiration.

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Afterwards, infected and control animals were isolated in separate rooms, kept under standard conditions (temperature, feeding, light and water), and were daily examined applying a supervision protocol. This model of mouse HSV-1 infection resembles the human pattern of HSE [26–28]. During the first two weeks postinfection, BALB/C mice were clinically scored on appearance (ruffled or normal coat), posture (hunched or normal), feeding habits, and neurological signs (e.g., seizures, circling), twice daily. After 30 days postinfection, HSV-1 latent state is established [26–29]. Brain tissue processing Mice were euthanized by overdoses of sodium pentabarbitone at 7, 15 and 60 days after inoculation and brains were aseptically removed considering Universidad Austral de Chile Biosafety committee and CONICYT guidelines 2008 recommendations to pathogens infected biological material procedures. For immunohistochemistry, trigeminal ganglia (TG) and one hemisphere of the brain of each mouse was fixed in 10% (v/v) formalin, embedded in paraffin, and sectioned (6 mm). The other brain hemisphere was dissected in two only the rostral area was used for RNA extraction. Residual biological material were disposed in containers with Clorox and afterwards burned in the institutional incinerator. Immunohistochemistry For immunoperoxidase localization, tissue sections were deparaffinized with xylene and rehydrated through a series of graded ethanol. Endogenous peroxidase was quenched in a 0.3% (v/v) H2 O2 /methanol bath for 5 min followed by several washes with PBS 1x pH 7.4. Slides were incubated for 60 min at room temperature in 5% (w/v) BSA-PBS pH 7.4, followed by incubation overnight at 4◦ C with primary antibodies (1 : 50–1 : 100 dilution) in 1% (w/v) BSA-PBS pH 7.4 and 0.3% (v/v) Triton X-100. Antibodies used were: anti-ICP4, anti-phospho (S396)-Tau, antiTauC3, anti-TLR4 (Santa Cruz Biotechnology, Inc., CA, USA) and anti-phospho-IRF3 (Cell Signaling Technology, Inc., MA, USA). Tissues were washed and incubated with biotinylated secondary antibodies (anti-mouse, anti-rabbit, and anti-goat IgG), and afterwards with avidin-horseradish peroxidase conjugates using the reagents provided by the manufacturer (Kit LSAB systems K0690, DAKO). Immunostaining was developed using 0.05% (w/v) diaminobenzidine and 0.03% (v/v) H2 O2 . Sections incubated without

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primary antibodies or with preimmune serum were used as controls. All the markers were evaluated in the same cortex area (piriform area), a region near to the olfactory bulb, which corresponds to the CNS entry site of HSV-1 during intranasal inoculation. Stained slides were examined with a Zeiss Axioskope II microscope equipped with a digital video camera (NikonDXM1200). The images obtained were processed with Adobe Photoshop 6.0. Positively stained cells were counted in slides obtained from the same cortex region in control and infected animals in an area of 200 ␮m2 of the captured images using Image J program. The mean number of immunoreactive cells for each antibody at the different times post infection was determined in triplicate. RNA isolation and RT-PCR analyses The rostral area of brains were immediately lysed for total RNA extraction by the addition of 1 ml of RNA reagent (OMEGA, Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer’s instructions. Then, RNA samples were digested with RNase-free DNase (20 kU/␮L) for 15 min to eliminate genomic DNA contamination. RNA was quantified by absorbance at 260 nm and used immediately or stored precipitated in ethanol at −70◦ C until use. To evaluate integrity, total RNA (30 ␮g per sample) was incubated in 1x MOPS buffer, 2.2 mM formaldehyde and 50% (v/v) deionized formamide for 10 min at 65◦ C, mixed with formaldehyde loading buffer [50% (v/v) glycerol, 1 mM EDTA pH 8.0, 0.25% (w/v) bromophenol blue and 0.25% (w/v) xylene cyanol] and separated by electrophoresis in 1% (w/v) agarose gel containing 6% (v/v) formaldehyde. Standard RT-PCR analyses were performed using 1 ␮g of total RNA for reverse transcription, followed by PCR amplification with Superscript III One Step RTPCR system (Invitrogen, Carlsbad, CA, USA) using the specific primers and conditions previously described [21]. GADPH was used to normalize the relative expression of the different transcripts. Agarose gels were scanned with a BIO-RAD Molecular Imager FX Pro System, and the pixel intensity of the bands (same area) was measured using the Quantity One program for Windows. Total counts (arbitrary units) from each band were obtained after subtracting the background. Statistical analysis All the results are representative of at least three different animals. Results were analyzed by one-way ANOVA and the data were expressed as

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Fig. 1. Clinical parameters and viral ICP4 protein detection in the brain of HSV-1 infected mice. A) Clinical parameters of HSV-1 and Mock infected mice were evaluated at different days post-infection. B) Samples of cortex and trigeminal ganglia of HSV-1 and Mock1 infected mice were fixed at 7, 15, and 60 dpi and stained with specific antibody against to ICP4 HSV-1 protein. Magnification 100× and 250× for inserted images. 1 Samples of Mock infected tissues were immune-negative at all times analyzed.

means ± standard error of the mean (SEM) using Grad Phad Prism 5.0 program. The p values were reported in each case; p < 0.05 was considered significant. RESULTS Clinical and immunohistological evaluation during acute and latent HSV-1 infection To study the pathogenesis of neuronal HSV-1 infection in BALB/C mice after intranasal inoculation of HSV-1 (strain F), three experimental units were carrying out. In each experimental unit, a total of nine mice were infected, all of them showed severe weakening with weight loss, trembling, skin lesions, hair loss, and the symptoms of severe encephalitis such as paralysis, movement disorders and/or seizures at 7–15 dpi (Fig. 1A). After 15 dpi, all mice were recovered and were apparently normal, displaying normal feeding and grooming behaviors until they were sacrificed. PBS-inoculated mice showed no lesions or neurological symptoms during the whole period ana-

lyzed. We confirmed HSV-1 infection in cortex and TG by immunohistochemistry using anti-ICP4 antibody. As shown in Fig. 1B, HSV-1 antigen was highly expressed at 7 and 15 dpi in all animals, and also at 60 dpi in neurons of cortex and TG of three infected asymptomatic mice, but not in mock-infected mice at all times analyzed. According to their characteristic morphology (large-diameter, etc.), many of the HSV-1 antigen-positive cells localized in the TG region would correspond to sensory neurons, which are the target for HSV-1 persistent latent infection. In addition, some cortical neurons also showed intense ICP4 reactivity. Interestingly, as mentioned previously, viral antigen expression was present in neurons of some asymptomatic mice 2 months after infection and persisted even after 220 dpi (data not shown). Taking in consideration that it has been previously demonstrated that latency is established in this mouse model of HSE after 30 dpi [27–29], our results showing HSV-1 ICP4 protein expression after 60 dpi would most probably correspond to a viral reactivation episode during asymptomatic latent infection.

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Fig. 2. Effects of HSV-1 infection on brain TLRs and IFN-I transcripts levels. Total RNA extracted from mock and HSV-1 infected mice brain tissue at 7, 15 and 60 dpi were analyzed by RT-PCR with specific primers. After gel separation, the relative expression levels of the studied markers were normalized with GAPDH mRNA levels by densitometric analysis (n = 3). ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

TLR expression and activation during productive and latent infection To evaluate the innate immune response in the brain during the course of HSV-1 infection, we analyzed the mRNAs levels of TLRs (2, 3, 4, 7, 9), and also of TLR-dependent pathway effectors such as interferon regulated factor 7 (IRF7) and interferon ␣ and ␤ (IFN␣, IFN␤) in the brains of mice that expressed the viral antigen ICP4. The results showed an initial decrease at 7 dpi of all the transcripts analyzed followed by an increase over basal levels especially of

TLR2 and TLR3 mRNA levels at 15 dpi (Fig. 2). At the same time, upregulation of IRF7, IFN␣, and IFN␤ mRNA was observed, demonstrating that TLRs activation was induced during productive infection. Accordingly, increased levels of activated IRF3 (pIRF3) was observed at 15 dpi in agreement with TLRs activation during HSV-1 neuronal infection (Fig. 3). In contrast, TLR4 transcript was only increased at 60 dpi during the asymptomatic phase of infection in parallel with IFN␣ mRNAs. However at the protein level, TLR4 was observed significantly increased in sensory neurons of the TG already at 15 dpi (Fig. 4)

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the difference was also significant even at 60 dpi. These results are in agreement with the persistence of the neuroinflammatory response evidenced by the upregulation of TLR4 and p-IRF in some asymptomatic mice during the latency phase of infection. DISCUSSION

Fig. 3. Activation of TLR-dependent pathways in the brain of HSV1 infected mice. Western blot analyses were performed to evaluate the levels of phosphorylated IRF3 (p-IRF3) protein extracted from mock and HSV-1 infected mice brain tissue at 7, 15 and 60 dpi. Levels were normalized using ␤-tubulin as constitutive protein (n = 3). ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

and remained high during the asymptomatic phase (60 dpi). Additionally, clear evidence of astrogliosis was observed in the cortex of these mice (data not shown). These results suggested the possibility of neuroinflammation triggered by HSV-1 not only during the acute phase but also during an asymptomatic reactivation episode. Early neurodegenerative markers during productive and latent infection With the aim to establish the effects of HSV-1 during acute infection and latency on the appearance of early neurodegenerative markers such as phospho-tau at serine 396 (p-tau) and tau cleavage by caspase 3 at aspartate 421 (TauC3), we determined the number of cells immunodetected with the corresponding specific antibodies in TG and cortex of HSV-1 infected mice. The results showed an increased number of positive cells per ␮m3 for p-tau (Fig. 5) and TauC3 (Fig. 6) especially in TG of HSV-1-infected mice in comparison with the mock-inoculated animals. Although the staining was less intense in the cortex of these animals,

Neuroinflammation triggered by central nervous system pathogens involve an initial immune innate response characterized by activation of TLRs and other pattern recognition receptors. TLR activation during neuronal HSV-1 infection has been clearly demonstrated by different groups [30–34]. Using intranasal inoculation of HSV-1 in MyD88 KO mice, Mansur et al. [32] showed that all the animals developed lethal encephalitis after viral inoculation, highlighting the relevance of TLR signaling in the control of viral infection [32, 34]. Previously, Aravalli et al. [31] had shown that TLR2 signaling is important for the production of the proinflammatory cytokines IL-1␤, IL-6, and TNF␣, in response to HSV-1 infection. Similarly, Wang et al. [35] showed that TLR2 KO mice had a significantly increased survival rate following intracranial inoculation of HSV-1, compared to wild type and TLR9 KO mice. Likewise, using TLR2, TLR9, and TLR2/9 KO mice, Sørensen et al. [33] concluded that TLR2 and TLR9 synergistically stimulate innate antiviral activities, thereby protecting against HSV infection. All these studies were focused on determining the contribution of TLRs and the neuroinflammatory response during productive infection associated to encephalitis. In a previous study, we reported that in vitro productive HSV-1 infection of mice astrocyte primary cultures induced strong TLR2 and TLR4 expression and activation, probably through IRF3 and IRF7 dependent pathways [21]. In this study, our group aimed to evaluate if asymptomatic neuronal reactivation of HSV-1 infection could occur in a mouse model of intranasal inoculation and also if these reactivation episodes could be associated with the increase in early neurodegeneration markers, in an attempt to establish a possible link between neuroinflammation triggered HSV-1 neuronal reactivation and deterioration of neuronal functions in infected individuals. In agreement with previous reports, in the present study we showed that TLR2, TLR3, and TLR9 transcripts significantly increase their levels at 15 dpi. This increase was also observed for IRF7 mRNA and phospho-IRF3; events associated with TLRs dependent signaling pathways activation involved in

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Fig. 4. HSV-1 induces TLR4 upregulation during latent infection. Samples of cortex and trigeminal ganglia of mock and HSV-1 infected mice fixed at 7, 15, and 60 dpi were staining with: anti-TLR4 specific antibody. The graphic shows the number of positive cells/␮m2 for TLR4 (n = 3). Magnification 100x. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

interferon production. However, the early reduction of the mRNAs corresponding to these markers observed at 7 dpi might be explained by an initial negative regulation of the innate immune response triggered by the virus as a means to survive enough time within the neuronal cells to establish latency [36]. The virion host shutoff protein of HSV, which is a ribonuclease encoded by the UL41 gene, is synthesized during productive HSV-1 infection and is known to preferentially degrade mRNA species [37–40]. It would be interesting to evaluate in the future if the reduction of TLR and IFN mRNAs observed during early HSV-1 infection is due to such a mechanism. Perhaps the most important finding of this study is the upregulation of TLR4 protein observed in TG and cortical neurons of some HSV-1-infected mice at 60 dpi, which should correspond to the latent phase of infection. This upregulation was detected only in animals that showed clear expression of the early viral

protein ICP4 at this stage and was accompanied by an increase of p-IRF3 and INF expression with evident astrogliosis in the cortex and TG, indicative of a persistent neuroinflammatory process, most probably due to viral reactivation from latency. In a previous study, random testing of 3200 CSF samples revealed HSV-1 DNA in 26 and HSV-2 DNA in 36 samples from subjects without symptoms of HSV activity [41]. This is an important finding supporting the possibility of frequent asymptomatic reactivation of HSV at neuronal level since detection of viral DNA in the CSF clearly demonstrates that even during clinically silent infections, virus replicates in the CNS [42]. Hua et al. [43] have shown ischemic upregulation of TLR4 in activated microglia of wild type mice, whereas less neuronal damage and activated microglial cells were observed in the ischemic area of the brains of TLR4 KO mice [43]. The authors suggested that activation of TLR4 in microglia contribute

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Fig. 5. HSV-1 increases tau phosphorylation in cortex and trigeminal ganglia. Samples of cortex and trigeminal ganglia from mock and HSV-1 infected mice were staining at 7, 15 and 60 dpi with anti-p-tau (Ser396) specific antibody. The graphic shows the number of positive cells/␮m2 (n = 3). Magnification 100×. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

to neuronal death, playing a key role in the pathogenesis of cerebral injuries [43, 44]. In addition, Balistreri et al. [45] described the involvement of TLR4 in age-related diseases such as neurodegenerative diseases, suggesting a crucial role of molecules of innate immunity in pathophysiology of these diseases. Further, treatment of primary murine neuronal cells of TLR4 KO mice versus wild type neuronal cells with supernatants of amyloid peptide-stimulated microglia demonstrated that TLR4 contributes to amyloid peptide-induced microglial neurotoxicity [46]. Also, an important finding of these authors was a marked upregulation of TLR4 mRNA in the brain of A␤PP transgenic mice, and an increased expression of TLR4 in AD brain tissue associated with amyloid plaque deposition, suggesting a role of this key innate immune receptor in neuroinflammatory pro-

cesses in AD [46]. Furthermore, some authors have suggested that TLR4 has emerged as a new susceptibility marker for AD [47, 48]. Accordingly, reduced TLR4 signaling in response to lipopolysaccharide has been associated with a common mutation of TLR4 gene (Asp299Gly) characterized by declined ability to induce inflammation [47, 49]. Additionally, the Asp299Gly polymorphism has been also associated with a decreased risk of late-onset AD in an Italian population cohort, independent of the susceptibility gene APOE ε4 [50]. Coincidently with these findings, we have recently shown increased transcripts encoding TLR2, TLR4, and one of their endogenous ligands, SAA3, in HSV1-infected astrocytes, suggesting that TLR activation could not only be triggered by the virus but also amplified by this locally produced danger signal [21]. As

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Fig. 6. Tau cleavage by caspase 3 in cortex and trigeminal ganglia of HSV-1 infected mice. Samples of cortex and trigeminal ganglia from mock and HSV-1 infected mice were staining at 7, 15 and 60 dpi with anti-TauC3 specific antibody. The graphic shows the number of positive cells/␮m2 (n = 3). Magnification 100×. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

SAA3 corresponds to an acute phase protein, induced expression of SAA3 transcript has also been demonstrated following exposure to different stimuli, such as oropharyngeal administration of lipopolysaccharide or during cerebral ischemia in mice [51, 52]. Therefore a possible hypothesis is that the local induction of SAA3 by different triggers could contribute to develop chronic neuroinflammation processes in individuals where HSV-1 has already established latency in the CNS. In a previous study, we had shown the induction of early neurodegenerative markers such as hyperphosphorylated and cleaved tau protein (p-tau and TauC3 markers, respectively) in an in vitro model of HSV-1 neuronal infection [21–23]. These post-translational modifications of tau are considered important neurodegenerative markers, initially associated to neurodegenerative disorders such as AD, frontotemporal dementia, and Parkinson’s disease

[53]. Here we show for the first time increased levels of these markers during in vivo HSV-1 neuronal infection. This constitutes a relevant finding since recent studies indicate that neuronal dysfunction precedes the formation of tau insoluble fibrillar deposits, suggesting that earlier tau dysfunction is sufficient to cause neurotoxic effects and neurodegeneration [54]. Additional support for this idea comes from previous evidence showing severe spatial memory deficits and chronic lesions derived from decreased brain volume, neuronal loss, activated astrocytes, and glial scar formation to severe atrophy in HSE surviving animals during latent infection [55, 56]. All these findings contribute to support the hypothesis that the presence of HSV-1 in the CNS could promote chronic neuroinflammation by recurrent reactivation episodes which trigger TLRs activation and as a result could constitute a risk factor of neurodegenerative processes.

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ACKNOWLEDGMENTS This study was supported by grants: FONDECYT INITIATION 11080067 and FONDECYT REGULAR 1120464 and DID-UACH S-2009-40. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1996). REFERENCES [1]

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