HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/7/863 03-0764fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by HANS, A. Articles by GONZALEZ-DUNIA, D. Search for Related Content PubMed PubMed Citation Articles by HANS, A. Articles by GONZALEZ-DUNIA, D.

Persistent, noncytolytic infection of neurons by Borna disease virus interferes with ERK 1/2 signaling and abrogates BDNF-induced synaptogenesis¹

AYMERIC HANS^*, JEFFREY J. BAJRAMOVIC^*, SYLVIE SYAN^*, EMMANUELLE PERRET, IRÈNE DUNIA, MICHEL BRAHIC^* and DANIEL GONZALEZ-DUNIA^*,,2

^* Unité des Virus Lents, CNRS URA 1930, Institut Pasteur, Paris, France;
Institut National de la Santé et de la Recherche Médicale U-563, Toulouse, France;
Centre d’Imagerie Dynamique, Institut Pasteur, Paris, France; and
Institut Jacques Monod CNRS, Université Paris VII, Paris, France

²Correspondence: Inserm U563, CPTP Bat B, CHU Purpan Place du Dr. Baylac 31059 Toulouse Cedex 3, France. E-mail: daniel.dunia{at}toulouse.inserm.fr

SPECIFIC AIM

Infection of the central nervous sytem by Borna disease virus (BDV) is a unique model to study the mechanisms whereby a persistent viral infection can impair neuronal function, leading to behavioral diseases reminiscent of neurobehavioral disorders in humans. The aim of this study was to assess the effect of BDV infection of hippocampal neurons, the main target for this virus in vivo, on the response to the neurotrophin BDNF.

PRINCIPAL FINDINGS

1. Infection with BDV interferes with BDNF-induced ERK 1/2 activation
BDV infected primary rat hippocampal neurons efficiently, spreading to the whole neuronal population within 10 days. Remarkably, the infection did not cause any morphological alteration or cytopathic effect. However, when neurons were treated with exogenous BDNF, the phosphorylation of ERK 1/2 was severely affected in BDV-infected neurons (P<0.0001, Student’s t test) as measured by quantitative immunocytochemistry and immunoblotting for the phosphorylated form of ERK 1/2 (Fig. 1 ). This was not the result of changes in the expression of TrkB receptor, the main mediator of ERK 1/2 activation after treatment with BDNF, whose level of expression was the same in control and infected cultures (P=0.4332, Student’s t test). Taken together, these data demonstrate that BDV interferes with neurotrophin-triggered ERK 1/2 phosphorylation downstream of TrkB. This interference appeared to be specific to the neurotrophin signaling pathway since activation of ERK 1/2 was the same in control and BDV-infected neurons when the neurons were treated for 10 min with 200 µM H₂O₂, another stimulus that activates ERK 1/2.

View larger version (30K): [in this window] [in a new window]   Figure 1. ERK 1/2 activation is impaired in BDV-infected neurons. A) Confocal immunocytochemistry for dually phosphorylated ERK 1/2 (pERK 1/2) and TrkB. Control (top row) or BDV-infected neurons (two bottom rows) were treated with BDNF and stained for pERK 1/2 or TrkB (left panels), in combination with BDV nucleoprotein or Tau (two right panels) for, respectively, infected or control neurons. Cultures were fixed early after infection to display both infected and non-infected neurons in the same field. B) Quantitation of pERK 1/2 and TrkB. Levels of pERK 1/2 were measured on a minimum of 100 neurons, in control and BDV-infected cultures (11 days postinfection) after treatment with BDNF (50 ng/mL) for 10 min. The expression of TrkB was measured in control and infected neurons under the same conditions. Statistical analysis of signal intensities was done with Student’s t test. Three independent experiments gave similar results. C) Immunoblots for pERK 1/2 and total ERK 1/2 in control (NI) or infected (BDV) cultures before (–) and after (+) exposure to BDNF, showing results similar to those obtained with confocal immunocytochemical quantification.

2. BDV blocks BDNF-induced synaptic protein expression
Given the role of neurotrophins in promoting synaptogenesis, we next analyzed the effect of BDV infection on the expression level of synapsin I, synaptophysin, synaptobrevin (VAMP-2), and syntaxin I before and after treatment with BDNF. Immunocytochemical and Western blot analysis revealed that basal expression levels of these proteins were not significantly changed by BDV infection. When uninfected neurons were treated with BDNF for 4 days, there was a 30–80% increase in the expression of synaptic proteins. In contrast, there was no increase when cultures were infected with BDV. Expression levels of other neuronal markers such as acetylated Tubulin or TrkB receptor were not altered by BDV infection. We also observed that long-term exposure to BDNF increased the level of viral proteins in neurons by 25%, consistent with previous demonstrations that neurotrophins enhance BDV production in persistently infected cells.

3. BDV infection abrogates BDNF-induced synaptogenesis and causes defects in synaptic ultrastructure
We next investigated whether the down-regulation of synaptic vesicle protein levels observed in BDV-infected neurons affected the number of synapses and their size in the cultures. The number of synapses was determined using double label immunofluorescence for presynaptic marker VAMP-2 and the postsynaptic marker PSD-95 (Fig. 2 A). The number of VAMP-2/PSD-95 double positive boutons was determined in control and infected cultures before and after treatment with BDNF. There was no obvious effect of viral infection on the number of synapses before exposure to BDNF. In contrast, BDNF-induced synaptogenesis, which was clearly seen in control neuronal cultures, was totally abrogated in BDV-infected cultures (Fig. 2B ). We also measured the size of double-labeled synaptic boutons in control and virus-infected cultures not treated with BDNF (Fig. 2C ). Although their number was not affected, the synapses were significantly smaller in BDV-infected neurons, even in absence of BDNF treatment. The smaller size of the synaptic boutons suggested that the infection disorganized the architecture of the synapses.

View larger version (24K): [in this window] [in a new window]   Figure 2. BDV infection decreases the number of synapses and their size. A) Immunofluorescence analysis of synaptic boutons 11 days after BDV infection. Cells were infected with BDV on day 1 and stimulated with BDNF from day 7 to 11. Synapses were identified by the colocalization of VAMP-2 staining (presynaptic marker, green) and PSD-95 staining (postsynaptic marker, red). B) Quantitative analysis of the number of synapses present in control and BDV-infected cultures with and without BDNF treatment. Synapses identified as described above were counted on a minimum of 10 randomly selected fields. Results from 3 separate experiments were combined to estimate the mean number of synapses per field for each experimental condition. NI-BDNF and BV-BDNF refer to control and BDV infected cultures exposed to BDNF for 4 days prior to analysis. Mann Whitney statistical test: * P < 0.05; **P < 0.01. C) Analysis of synapse size distribution, as assessed with the Simple PCI image analysis software in noninfected and BDV-infected neurons. Note that this distribution is shifted to the left in BDV-infected neurons, indicating a reduction in the average size of the synapses.

We examined this possibility using transmission electron microscopy. In control cultures, the typical ultrastructure of synapses (i.e., the clustering of synaptic vesicles in the vicinity of electron dense postsynaptic densities) was conspicuous. After treatment with BDNF, the number of synapses increased in sections from control samples. This contrasted with the situation observed in infected cultures, where the synapses were smaller and there was no detectable increase in the number of synapses. Moreover, there were striking alterations in the distribution of synaptic vesicles in the infected neurons. They were often found in clusters, accumulating in the synaptic bouton. Most of the synaptic vesicles appeared to be still associated with the actin cytoskeleton and were aggregated in crystal-like structures. Since we showed that BDV decreases the expression of proteins that play important roles in synaptic vesicle cycling, altered cycling could lead to the accumulation of synaptic vesicles in the synaptic bouton. In addition, the clustering of synaptic vesicles observed in BDV-infected cultures could be a consequence of the inhibition of ERK 1/2 phosphorylation by the virus. Since the release of the vesicles from actin filaments is triggered by the phosphorylation of synapsin I by pERK 1/2, the infection may prevent this release and cause an accumulation of actin-bound vesicles.

CONCLUSIONS AND SIGNIFICANCE

The novel findings of this study designed to examine the impact of persistent infection by BDV on neurotrophin responsiveness and synaptogenesis in primary cultures of hippocampal neurons are 1) BDV infection of neurons is very efficient, with a remarkable lack of cytopathic effect; 2) the virus interferes specifically with the BDNF-induced phosphorylation of ERK 1/2, without altering the level of TrkB; 3) after chronic exposure to BDNF, BDNF-induced synaptic protein synthesis and synaptogenesis were both abrogated. The ultrastructure of synapses—in particular, the distribution of synaptic vesicles within the boutons—was altered in infected neurons. To our knowledge, this is the first description that a persistent viral infection can selectively interfere with neurotrophin-regulated neuronal plasticity.

We propose that the virus diverts the neurotrophin signaling pathway to enhance its replication, inhibiting normal protein expression or synaptogenesis in response to BDNF. In support of this hypothesis, we showed that BDNF stimulation increased the level of BDV protein production. Like all negative strand RNA viruses, BDV encodes a phosphoprotein (P), which is an important component of the viral replication complex. The phosphorylation of BDV P is performed by cellular kinases, including protein kinase c-epsilon and casein kinase II. It is conceivable that BDV could also use kinase(s) located upstream of ERK 1/2 in the BDNF signaling pathway to phosphorylate P.

Neurotrophins such as BDNF play a major role in regulating synaptogenesis, both during development and in the adult CNS, as illustrated by the abnormal synaptogenesis observed in BDNF knockout mice. Impaired synaptogenesis can affect all brain functions, including cognition and behavior; impaired synaptic activity is now considered to cause the early cognitive deficits of many behavioral or neurodegenerative diseases in humans. In most cases, the cause of such synaptic alterations is unknown. We describe a novel type of interaction between a persistent virus and neurons and provide clues to better understand the basis of neuronal impairment caused by BDV, which may be important evidence suggesting that BDV or a BDV-like virus can infect and persist in the human brain with still unresolved clinical consequences.

View larger version (20K): [in this window] [in a new window]   Figure 3. A model of BDV interference with the BDNF signaling cascade and adaptive responses in neurons. BDNF binding to its TrkB receptor normally triggers a series of sequential phosphorylations leading to ERK 1/2 phosphorylation. We propose that BDV diverts this phosphorylation cascade, presumably through enhanced phosphorylation of its phosphoprotein, thereby enhancing viral replication in neurons at the expense of normal responses to neurotrophins. This model provides a working hypothesis to explain the effects of BDV infection on neuronal physiology, a novel type of viral interference with neurons without direct cytopathic effects.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0764fje

This article has been cited by other articles:

				D. L. Montgomery, A. Van Olphen, H. Van Campen, and T. R. Hansen The Fetal Brain in Bovine Viral Diarrhea Virus-infected Calves: Lesions, Distribution, and Cellular Heterogeneity of Viral Antigen at 190 Days Gestation Vet. Pathol., May 1, 2008; 45(3): 288 - 296. [Abstract] [Full Text] [PDF]

				R. Volmer, C. M. A. Prat, G. Le Masson, A. Garenne, and D. Gonzalez-Dunia Borna Disease Virus Infection Impairs Synaptic Plasticity J. Virol., August 15, 2007; 81(16): 8833 - 8837. [Abstract] [Full Text] [PDF]

				M. V. Ovanesov, C. Sauder, S. A. Rubin, J. Richt, A. Nath, K. M. Carbone, and M. V. Pletnikov Activation of Microglia by Borna Disease Virus Infection: In Vitro Study J. Virol., December 15, 2006; 80(24): 12141 - 12148. [Abstract] [Full Text] [PDF]

				P. Ashwood, S. Wills, and J. Van de Water The immune response in autism: a new frontier for autism research J. Leukoc. Biol., July 1, 2006; 80(1): 1 - 15. [Abstract] [Full Text] [PDF]

				D. Mayer, H. Fischer, U. Schneider, B. Heimrich, and M. Schwemmle Borna Disease Virus Replication in Organotypic Hippocampal Slice Cultures from Rats Results in Selective Damage of Dentate Granule Cells J. Virol., September 15, 2005; 79(18): 11716 - 11723. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/7/863 03-0764fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by HANS, A. Articles by GONZALEZ-DUNIA, D. Search for Related Content PubMed PubMed Citation Articles by HANS, A. Articles by GONZALEZ-DUNIA, D.