Borna Disease Virus Phosphoprotein Impairs the Developmental Program Controlling Neurogenesis and Reduces Human GABAergic Neurogenesis

It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.     

Synaptic pathology in Borna disease virus persistent infection

Borna disease virus (BDV) infection of newborn rats leads to a persistent infection of the brain, which is associated with behavioral and neuroanatonomical abnormalities. These disorders occur in the absence of lymphoid cell infiltrates, and BDV-induced cell damage is restricted to defined brain areas. To investigate if damage to synaptic structures anteceded neuronal loss in BDV neonatally infected rats, we analyzed at different times postinfection the expression levels of growth-associated protein 43 and synaptophysin, two molecules involved in neuroplasticity processes. We found that BDV induced a progressive and marked decrease in the expression of these synaptic markers, which was followed by a significant loss of cortical neurons. Our findings suggest that BDV persistent infection interferes with neuroplasticity processes in specific cell populations. This, in turn, could affect the proper supply of growth factors and other molecules required for survival of selective neuronal populations within the cortex and limbic system structures.     

1-beta-D-arabinofuranosylcytosine inhibits borna disease virus replication and spread

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.     

Activation of microglia by borna disease virus infection: in vitro study

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to the certain neuronal populations. Since persistent BDV infection of neurons in vitro is noncytolytic and noncytopathic, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brain have not been investigated. To address these issues, activation of primary rat microglial cells was studied following exposure to purified BDV or to persistently BDV-infected primary cortical neurons or after BDV infection of primary mixed neuron-glial cultures. Neither purified virus nor BDV-infected neurons alone activated primary microglia as assessed by the changes in cell shape or production of the proinflammatory cytokines. In contrast, in the BDV-infected primary mixed cultures, we observed proliferation of microglia cells that acquired the round morphology and expressed major histocompatibility complex molecules of classes I and II. These manifestations of microglia activation were observed in the absence of direct BDV infection of microglia or overt neuronal toxicity. In addition, compared to uninfected mixed cultures, activation of microglia in BDV-infected mixed cultures was associated with a significantly greater lipopolysaccharide-induced release of tumor necrosis factor alpha, interleukin 1beta, and interleukin 10. Taken together, the present data are the first in vitro evidence that persistent BDV infection of neurons and astrocytes rather than direct exposure to the virus or dying neurons is critical for activating microglia.     

Borna disease virus phosphoprotein binds a neurite outgrowth factor, amphoterin/HMG-1

The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.     

博尔纳病病毒感染对神经元可塑性影响的研究进展

博尔纳病病毒(Borna Disease Virus,BDV)是一种具有高度嗜神经性的病毒。近年,有大量研究证实该病毒感染与人神经精神疾病的发生有关。但其确切机制仍未明了。一些研究认为BDV感染对中枢神经系统神经元可塑性的影响可能是其致病的重要基础。近年许多学者通过对沙鼠、小鼠、大鼠及转基因鼠等各种BDV感染模型的研究,进一步揭示了BDV感染对神经元可塑性影响的分子机制。结果发现BDV感染主要通过对星形胶质细胞功能的影响、干预HMGB 1蛋白以及神经营养因子信号转导等途径干预神经元的可塑性,影响脑内神经元的功能及其存活和发育,从而引起脑功能损害,导致宿主精神、行为异常。今后随着新的BDV转基因模型的成功建立将进一步揭示BDV感染对神经元可塑性影响的分子机制,给临床预防和治疗博尔纳病提供理论基础。     

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.     

Real time imaging of human progenitor neurogenesis

Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer's disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2(+) neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their identities when placed in diagnostic or transplantation environments.     

Borna disease virus blocks potentiation of presynaptic activity through inhibition of protein kinase C signaling

Infection by Borna disease virus (BDV) enables the study of the molecular mechanisms whereby a virus can persist in the central nervous system and lead to altered brain function in the absence of overt cytolysis and inflammation. This neurotropic virus infects a wide variety of vertebrates and causes behavioral diseases. The basis of BDV-induced behavioral impairment remains largely unknown. Here, we investigated whether BDV infection of neurons affected synaptic activity, by studying the rate of synaptic vesicle (SV) recycling, a good indicator of synaptic activity. Vesicular cycling was visualized in cultured hippocampal neurons synapses, using an assay based on the uptake of an antibody directed against the luminal domain of synaptotagmin I. BDV infection did not affect elementary presynaptic functioning, such as spontaneous or depolarization-induced vesicular cycling. In contrast, infection of neurons with BDV specifically blocked the enhancement of SV recycling that is observed in response to stimuli-induced synaptic potentiation, suggesting defects in long-term potentiation. Studies of signaling pathways involved in synaptic potentiation revealed that this blockade was due to a reduction of the phosphorylation by protein kinase C (PKC) of proteins that regulate SV recycling, such as myristoylated alanine-rich C kinase substrate (MARCKS) and Munc18-1/nSec1. Moreover, BDV interference with PKC-dependent phosphorylation was identified downstream of PKC activation. We also provide evidence suggesting that the BDV phosphoprotein interferes with PKC-dependent phosphorylation. Altogether, our results reveal a new mechanism by which a virus can cause synaptic dysfunction and contribute to neurobehavioral disorders.     

Mutation of the Protein Kinase C Site in Borna Disease Virus Phosphoprotein Abrogates Viral Interference with Neuronal Signaling and Restores Normal Synaptic Activity

Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV) represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS) and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC)–dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P) may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.     

Endogenous neurogenesis following ischaemic brain injury: Insights for therapeutic strategies

Ischaemic stroke is among the most common yet most intractable types of central nervous system (CNS) injury in the adult human population. In the acute stages of disease, neurons in the ischaemic lesion rapidly die and other neuronal populations in the ischaemic penumbra are vulnerable to secondary injury. Multiple parallel approaches are being investigated to develop neuroprotective, reparative and regenerative strategies for the treatment of stroke. Accumulating evidence indicates that cerebral ischaemia initiates an endogenous regenerative response within the adult brain that potentiates adult neurogenesis from populations of neural stem and progenitor cells. A major research focus has been to understand the cellular and molecular mechanisms that underlie the potentiation of adult neurogenesis and to appreciate how interventions designed to modulate these processes could enhance neural regeneration in the post-ischaemic brain. In this review, we highlight recent advances over the last 5 years that help unravel the cellular and molecular mechanisms that potentiate endogenous neurogenesis following cerebral ischaemia and are dissecting the functional importance of this regenerative mechanism following brain injury.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Effects of neuroinflammation on the regenerative capacity of brain stem cells

In the adult brain, neurogenesis under physiological conditions occurs in the subventricular zone and in the dentate gyrus. Although the exact molecular mechanisms that regulate neural stem cell proliferation and differentiation are largely unknown, several factors have been shown to affect neurogenesis. Decreased neurogenesis in the hippocampus has been recognized as one of the mechanisms of age-related brain dysfunction. Furthermore, in pathological conditions of the central nervous system associated with neuroinflammation, inflammatory mediators such as cytokines and chemokines can affect the capacity of brain stem cells and alter neurogenesis. In this review, we summarize the state of the art on the effects of neuroinflammation on adult neurogenesis and discuss the use of the lipopolysaccharide-model to study the effects of inflammation and reactive-microglia on brain stem cells and neurogenesis. Furthermore, we discuss the possible causes underlying reduced neurogenesis with normal aging and potential anti-inflammatory, pro-neurogenic interventions aimed at improving memory deficits in normal and pathological aging and in neurodegenerative diseases.     

Pathogenesis of Borna Disease Virus: Granulocyte Fractions of Psychiatric Patients Harbor Infectious Virus in the Absence of Antiviral Antibodies

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.     

Antiviral CD8 T cells recognize borna disease virus antigen transgenically expressed in either neurons or astrocytes

Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.     

Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain

Background

Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.

Methodology/Principal Findings

We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.

Conclusions

MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.     

Borna disease virus persistence causes inhibition of glutamate uptake by feline primary cortical astrocytes

Borna disease virus (BDV), a nonsegmented, negative-stranded (NNS) RNA virus, causes central nervous system (CNS) disease in a broad range of vertebrate species, including felines. Both viral and host factors contribute to very diverse clinical and pathological manifestations associated with BDV infection. BDV persistence in the CNS can cause neurobehavioral and neurodevelopmental abnormalities in the absence of encephalitis. These BDV-induced CNS disturbances are associated with altered cytokine and neurotrophin expression, as well as cell damage that is very restricted to specific brain regions and neuronal subpopulations. BDV also targets astrocytes, resulting in the development of prominent astrocytosis. Astrocytes play essential roles in maintaining CNS homeostasis, and disruption of their normal activities can contribute to altered brain function. Therefore, we have examined the effect of BDV infection on the astrocyte's physiology. We present here evidence that BDV can establish a nonlytic chronic infection in primary cortical feline astrocytes that is associated with a severe impairment in the astrocytes' ability to uptake glutamate. In contrast, the astrocytes' ability to uptake glucose, as well as their protein synthesis, viability, and rate of proliferation, was not affected by BDV infection. These findings suggest that, in vivo, BDV could also affect an important astrocyte function required to prevent neuronal excitotoxicity. This, in turn, might contribute to the neuropathogenesis of BDV.     

Interplay between Hsp90 and TPR domain-containing proteins in steroidogenic signaling

EGFL7 drives the formation of neurons from neural stem cells. In the embryonic and adult brain this process is essential for neurogenesis and homeostasis of the nervous system. The function of adult neurogenesis is not fully understood but maybe it supports life-long learning and brain repair after injuries such as stroke. The transition of neural stem cells into mature neurons is tightly regulated. One of the essential signaling pathways governing this process is the Notch pathway, which controls metazoan development. In a recent publication, we identified a novel non-canonical Notch ligand, EGFL7, and described its impact on neural stem cells.¹ We explored the molecular mechanisms, which this molecule affects to regulate the self-renewal capacity of neural stem cells and to promote their differentiation into neurons. In this review, we discuss the implications of our findings for adult neurogenesis and illustrate the potential of EGFL7 to serve as an agent to increase neurogenesis and the self-renewal potential of the brain.