Identification of viral determinants and molecular mechanisms involved in Borna disease virus interference with human neurogenesis

Le virus de la maladie de Borna (BDV) est un virus persistant dans le système nerveux central responsable de troubles du comportement chez l’animal et possiblement chez l’homme. En utilisant des cellules progénitrices neurales humaines, des travaux antérieurs à mon arrivée au laboratoire ont montré que BDV altère la neurogenèse humaine. Les objectifs de ma thèse étaient d’identifier les déterminants viraux responsables de cette altération et de caractériser les mécanismes moléculaires impliqués. Nous avons montré que la phosphoprotéine (P) et la nucléoprotéine (N), mais pas la protéine X, induisent une inhibition spécifique de la neurogenèse humaine, la genèse des astrocytes n’étant pas altérée. Ensuite, focalisant notre étude sur P, nous avons montré qu’elle affectait particulièrement la genèse des neurones GABAergiques. La caractérisation moléculaire a ensuite révélé une diminution de l’expression de gènes impliqués dans la spécification (ApoE et Noggin) et dans la maturation (SCG10/Stathmin2 et TH) neuronale. En conclusion, nos résultats démontrent, pour la première fois, qu’une protéine virale perturbe la neurogenèse GABAergique humaine, un processus connu pour être dérégulé dans certaines maladies psychiatriques. Ils améliorent ainsi notre compréhension de la pathogenèse de ce virus persistant et de son rôle possible dans les maladies psychiatriques chez l’homme.Borna disease virus (BDV) is a persistent neurotropic virus causing neurobehavioral disorders in animals and possibly humans. Using human neural progenitor cells, it had been shown, before my arrival in the laboratory, that BDV induces an alteration in human neurogenesis. Here, we aimed at identifying the viral determinants involved in BDV-induced impairment of neurogenesis and at characterizing the underlying molecular mechanisms. We demonstrated that the phosphoprotein (P) and the nucleoprotein (N), but not the X protein, reduce neurogenesis. Focusing on the role of P, we evidenced an impairment of GABAergic neurogenesis. Then, seeking for the molecular mechanisms responsible for P-induced inhibition of neurogenesis, we showed that it induces a decrease in the expression of cellular factors involved in either neuronal specification (ApoE, Noggin) or maturation (SCG10/Stathmin, TH). Thus, in this study, we demonstrated for the first time that a viral protein is capable of inhibiting GABAergic neurogenesis, a process that is dysregulated in some psychiatric diseases. Our results improve our understanding of the pathogenesis of this persistent neurotropic virus and of its possible role in psychiatric disorders

La phosphoprotéine P du virus de la maladie de Borna altère le développement des neurones GABAergiques humains

National audienc

Identification of viral determinants involved in Borna Disease Virus interference with human neurogenesis

National audienc

Identification of viral determinants involved in Borna Disease Virus interference with human neurogenesis

National audienc

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

International audienceIt 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 lenti-viral 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. PLOS Pathogens

<i>bdv-p</i> alters the expression of <i>ApoE</i> and <i>Noggin</i>.

<p><i>bdv-p</i>- and <i>bdv-x</i>-expressing-hNPCs and their matched NT controls were induced to differentiate for 0, 4 or 14 days before RNA and protein analyses. <i>ApoE</i> expression was measured by RT-qPCR at (A) day 4, (B) day 0 and (C) day 14. (D) Western blot analysis showing ApoE level. It was normalized to actin. <i>Noggin</i> expression was measured by RT-qPCR at (E) day 4, (F) day 0 and (G) day 14. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001.</p

Lentiviral transduction and establishment of transgenic hNPCs.

<p>(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. <i>ns</i>, non-significant (p > 0.5). Scale bar, 20 μm.</p

The S26/28 phosphorylation site is not necessary for <i>bdv-p</i>-induced reduction in GABAergic neurogenesis.

<p>(A) Schematic representation of P and Paass. Paass is mutated in S26/28 residues and thus lacks the corresponding phosphorylation site. (B) hNPCs were transduced with lentiviral vectors bearing the <i>bdv-p</i> or <i>bdv-p</i>_<i>aass</i> gene and immunostained at the undifferentiated stage (day 0) with an antibody directed against the P protein (green). Nuclei were stained with DAPI (blue). Transduction efficiency was evaluated by enumeration of P-positive cells (right panel). (C) Western blot showing the level of P in <i>bdv-p</i> and <i>bdv-p</i>_<i>aass</i>-expressing hNPCs at 14 days of differentiation. P is normalized to actin. (D) Transgenic hNPCs and their NT matched controls were induced to differentiate for 14 days and GABAergic neurons were quantified. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p = 0.002. Scale bar, 20 μm.</p

Expression of <i>bdv-p</i> or <i>bdv-x</i> gene does not alter hNPCs at the undifferentiated stage.

<p>RNAs from <i>bdv-p-</i> and <i>bdv-x</i>-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of <i>bdv-p</i> and <i>bdv-x</i>-expressing hNPCs was analyzed by BrdU labeling (C) and by a mitochondrial dehydrogenase activity-based assay (D and E) in the presence of growth factors and by enumeration of DAPI-positive cells (F and G) in the absence of growth factors. Results in A and B are representative of two independent experiments performed in triplicate. Results in C represent the mean of two independent experiments performed in quintuplicate. Results in D and E are representative of 2 independent experiments performed in quintuplicate. Results in F and G are from 1 experiment performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. <i>ns</i>, non-significant (p > 0.5).</p

Expression of the <i>bdv-p</i> but not <i>bdv-x</i> gene in hNPCs impairs neuronal differentiation.

<p>Transduced hNPCs expressing <i>gfp</i>, <i>bdv-p</i> or <i>bdv-x</i> genes and their matched NT controls were induced to differentiate for 14 days. (A) Immunostaining with antibodies directed against βIII-Tubulin, a neuronal marker (red), or GFAP, an astrocytic marker (green). Nuclei were stained with DAPI (blue). For panel homogenization, GFAP immunostaining performed in <i>gfp</i>-expressing hNPCs was re-colored in green. The percentage of neurons and astrocytes was determined based on enumeration of βIII-Tubulin-, GFAP- and DAPI-positive cells in (B) <i>gfp</i>-expressing hNPCs, (C) <i>bdv-p</i>-expressing hNPCs and (D) <i>bdv-x</i>-expressing hNPCs. Results in B, C and D are representative of 2, 5 and 2 independent experiments, respectively. All experiments were performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001, ns, non-significant (p > 0.5). N, neurons. As, astrocytes. Scale bar, 50 μm.</p